Minicomputer monitored social behavior of mice with hippocampus lesions

BEHAVIORAL BIOLOGY 16, 1-29 (1976), Abstract No. 5139

Minicomputer Monitored Social Behavior of Mice with Hippocampus Lesions I DANIEL L. ELY 2

Department of Physiology, University of Southern California, Los Angeles, California 90007 ERNEST G. GREENE

Department of Psychology, University of Southern California, Los Angeles, California 90007 and J A M E S P. H E N R Y

Department of Physiology, University of Southern California, Los Angeles, California 90007 A detailed behavioral analysis (24 hr/day) was made of four groups of socially interacting male CBA mice in a population cage during a 24-day period. The four groups were: hippocampus lesioned animals, cortical lesioned control animals, sham operated, and unoperated control animals. Each male was magnetically tagged, and individual transactions were detected using Hall Effect sensors located at the portals to each of the eight chambers in the population cage. The sensors interfaced with a minicomputer and teletype that produced 6-hr behavior profile reports for each male. The animals with hippocampus lesions exhibited significantly higher locomotor activity and patrol patterns that peaked on day 8 at a level five times that of the control groups. They also showed greater consistency of circadian activity rhythm over the 24-day period as well as greater cresttrough amplitude as compared to control groups. The animals with hippocampal lesions spent more time in the food chamber and inc/uded it more in their patrols than the control animals. Less aggression was observed in this group, and they failed to develop a social hierarchy with dominantsubordinate relationships. However, with time locomotor activity returned to normal, and increased flight responses were observed. The evidence suggests that the hippocampus may indeed be involved with the experience and control of emotion and social behavior. 1This research was supported by NIMH Grants MH 19441 and MH 26155. The authors would like to acknowledge the technical assistance of John Henry for design and maintenance of the electrical hardware and Patricia Stephens for preparation of the figures. 2present address: Department of Biology, University of California, Riverside, Calif. 92502.

Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

ELY, GREENE AND HENRY The current impetus and enthusiasm for research in the limbic system comes from the long held theory that it is involved in the experience and control of emotion. Papez (1937) first traced out a set of anatomical structures that included the hippocampus and proposed that it might act with these structures to modulate and control the animal's drive states. MacLean (1949) further amplified this concept by noting that epileptic seizures are frequently associated with visceral, emotional, and other sensory manifestations. In animals, hippocampal stimulation or seizure may produce a variety of autonomic responses, including salivation, pupillary dilation, gagging, retching, piloerection, as well as various movements that might be interpreted as "defensive behavior" (Green, Clemente, and deGroot, 1957). In more recent work, a few theorists have supported the idea that the hippocampus is active in the regulation of drives or in the experience of emotion (Brady, 1958; Pribram, 1960; deGroot, 1965; Jarrard, 1973). It is interesting that a concept once widely accepted would now be widely rejected, each stand being taken on the basis of very little firm evidence. True enough, there are a number of contradictory reports on the effect of" hippocampal lesion. While Spiegel, Miller, and Oppenheimer (1940) and Rothfield and Harman (1954) reported rage tendencies following lesions of hippocampus and fornix, the results of Kluver and Bucy (1937) and Bard and Mountcastle (1948) suggested that the animals were tamer and more friendly following damage that included the hippocampus. The existence of contradictory effects does not seem an adequate basis for rejecting the theory of an emotional role for the hippocampus, since the anatomical projections to, from, and within this structure are complex and involve inhibitory as well as excitatory interactions. More important were the subsequent studies showing that other areas were active in the control of aggression. The "taming" noted by Kluver and Bucy (1937) followed removal of the entire temporal lobe, including neocortex, pyriform cortex, amygdala, and hippocampus. When several laboratories demonstrated that taming and changes in social dominance could be produced by damage to the amygdala or pyriform lobe (e.g. Rosvold, Mirsky, and Pribram, 1954; Green, Clemente, and deGroot, 1957), there was a tendency to discount any contribution on the part of the hippocampus. It was the intent of our study to reexamine the question of the role of the hippocampus in the control of emotional responses and social behavior. In the interest of better examining the classic suggestions of Papez and MacLean, we have monitored patterns of social behavior such as: territorial defense, eating and drinking behavior, aggression, and dominant-subordinate relationships by utilizing a computerized version of a recently developed behavioral monitoring system (Ely, Henry, Henry, and Rader, 1972).

HIPPOCAMPUS AND SOCIAL BEHAVIOR METHODS

Subieets All animals lived under the same light-dark cycle and level of illumination (light, 0600-1800hr; dark, 1800-0600 hr) and a constant room temperature of 68-72°F. All were reared with their mothers until 3 weeks of age and then weaned, sexed, and kept together as siblings in a standard laboratory cage (23 × 11 X 11 cm) for 13 weeks. Twenty males were assigned to one of four colonies depending upon the surgical treatment received (five males per colony). Littermates were not assigned to the same colony. In addition, each colony contained 10 CBA females of the same age, who received no surgical treatment, nor was their behavior monitored.

Surgery All operations were performed in one stage using surgical technique, an aspiration pump, mad a Bausch & Lomb dissection-scope. Each mouse was anesthetized intraperitoneally with Nembutal, using an initial dose of 1.5/~g/g body wt and additional small doses given as needed to maintain anesthesia. At the end of surgery a 20-/al dose of Metrazol was given to bring the mice out of the anesthetic more rapidly. The mice were assigned to groups that received one of four treatments: (a) lesion of the hippocampus and overlying cortex (Hippocampals), (b) lesion of the cortex, but sparing the hippocampus (Cortical Controls), (c) removal of the bone over the cortex, but inflicting no lesions (Shams), and (d) no surgical intervention (Controls). To perform the operations, the head was shaved, an incision was made above the midsaggital suture, and the skin was retracted and held with hemostats. An opening was made in the posterior half of the parietal plate. For the animals of the Sham group this was the extent of the intervention. The skin was then closed with sutures using interrupted stitches. For the animals of the Cortical Control group, the dura was cleared from over the cortex, and the tissue above the hippocampus (including corpus callosum) was removed using the suction pump (Gomco aspiration pump) and a 21-gauge needle for the aspiration tip. Among the animals of the hippocampal lesion group, both the cortex and also all portions of the hippocampus that could be visually identified were removed. The wound was rinsed with saline until the major bleeding had stopped, then the skin was sutured closed. One hippocampal male died on day 1 of colony interaction and was not replaced since this would have disrupted the social behavior of the resident males.

ELY, GREENE AND HENRY The surgical procedure differed somewhat from the methods commonly used with rats (vide Isaacson, Douglas, and Moore, 1961; Kimble, 1963; Greene, 1971). In the rat, the temporal muscle is retracted from over the temporal skull plate, and the opening is made in the temporal as well as the parietal plate, In the mouse the temporal ridge is quite lateral, and the entire procedure can be accomplished from the top of the skull. Hence the site of cortical damage is less lateral than is common in studies using rats, but the extent of the hippocampal damage is comparable (see Histological Results). The mice were allowed to recover for 2 weeks following brain surgery before undergoing surgery for magnet placement, which entailed ether anesthesia and implantation with either one or two magnets. The Alnico VIII magnets (measuring 2 X 9 mm) were dorsally or ventrally implanted in the demagnetized state in invaginations of muscle tissue (Ely et al., 1972). When subsequently magnetized in a 300-kG field, the direction of polarity of the dorsal or ventral magnets was used in an eight-combination binary code to identify each of the mice. Five thousand units of crystalline penicillin G (IM) was given after the operation, and the animals were placed into the population cages approximately 1 week post surgery (3 weeks after the brain surgery).

~3ehavioral Monitoring Equipment The population cage consisted of a 270 X 270 × 13-cm open field area to each side of which were attached two 23 × 11 × 11-cm standard polycarbonate cages (Fig. 1). The eight specific cages included: a feeding area, drinking area, activity wheel area, three living areas, and two female nest box areas. Each of the tubes connecting the areas with the open field passed through a magnetic detection portal. Hall generators were placed in the upper and lower grooves of the portal so that as a mouse traversed it, an electric signal was generated. An interfacing electronic circuit converted the signals into a binary code that specified the identity of the animal passing through the portal. This information was sent into computing equipment consisting of a Cincinnati Milicron (CIP/2100) minicomputer, an ASR33 teletype unit, and a parallel teletype controller. The computer routines provided a record of each mouse transaction (i.e., passing a portal to enter or leave one of the chambers), specifying both the time and location of the transaction. Summaries of colony activity were provided at 6- and 24-hr intervals. Several parameters of activity were tallied, some being redundant as indicators of colony behavior, and others containing information provided in greater detail by processing a paper-tape record of individual transactions using an IBM 370 computer. In addition to the continuous minicomputer summaries, paper-tape recordings were made for three 24-hr periods-days 4, 11, and 22. In these records, each transaction is punched onto a paper tape for subsequent analysis by an IBM 370, Model 158 computer. The transactions can be evaluated in

HIPPOCAMPUS AND SOCIAL BEHAVIOR FEMALE

5

FEMALE

MALE

[ 1 II-OPEN FIELD

WATER

MALE

30 cm MALE

FOOD

Fig. 1. Eight box open field population cage. various ways to show qualitative and quantitative differences in colonyactivity. In this study, we will report: (a) the number of transactions per hour and percentage time spent in each of the colony-chambers; (b) frequency and length of patrol behavior (entry into three or more chambers with less than 1 rain between chambers; (c) which colony-chambers tended to be included in patrols; (d) frequency of uninterrupted transactions between food and water chambers; (e)the circadian activity rhythm; (f) frequency of revisits to a chamber the animal had just left; (g)the incidence of displacement among animals (one animal leaving a chamber when another enters); and (h)ethograms, which provide an illustration of individual behavior patterns. Behavior patterns between the food and water areas were measured by calculating the humber of times a male went from either the food box directly to the water box, or vice versa, as opposed to going to another box. On days 4, l l , and 22, 10 food or water transactions were recorded starting at 1200hr and continuing until 10 such events occurred. The results were expressed as the percentage of time the animal went from food-water or vice versa as compared to going to another area from the food or water chamber. Reentry patterns were examined by tabulating the frequency of exits and reentries into the same box in a 24-hr period expressed as a percentage of the total activity. Displacement activity was measured by counting the frequency of male entries into a box from which another male had been

6

ELY, GREENE AND HENRY

displaced (left box) within 30 sec. The scores are expressed as a percentage of the total activity for that period. The current behavioral monitoring system can record two colonies simultaneously. To conduct the present study we first tested the Hippocampal and Cortical Control groups. The males with hippocampus lesions were placed in one of the population cages along with the unoperated females, while Cortical Control males and unoperated females were placed in the other. Male social rank was determined by a previously described method that uses physical appearance, size of territory, and number of female associations per unit of time (Ely and Henry, 1974). The animal with the highest total score was classified as Rank 1 or dominant and descending ranks required a point separation of two.

Histology At the end of the experiment, the animals with lesions were sacrificed with Nembutal and perfused via the heart with a cold solution of 1% DMSO-10% glycerol. The brains were then removed, frozen, and sectioned in an IEC cryostat to verify lesion placement. Sections were taken every 100/am, beginning at the level of the fornix. Following this the sections were fixed in 4% formalin for a minimum of 1 day and then washed and dehydrated through a sequence of ethyl alcohol and xylene. The sections were then rehydrated, stained in 0.5% cresyl violet, refixed in acetic formalin, and dehydrated in changes of 95% ethanol, 100% ethanol, and xylene. The sections were then mounted with coverslips. Placement of the lesions was examined using a dissection microscope. RESULTS The data were analyzed primarily using a Multivariate Analysis of Variance (MANOVA) program, developed at the University of North Carolina and modified for use on the USC 370 computer. This program renders an overall test for significance among the dependent measures, as well as a univariate analysis on each of the measures. In most cases, post-hoc paired comparisons were not calculated, since the source of differences among the groups could be determined by inspection of figures or tables. However, Student's two-tailed t tests were calculated for: inactivity time, number of boxes in a patrol, and specific chambers included in a patrol. The term control groups is used in the text to- designate the Cortical Controls, Shams, and Normal Controls.

Locomotor Activity Figure 2 shows the general activity rates for the four groups of animals

HIPPOCAMPUS AND SOCIAL BEHAVIOR ENTRY-EXITS/HOUR /ANIMAl. 60-

GENERAL ACTIVITY PATTERN

20

o • [] •

0 0

7

I

I

I

4,

8

12

I

I

16

20

CONTROLS SHAMS CORTICALS HIPPOCAMPALS

I4 2

DAY

Fig. 2. Locomotor activity in the four groups of males.

over the 24-day test period. The males with hippocampus lesion exhibited higher overall activity, which peaked on day 8 at a level approximately five times higher than tile activity of the control groups. Comparing the three control groups (summed across days) it was found that they did not differ significantly from one another (P= 0.858), though taken together they did score significantly lower than the group with lesions of the hippocampus (P= 0.01). Univariate analysis for trends showed significant decreasing activity for the Hippocampus Lesion Group (P= 0.001), while the other three groups showed no significant change over time (P=0.891). The trend difference between the Hippocampus Lesion Group and the control groups was also significant (P = 0.002). Figure 3 shows the circadian activity rhythms of the four groups expressed as the percentage of activity that occurred in each of the four 6-hr intervals. In comparing the rhythms, the most striking characteristic is the consistency of the activity pattern of animals with hippocampal lesions, whereas the other three groups showed marked temporal variation in crest and trough activity across days. On most of the test days, the Hippocampal Lesion Group had a peak of activity at 1800-2400 hr (evening) and minimal activity at 0600-1200hr (morning). Values for the four periods of activity were collapsed across days and compared statistically. Overall there was no significant difference in the circadian activity among the three control groups (P = 0.187), although univariate tests at each time period yielded a marginally significant difference at 1200-1800hr that was due to abnormally high activity among the corticals. The Hippocampal Lesion Group differed from control groups as indicated by the MANOVA test (P= 0.03), and the univariate probabilities were also significant on the following time periods: 1200-1800 hr (P= 0.08); 1800-2400 hr (P = 0.007); 0600-1200 hr (P = 0.006). They also differed from control groups in the occurrence of periods in which no animal was active in the colony for 15 min or more. Table 1 shows

8

ELY, GREENE AND HENRY CIRCADIAN ACTIVITY RHYTHM %ACTIVITY/6HOUR TIME BLOCK 504050- ~ 0

o CONTROLS • SHAMS

20.

I0.

0

,

i

i

i

i

i

i

i

t

i

i

i

i

t

E

i

]

~

I

,

i

i

t

I1~

3o-J~"A 20-

13 CORTICALS • HIPPOCAMPALS

I00

18 214 13 12

HOUR

8

DAY 4

12

16

20

24

TIME

Fig. 3. Circadian activity rhythms of the four groups expressed as the percentage of activity occurring in 6-hr intervals.

TABLE 1 Average Inactivity Times during a 24-hr Perioda Day 4 Number of inactivity periods

Day 11

Day 22

Inactivity time (rain)

Number of inactivity periods

Inactivity time (min)

Number of inactivity periods

Inactivity time (rain)

9

24 -+2.9b

16

22 _+1.4

19

34 _+3.7

4

22 -+2.5

11

22 -+4

16

27 -+4.5

Cortical Controls

14

32 -+5.6

6

28 -+5.0

10

22 _+2.5

Hippocampus Lesions

6

51 -+16.7

5

6

59 -+13.1

Group Shams Controls

aNo activity in the colony for 15 min or more. b Sta n dard error of the mean.

87 _+21

HIPPOCAMPUS AND SOCIAL BEHAVIOR the frequency and duration of such inactive periods. Although they had fewer inactive periods, they were significantly longer than the duration of control groups on days 11 and 22 (P = 0.01 for both days). As might be expected, the greatest number of these inactive periods occurred during the period of minimal circadian activity (0600-1200 hr). The degree of interaction with each of the five functional areas (water, food, male, female, and activity-wheel) was examined in terms of the activity rate (number of entries and exits) as well as the time spent in the area. The overall comparison of activity rates in the five functional areas (using MANOVA) did not indicate a significant difference between hippocampal lesion and control groups, although the univariate tests indicated a significantly higher activity rate by the Hippocampal Lesion Group in the activitywheel area (P = 0.02), and the difference approached significance in the female area (P=0.07). There was no indication o f an overall trend difference between hippocampal lesion and control groups. Table 2 shows the percentage of time per day that each group spent in the functional areas. There was a significant difference among the control groups (P= 0.004) due primarily to the amount of time spent in the activity-wheel area (P= 0.02). The hippocampal lesion animals differed from the control groups in that more time was spent in the food area (P= 0.01), expecially on day 2, and less time was spent in the male area (P = 0.05). Analysis of the number of patrols (entry into three or more areas with less than 1 rain of latency between areas) showed that across days the Hippocampal Lesion Group patrolled significantly more than the other three groups (P = 0.014, Fig. 4A). No trends were evident among the control groups (P= 0.843). Like overall activity, the number of patrols by animals with lesions of the hippocampus peaked at day 8, and returned to control levels by day 16. Overall this was a decfining trend that was significant when they were considered alone (P= 0.041) or when compared to the control groups (P= 0.005). Figure 4B shows that the patrols of the Hippocampal Lesion Group included significantly more chambers than the other three groups on days 2 and 11 (P = 0.01 for each day). An analysis was made of the frequency with which particular functional areas were included in a patrol. The only significant finding was that the Hippocampal Lesion Group included the food area in their patrols far more often than did the control groups on day 2 (22 vs 9%, P = 0.01, Fig. 4C). Behavior Patterns

Abnormally high interaction with the food chamber was further suggested in the detailed daily records in which percentage of food-chamber time was monitored for the hippocampal lesion animals. It must be noted that with our measurement technique, the amount of food consumed was not directly measured, nor was the actual amount of time spent eating even though the

9-+1.5

Hippocampus Lesions 5 -+ 1.5

10-+1.0

10_+2.7

49_+8.0

19_+2.5

20-+4.0

1 5 - 3.7

2

16_+2.5

17_+4.5

20+5.0

12±5.0

Day 11

Food

25-+5.0

15+-4.5

19--7.0

35-+ 3.5

22

Day 11

5.0

7.0

4_+ 1.5

21-+

29+

8 -+ 4.0

12_+ 3.0

2 1 -+ 5.5

46-+ 10.0 43-+ 10.0

2

Male

Chamber

5.2

32_+ 4.0

27_+ 3.6

20-+11.0

21-+

22

25-+2.0

34-+0.9

19+5.8

13-+5.7

2

3.5

26-+

5.5

27 + - 5.4

16+

26-+ 15.0

Day I1

Female

aThe totals for the five areas do not total 100% for each group because sometime is spent between areas and in the open field. bStandard error of the mean.

16-+5.4

i 3 _+2.2

Cortical Controls

5..0.3

13_+3.0

31-+8.5

12

2 0 ± 1.5

5 _+ 1.1 b

11 -+ 3.1

Day 11

Shams

2

Controls

Group

Water

TABLE 2

2.7 17_+ 0.5

21-+

39+12.5

51-+ 10.4

22

Elapsed Time Spent in Each o f the Five Functional Areas Expressed as a Percentage of Time per 24 hr a

3_+0.7

2-+1.8

13--5,0

2.4-+ 1.0

2

3_+1.5

3-+0.9

3±1.5

0

Day 11

Activity

3_+1.5

3-+1.8

1+0.9

0

22

Z

> Z

t~

O

HIPPOCAMPUS AND SOCIAL BEHAVIOR

11

PATROL ANALYSIS

TOTALPATROLS/24HR /ANIMAL 160 -

A 80

0 DAY NUMBER OF BOXES IN PATROL/ANIMAL 20.

B

I£

0 DAY 2

I~l

2'2

*/.TIME FOOD BOX IN PATROL/ANIMAL 50-

20C

oCONTROL eSHAM BCORTICALS AHIPPOCAMPALS

I0-

0 DAY '~

I'1

~2

Fig. 4. Patrol analysis for the four groups of males. (A)total patrols (entry into three or more boxes in less than 1 rain between boxes), (B)the number of boxes included in a patrol, (C) the percentage of time the food box was included in a patrol. amount o f time in the food chamber was measured. However, from direct observation of the colonies, we k n o w that most of the time in the food area is spent feeding. Figure 5A shows that across the period of study the Hippocampal Lesion Group spent more time in the feeding area than did the other three groups ( P = 0.002), with the effect being most pronounced on days 4 and 8. There were no significant trends among the control groups, but the decline in food-chamber time o f the Hippocampal Lesioaa Group was significant as compared to control groups (P = 0.002). Figure 5B shows the mean frequency of transactions between the food and water area made by each o f the groups (passing directly from the water

12

ELY, GREENE AND HENRY ANALYSIS OF FEEDING BEHAVIOR

%TIME IN FEEDING AREA/ANIMAL 50" 4030-

A

20,

0 DAY

% FOOD- WATER LINKAGES 7560.42-

B

~

28140 DAY 2

III

212

%TIME OTHER MALES FEEDING WITH DOMINANT 75.

C4530. ~ 0

o DONTROLS • SHAMS D CORTICALS • HIPPOCAMPALS

L5.

18oo

zgoo

odoo

labo

IBO0 HOURS

DAY II

Fig. 5. Feeding behavior analysis. (A) the percentage of time spent in the feeding area, (B) the percentage of time that a transaction into the food area was followed by a transaction to the water area or vice versa, (C) the percentage of time at least one other male was feeding with the most active male (dominant). chamber to the food chamber or vice versa). On day 2, 73% o f the food chamber transactions involved food-water linkages, while the average o f the other three groups was 46%. Subsequently, the number of linkages among the Hippocampal Lesion Group dropped precipitously to 28%, then to 23% on days 11 and 22, respectively, while the other three groups remained relatively stable around 45-50%. There were no significant differences among the control groups, but the Hippocampal Lesion Group was significantly different from the control groups ( P = 0.013). Further analysis of feeding behavior showed that there was more group feeding among the Hippocampal Lesion Group than in the other groups. When the most active animal (dominant) in

HIPPOCAMPUS AND SOCIAL BEHAVIOR

13

the control colonies was feeding, there was a 20% chance that another male was also feeding; however, among the Hippocampal Lesion Group there was a 60% chance that others were feeding with the most active male. Two other aspects of behavior that show sustained departure from control values can be seen in Fig. 6. In Fig. 6A the animals were scored for the number o f times the entry of one male into a chamber led to the exit of another male within 30 sec, which may serve as a measure of "displacement activity." Although the animals with hippocampal lesions were normal in the frequency of displacement activity on days 2 and 11, by day 22 the frequency of displacement had increased 9-fold and provided an overall significant difference when compared to the control groups (P= 0.01). Figure 6B shows the frequency of revisits (returning to the chamber from which the animal just exited). While the frequency of revisits among the control groups was relatively stable across time, there was a sustained decline of revisit activity in the Hippocampal Lesion Group as compared to control groups (P= 0.001). ORGANIZATION OF BEHAVIOR DISPLACEMENTS % OF TOTAL ACTIVITY 3024. A

1812-

0 REVISITS %OF TOTAL ACTIVITY

504.0B

. :.'50-

o CONTROLS • SHAMS n CORTICALS • HIPPOCAMPALS

20-

I0o

~

ii

~z DAY

Fig. 6. (A) the percentage o f time that one male enters an area and displaces the resident male in less than 30 sec, and (B) the percentage o f time that a male leaves a box and reenters the same b o x w i t h no other transaction.

14

ELY, GREENE AND HENRY

Social Organization Social rank was evaluated on days 18 and 22 using a previously established procedure (Ely and Henry, 1974). Figure 7 shows the social rank score for each male in the four colonies. A finding that is central to the interpretation of other results in our study was the lack of social differentiation among the animals with hippocampus lesions. In each of the other three groups a def'mite dominant (rank 1) male emerged with a social ranking score of 14 out of a possible total of 15. A point separation of two is required for classification into a descending rank (Ely and Henry, 1974), and in each of the three control groups there were at least two males with very low scores (subordinates), and in the Sham and Cortical Control groups there were four males with low scores (see Fig. 7A, B, C). Among the males with hippocampal lesions there was an absence of a definite dominant since three of the animals scored 12 and one scored 10 (see Fig. 7D). In each of the control groups it was only the dominant animal who increased his score between days 18 and 22, whereas all the animals in the Hippocampal Lesion Group increased their ranking scores. Another indicator of the development of a social dominance hierarchy is SOCIAL RANK

DETERMINATION

TOTAL SOCIAL RANKING SCORE CORTICALS

15-

15CONTROLS

9 C 3. 0 0

I

15-

3 7 ANIMAL NUMBER

8

0

SHAMS

l

15-

9-

3

7

9

HIPPOCAMPALS

9D

30

6

0 I

3

4

7

0

3

5

7 DAY 18 DAY 22

Fig. 7. Social rank determination for each male in each group on day 18 and day 22.

HIPPOCAMPUS AND SOCIAL BEHAVIOR

15

TABLE 3 Activity Differential Score for Entry Exits/hr and Patrols/24 hr Based on the Ratio of the Most Active Animal (A) in the Colony Minus the Next Most Active Animal (B) Divided by A and Multiplied by 100 (Dixon and Massey "Outlyer Procedure")a lstweek

2ndweek

3rdweek

4thweek

Average of 4weeks

Groups EE

P

EE

P

EE

P

EE

P

EE

P

Shams

73

90

79

98

87

95

71

89

77

93

Controls

67

55

68

88

77

81

93

97

76

80

Cortical controls

71

89

86

94

80

90

87

93

81

92

Hippocampus Lesions

34

43

27

47

7

0

28

56

24*

37**

Eight control colonies (averaged)

76

85

83

93

80

91

80

92

80

91

aEE=Entry exits/hr; P=patrols/24 hr. The higher the differential score, the greater the social differentiation in the colony. P < 0.05. **P < 0.001. the differential activity score. Dominant males are the most active and constantly patrol the colony. An activity differential can be calculated for total entry/exits or for the frequency o f patrols using the formula A - B X 100, where A and B represent the activities of the most and next most active animals. F o r instance, if animal A averages 40 entry/exits per hour, and animal B averages only five, the differential ratio is 87.5. Table 3 shows these ratios for each o f the groups in this experiment and for t h e average o f eight additional control colonies from other experiments (same age, composition, and social situation). It can be seen that the Hippocampal Lesion Group has low scores in each o f the 4 weeks o f measurement, which indicates a lack o f normal social differentiation. Using the " o u t l y e r " procedure of Dixon and Massey (1969), it was found that the score o f this group was significantly less than the scores o f the control groups for entry/exits per hour as well as for patrols (P = 0.02 and P = 0.001, respectively). Figure 8 consists of ethograms of activity within the colonies, showing the individual patterns o f traffic among the eight chambers. F r o m an inspection o f the figures it is clear that in each of the control colonies there is one male with a complex pattern of activity (Sham 11, Control 20, Cortical 13), the same animals that were judged to be dominant using the methods o f

16

ELY, GREENE AND HENRY SHAMS

17EE/HR

5EE/HR

FEMALE

WATER

FEMALE

MALEo

MALE

MOUSE II

MOUSE 13

2EE/HR

MOUSE I0

CONTROLS MALE

ISEE/HR

2EE/HR

FEMALE

WATER,FEMALE MALE

MALE MOUSE 20

MOUSE 28

IEE/HR

) o

MOUSE 24

Fig. 8. Ethograms (24 hr) of the most active males in each of the four groups on day 22. The Shams and Controls have ethograms of only three males due to a technical problem; however, in all cases the most active males aEe represented in the ethogram.

HIPPOCAMPUS AND SOCIAL BEHAVIOR CORTICAL CONTROLS

MALE W

A

23EE/HR

7EE/HR FEMALE

T

~

LE

MOUSE13

MOUSE17 I EE/HR

5EE/HR

/ MOUSEI0

MOUSEII

HIPPOCAMPALS I0 EE/HR MALE

IOEE/HR FEMALE

FEMALE

WATER

MALE

MALI~

MOUSE27

MOUSE20

5 EE/HR

3EE/HR

MOUSE23

MOUSE25

Fig. 8. Continued.

17

18

ELY, GREENE AND HENRY

scoring described above. However, there is far less differentiation among the behavior patterns and activity rates of hippocampal lesion males (Fig. 8D). In several ways the activity of each of the hippocampal lesion males was like that of the dominant animals in the other three colonies. In order to evaluate this aspect of the results statistically, the data of the dominant animals in each of the control groups were compared to the data for subordinate animals and for the Hippocampal Lesion Group. In overall activity across days the dominants did not differ significantly from hippocampal lesion animals (P=0.959), nor was there a difference in the activity trends (P= 0.221). Dominant and hippocampal lesion animals both differed significantly from subordinate animals in overall activity (P= 0.001, P = 0.026, respectively). Figure 9 shows the number of transactions in each of the functional areas of the colony. On day 2 the Hippocampal Lesion Group had higher activity scores than dominants or subordinates in all but the male chambers. Averaged across the period of the experiment, there was no significant difference between hippocampal lesion and dominant animals (P = 0.314), but both alike had higher transactions than subordinates animals in the five functional areas (P=0.001 and P = 0 . 0 2 6 , respectively). Univariate tests showed the greatest hippocampal-subordinate difference to be in the number

DOMINANT, SUBORDINATE AND HIPPOCAMPAL LOCOMOTOR ACTIVITY

ENTRY-EXI HOUR TS I!1 ~ W A T E ARREA

i

I01 ~ o ~

1 ~~--------~EMALE° ~3 AREA -o

t

i

i

t

E

i

i

i

~ AERAM

i

A

L~20 E OVERALL ACTIVITY

?

~I DAY

,

AoHI PPOCAMPALS n=4 NANTS oDOMI SUBORDI Nn=3 ATES n=12

Fig. 9. Comparison of locomotor activity between hippocampals, dominants, and subordinates in each of the functional areas.

HIPPOCAMPUS AND SOCIAL BEHAVIOR

19

of transactions with the male, female, and activity-wheel areas ( P = 0.002, P = 0.001, and P = 0.001, respectively). Allocation of time to the functional areas was also evaluated. Overall, the hippocampal lesion animals did not differ from dominant animals in the time they spent in the five areas (P= 0.284), though univariate analyses did indicate that they spent significantly less time in the male area (P= 0.001). There was a marginally significant difference in time allocation between the hippocampal lesion and subordinate animals (P= 0.006). Dominant animals did not differ from subordinate animals in overall pattern of time allocation nor when the functional areas were considered individually. In general, hippocampal lesion animals were more like dominant animals in their activity, showing significantly more transactions with most of the colony areas and far more patrols than produced by subordinate animals. However, inspection of Fig. 9 d o e s show that by the end of the experiment the transaction rates were very close to subordinate animal levels. Hippocampal lesion animals were unlike dominant animals in that they did not inflict injuries upon the other animals of the colony. i

Fig. 10. Photomicrographs of a representative hippocampal lesion showing anterior and posterior locations. Dorsal hippocampus has been removed completely, but portions of ventral hippocampus remain.

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Histology Results The hippocampal lesions of the present experiment were quiet comparable to those commonly used with rats (Fig. 10). Less cortex lateral to the hippocampus was removed, and this tissue was folded in against the thalamus, filling the space normally occupied by the hippocampus. Dorsal hippocampus was removed entirely. There was some sparing of posterior hippocampus, and the ventral quarter of the hippocampus was commonly intact. Cingulate cortex, cingulum, thalamus, stria terminalis, and amygdala were totally undamaged. The amount of neocortex removed was comparable for the hippocampal lesion and for the cortical lesion control animals. Among the Cortical Control animals, it was common for the upper layer of the hippocampus to show the effects of abrasion against the skin covering the opening in the skull. The skull of the mouse is extremely thin, and at autopsy the exposed hippocampus appeared to be in contact with the skin. Damage to the hippocampus was not extensive but included loss of the superficial regions of alveas and pyramidal layer in the dorsal quarter of the hippocampus.

DISCUSSION One of the most conspicuous effects of the hippocampal lesion was to change the activity level of the animals during the first 2 weeks of colony interaction. This was reflected both in terms of the total frequency of entries and exists into the various chambers of the colony and in the number of patrols executed by the animals. At least 75% of the animals with hippocampal lesions patrolled 20 or more times per day over the 24-day period, whereas only the dominant animal patrolled at this rate in the other three groups. A number of studies have reported hyperactivity in animals with hippocampal lesions (see reviews by Douglas, 1967; Kimble, 1968; Jarrard, 1973), but the reason for the change is not clear. Some aspects of the behavior suggest that general drive or arousal level has been affected by the lesion (Jarrard, 1-973). However, it has also been suggested that the change may represent an absence of "habituation," in that active, exploratory responses to a novel environment do not decline as a function of time and experience (Douglas, 1967). After a period of initial exploration, a normal animal becomes familiar with its surroundings and eventually settles down to groom or to rest. Under the same conditions the hippocampal lesioned animal may continue to be active, and one can argue that its behavior has been "captured" by the cues of the novel environment, which implies an attentional dysfunction. On the other hand, the animal might be continuing to explore because it has not yet gained adequate familiarity with the environ-

HIPPOCAMPUS AND SOCIAL BEHAVIOR

21

ment, which implies a memory dysfunction (Kimble and Greene, 1968; Henderson, Henderson, and Green, 1973). Similar explanations of the hyperactivity can be offered in terms of motor dysfunction or the lack of organization of voluntary movements (McCleary, 1966; Vanderwolf, 1969; Wishaw, 1972; Teitelbaum and Milner, 1963; Greene, Saporta, and Walters, 1970; Saporta and Greene, 1974). It seems that hyperactivity is especially marked when the animal interacts with a novel environment and is less often seen in small chambers or in activity wheels (Isaacson, 1974). However, Jarrard (1968) and Kim, Choi, Kim, Chang, Park and Kang (1970) have noted increased nocturnal home-cage activity in hippocampal lesioned rats, and Jarrard (1973) found that this nocturnal activity persisted for up to 4 months after surgery when measured either in the home cage or in an activity wheel. In the present study the circadian timing of the hyperactivity was in agreement with these reports, but the very large changes in activity level dropped off by the end of the second week of observation. By the end of the month the activity of the hippocampal lesion animals was still slightly elevated and possibly would have remained at this level if the observations had been continued. Even so, it seems likely that the major portion of the high transaction rate of the Hippocampal Lesion Group was a function of abnormal exploratory behavior. Perhaps the animals were impaired in their ability to form an adequate "map" of the spatial position of the chambers, as will be discussed subsequently. Several investigators have suggested that the hippocampus is involved in the regulation of food and water consumption (MacPhial, 1968; Fisher and Courey, 1962; Courey, 1967; Kimble and Coover, 1966; Jarrard, 1973). Although we did not directly monitor how much food or water was consumed by the animals of the present experiment, measures of the number and duration of visits seem a reasonable way to establish the existence of abnormal motivational states. Using these indicators, we found minimal evidence of preoccupation with water. There was initially a high number of food-water linkages among animals with hippocampus lesions (moving directly from the food chamber to the water chamber, or vice versa), but by the second week this had declined to an abnormally low level. In terms of the number of trafisactions with the food chamber and the amount of time spent in that area, there was evidence that this group were abnormally preoccupied with food. As was the case for overall activity, the amount of time spent in the food area decreased precipitously during the first 2 weeks; it is possible that the animals simply had to eat more to supply the extra calories needed to maintain their high level of activity. This is consistent with the observation by Kimble and Coover (1966) that while the amount of food consumed by rats with hippocampal lesions is significantly higher than normal, the animals remain normal in body weight. Considering the history of theory and speculation about hippocampal

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function, the most provocative result of the present study was the suggestion that hippocampal lesioned animals were abnormal in their pattern of social interaction, particularly with regard to aggression and dominance. The hippocampal lesioned animals had healthy coats with an absence of wounds, scarring, or discoloration of the hindquarters and tail. They had larger territories than is found among subordinate animals and did not show the differential activity that is seen between dominants and subordinates. Furthermore, there was a synchrony of feeding behavior in the hippocampal lesion colony, unlike a normal colony in which the subordinate males rarely will eat in the presence of the dominant animal. Also, no particular hippocampal lesioned animal was singular in displacing other animals as was the case in control colonies, but rather they displaced each other equally. In general the group did not exhibit much behavioral individuality; all appeared similar to dominant animals in terms of hyperactivity and patrols, yet they were completely unlike the dominant in terms of aggression. This is consistent with several reports of abnormal aggression and defense in animals with hippocampus lesions, for example, less biting of objects (Glickman, Higgins, and Isaacson, 1970), a decrease in shock-induced aggression (Eichelman, 1971), and impairment of defensive behavior (Kim e t al., 1971; Blanchard and Blanchard, 1972). Seigel and Flynn (1968) observed that electrical stimulation of the hippocampus could modify the threshold for aggression in cats. Their cats were induced to attack rats using stimulation of the ventromedial hypothalamus. Concommitant stimulation of ventral hippocampus reduced the latency of attack, and stimulation of dorsal hippocampus increased the latency of attack. As might be expected, lesions of the dorsal and ventral areas produced the converse effects. Consistent with this finding, Green, et al. (1957) noted that rage could be produced by electrolytic lesions of the hippocampus of cats, but it was always accompanied by seizure in the remaining hippocampal tissue. MacLean and Delgado (1953) and Kaada, Jansen, and Anderson (1953) have found rageful and defensive behavior from chemical or electrical stimulation of the hippocampus. In the period following seizure (i.e., during postictal depression of hippocampal activity), animals show an enhancement of "pleasure reactions" and are more tame (MacLean, 1957). There is reason to believe that postictal depression of the hippocampus produces a functional blockade similar to the effects of lesion (Greene, 1971; Greene and Lomax, 1970). Thus the observations of MacLean are congruent with the present results in suggesting a role for the hippocampus in the initiation and control of aggression. An argument might be made that the male with a hippocampus lesion that died could have eventually been the "dominant" male, consequently no hierarchy developed. However, we have two different lines of evidence opposing this idea. First, when a dominant male is removed from a control

HIPPOCAMPUS AND SOCIAL BEHAVIOR

23

colony, a new dominant emerges within 24-48 hr (Ely, 1971; Ely, Henry, and Jarosz, 1975). Secondly, in a folio,w-up set of experiments using the same basic experimental design, hippocampal lesioned males did not develop a social hierarchy when all five males were present (Ely, Greene, and Henry, in preparation), Also, in our follow-up study of the physiological responses of hippocampal lesioned animals (Ely, Greene, and Henry, in preparation), we have directly observed encounters between animals and have found that there was no aggressive activity among the hippocampal lesioned animals. They did not behave aggressively toward one another, nor did they attack an intruder that had been placed in the colony. Kolb and Nonneman (1974) report that hippocampal lesion in the rat reduces the amount of social contact in an open field and eliminates shock-induced aggression, though muricide was not affected by the lesion. Similar results have been reported by De Castro and Marrone (1974). Using cats, Nonneman and Kolb (1974) found that hippocampal lesion reduced the animals' response to threat gestures and reported that the hippocampal lesioned cats were submissive when paired with normal, unlesioned animals. This is in keeping with an earlier report (Fuller, Rosvold, and Pribram, 1957) that lesion of the pyriform-amygdala-hippocampal area reversed the social rank when given to the dominant member of dog-pairs. The lesion did not affect the animal's ability to fight but did abolish initiation of aggressive activity. It is generally assumed that damage to amygdala or pyriform lobe was responsible for the change, but given these recent results, it is possible that damage to the hippocampus was an important factor. One must consider the possibility that the effects of the hippocampal damage might be due to a release of inhibition in an anatomically related structure. In particular, one might propose that a release of septal influence might alter the animal's reactivity and response to social confrontation. A number of studies indicate that septal lesions increase shock-induced aggression (see review by Caplan, 1973). Seigel and Leaf (1969) noted an increase in mouse killing as a result of septal lesions in rats, and Thomas, Hostetter, and Baker (1968) reported that wild rat strains were "bolder" following septal damage. However, Clody and Carlton (1969) have found just the opposite (increased docility after septal lesions), and Lubar, Herrmann, Moore, and Shouse (1973) did not fmd any change in social dominance relationships as a result of septal damage. The existence of contradictory results makes it difficult to assert that the effects of hippocampal damage are due to a release of septal activity. Furthermore, even if consistent effects of septal damage could be shown, there would still be a question about whether the changes were produced by a release of inhibition at some other site. The ability of septal lesion to produce a change in aggressive behavior might just as well be due to a modification of hippocampal activity as the reverse. The most defensible position is that the hippocampus serves as part of a "system"

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involved in the control of aggression, and the results of the present study both indicate that damage to the hippocampus alters the level of aggression in a colony and eliminate the social dominance hierarchy. We believe that the modification of aggression may be a primary factor in producing several other of the behavioral changes. For example, in control colonies it is mainly the dominant male who patrols; if a subordinate male is encountered, he is chased back into his home box. The low overall activity of subordinate animals (and low patrols) probably is not a function of low basal metabolism, or laziness, or a lesser amount of curiosity about the new environment. More likely the low activity level is a defensive strategy to minimize the chances of being battered from an encounter with the aggressive dominant. If the dominant male is removed from the colony, another male will become dominant and show the same pattern of high activity and patrol sequences. If that animal is removed, another will become active. In other words, it appears that the low activity levels of subordinates is a result of conflict with the dominant animal, wherein the aggressive behavior of the dominant plays a prime role in determining the activity of the other males and the eventual development of a stable social hierarchy tends to minimize vicious aggressive encounters. By contrast, it appeared that the hippocampal lesioned animals had very little behavioral restraint and a minimal number of confrontations. The hippocampal colony appeared to be rather laissez-faire, with fairly undifferentiated high activity among all the males of the colony. Examination of the simultaneous activity of the animals shows that when one of the hippocampal lesioned animals was active, there was a tendency for all of them to be active, and vice versa. In a normal colony the activity of dominants and subordinates tends to vary inversely, which may explain the fact that the circadian rhythm in the control colonies was more variable than it was in the hippocampal colony. Furthermore, in the control colonies the subordinate males rarely were found eating in the presence of the dominant animal, while synchronous social feeding was common among the Hippocampal Lesioned Group. The only result that seems incongruent with the idea that the hippocampal lesioned colony was relatively laissez-faire was the development of abnormally high levels of "displacement activity." An animal may be displaced from a chamber for two main reasons: avoidance of a confrontation or hyperreactivity. It is possible that this group were undergoing CNS reorganization and recovery, and the displacement activity was an early indicator of aggression to come. However, the control colonies that all had aggressive dominant males did not show this high level of displacement. A more acceptable explanation stems from the many studies of active avoidance, passive avoidance, CER, and other tests of defensive strategy (Thomas, Hostetter, and Barker 1968; Blanchard and Blanchard, 1972). Many experiments have reported that animals with hippocampal lesion avoid threatening situations quite well when flight is the appropriate defensive strategy.

HIPPOCAMPUS AND SOCIAL BEHAVIOR

25

However, when freezing is required for successful performance (with shock or when confronted with a potential predator), then hippocampal lesioned animals perform poorly. If anything, such animals have a lowered threshold for flight, since the opposite defensive strategy of freezing has been eliminated. In the present experiment the lesion may have abolished the tendency of the mice to initiate aggression but may also have augmented the probability that they would flee from potential conflict. Therefore, it appears that initially the hippocampal lesioned animals were primarily exhibiting hyperactivity to a new physical environment that masked the escape behavior. However, as locomotor activity i subsided , the escape behavior was observed as increased frequency of displacements from a chamber. With respect to the recent work of Gellhorn (1970) and Isaacson (1972), the behavioral effects we have observed in the hippocampal lesioned animals could be explained by invoking Hess' (1949) concept of ergotrophic and trophotropic systems. These two systems are antagonistic, and although not identical to the sympathetic and parasympathetic nervous systems, they do have some similarities. A critical amount of ergotrophic activity is necessary for the initiation and maintenance of all behavior. Strong activity of the trophotropic system is related to a decrease in ongoing behavior and a tendency for rest. Isaacson (1972) has suggested that the hippocampal influence on the hypothalamus could be the inhibition of ergotrophic activity. Therefore, destruction of the hippocampus would lead to an exaggerated ergotrophic balance in the hypothalamus. This would lead to increased arousal and a sympathetic nervous system activity, which is what we observed in the hippocampal lesioned animals. From previous work we have shown that the dominant males have much higher sympathetic nervous system activity as measured by elevated systolic blood pressure and levels of the biosynthetic catecholamine enzymes, tyrosine hydroxylase, and phenol-ethanolamine N-methyl-transferase as compared to subordinates or controls (Ely, Henry, and Ciaranel]o, 1974). With time it appears that the trophotropic system brings the animal back into a homeostatic balance (ongoing studies are examining indicators of sympathetic nervous system activation). We have just examined the possibility that behavioral changes seen in the social colony were due to a modification of emotional control, as originally proposed by Papez (1937) and amplified by MacLean (1949). It is also possible that a spatial dysfunction was responsible for some aspect of the abnormal behavior. A number of previous articles suggest that the hipocampus is important in the animal's ability to map or retain spatial relationships (Kimble and Green, 1968; Samuels, 1972; O'Keefe and Dostrovsky, 1971; Henderson, Henderson, and Greene, 1973; Green and Stauff, 1974; Nadel and O'Keefe, 1974; DeCastro, 1974). If the animals were confused about the position of the colony chambers, due to perceptual, attentional, or memory dysfunction, they might be expected to maintain a high level of exploratory

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activity as well as a high rate until finally they gained some mastery of the situation. Furthermore, if an animal had faulty spatial mapping, he could fail to defend a territory and consequently aggression would not be initiated. The aggressive drive of the hippocampal lesioned animal may be intact, but the situation may not be perceived as appropriate for the initiation of aggression. Green et al. (1957) made a similar point with regard to territory and sexual behavior in cats that had sustained damage to the pyriform lobe and amygdala. These investigators argued that the animals were not hypersexual but were simply less discriminating about the appropriate place for initiation of sexual activity. Most of the theoretical constructs of hippocampal function indicate some type of behavioral inhibitory function whether it be modification of responsiveness to events in the environment as a function of homeostasis, spatial deficits, or internal inhibition of specific systems. As an alternative to such unitary views, we must keep in mind the possibility that the hippocampus may have many functions and that changes in food consumption, perserverative exploratory activity, and aggression may be the result of damage to several functionally distinct regions. A dorsal-ventral hippocampus difference and variations in aggressive behavior have already been mentioned (Siegel and Flynn, 1968). Other studies that suggest regional specialization include Kimura (1958), Nadel (1968), Jarrard (1973), Edinger, Siegel, and Troiano (1973), and Henderson and Greene (in preparation). Greene and Stauff (1974) suggest that ventral hippocampus may have a special role in the control of defensive behaviors, and that essential connections for mediating this function passes into the hippocampus via the subiculum. In this regard, in a recent review of limbic connections Powell and Hines (1974) have emphasized indirect communication between amygdala and hippocampus that pass via the entorhinal area, subiculum, and temporoammonic tracts The outcome of subsequent work should clarify which anatomical connections of the hippocampus are involved in the regulation of social aggression. For the present, this work has demonstrated the potential of ~the minicomputer for the study of complex social behaviors and has suggested that the classic hypothesis that the hippocampus serves in the experience and control of emotion merits further appraisal.

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Brady, J. Vo (1958). The paleocortex and behavioral motivation. In H. N. Harlow and C. N. Woolsey (Eds.), "Biological and Biochemical Bases of Behavior." Madison: Univ. of Wisconsin Press. Caplan, M. (1973). An analysis of the effects of septal lesions on negatively reinforced behavior. Behav. Biol. 9, 129-167. Clody, D. E,, and Carlton, P. L. (1969). Behavioral effects of lesions of the medical septum of rats. J. Comp. Physiol. Psychol. 67, 344-351. Courey, J. N. (1967). Neural correlates of food and water intake in the rat. Science 156, 1763-1765. DeCastro, J. M. (1974). A selective spatial discrimination deficit after fornicotomy in the rat. Behav. Biol. 12, 373-382. DeCastro, J. M., and Marrone, B. L. (1974). Effect of fornix lesions on shock-induced aggression, muricide and motor behavior in the albino rat. Physiol. Behav. 13, 737-743. deGroot, J. (1965). The influence of limbic sturctures on pituitary functions related to reproduction. In F. A. Beach (Ed.), "Sex and Behavior." New York: Wiley. Dixon, W. J., and Massey, F. J. (1969). "Introduction to Statistical Analysis," 3rd ed. New York: McGraw-Hill. Douglas, R. J. (1967). The hippocampus and behavior. Psychol. Bull. 67, 416-442. Edinger, H., Siegel, A., and Troiano, R. (1973). Single unit analysis of the hippocampal projections to the septum in the cat. Exp. Neurol. 41,569-583. Eichelman, B. S., Jr. (1971). Effect of subcortical lesions on shock-induced aggression in the rat. J. Comp. Physiol. Psych. 7, 331-339. Ely, D. L. (1971). Physiological and behavioral differentiation of social roles in a population cage of magnetically tagged CBA mice. Ph.D. dissertation, University of Southern California. Ely, D. L., and Henry, J. P. (1974). Effects of prolonged social deprivation on murine behavior patterns, blood pressure and adrenal weight. J. Comp. PhysioL Psychol. 87, 733-740. Ely, D. L., Henry, J. P., and Ciaranello, R. D. (1974). Long-term behavioral and biochemical differentiation of dominant and subordinate mice in population cages. Psychosom. Med. 36,463. Ely, D. L., Henry, J. P., and Jarosz, C. J. (1975). Effects of marihuana (zk9-THC) on behavior patterns and social roles in colonies of CBA mice. Behav. BioL 13,263. Ely, D. L,, Henry, J. A., Henry, J. P., and Rader, R. D. (1972). A monitoring technique providing quantitative rodent behavior analysis. Physiol. Behav. 9, 675-679. Fisher, A. E., and Courey, J. N. (1962). Cholinergic tracing of a central neural circuit underlying the thirst drive. Science 138, 691-693. Fuller, J. L., Rosvold, H. E., and Pribram, K. H. (1957). The effect on affective and cognitive behavior in the dog of lesions of the pyriform-amygdala-hippocampal complex. J. Comp. Physiol. Psychol. 50, 89-96. Gelhorn, E. (1970). The emotions and the ergotrophic and trophotropic systems. Psychol. Forsch. 34, 48-94. Glickman, S. E., Higgins, T., and Isaacson, R. L. (1970). Some effects of hippocampal lesions on the behavior of Mongolian gerbils. Physiol. Behav. 5, 931-938. Green, J. D., Clemente, C. D., and deGroot, J. (1957). Rhinencephalic lesions and behavior in cats. J. Comp. Neurol. 108, 505-536. Greene, E. (1971). Comparison of hippocampal depression and hippocampal lesion. Expo Neurol. 31, 313-325. Greene, E., and Lomax, P. (1970). Impairment of alternation-learning in rats following microinjection of carbachol into the hippocampus. Brain Res. 18, 355-359.

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Greene, E., and Stauff, C. (1974). Behavioral role of hippocampal connections. Exp. NeuroL 45, 141z160. Greene, E., Saporta, S., and Waiters, J. (1970). Choice bias from unilateral hippocampal or frontal lesions in the rat. Exp. NeuroL 29, 534-545. Henderson, J., Henderson, R., and Greene, E. (1973). Impairment of memory with administration of KC1 to the hippocampus. Behav. Biol. 9, 655-670. Hess, W. R. (1949). "Das Zwischenhirn." Basel: Schwabe. Isaacson, R. L. (1972). Neural systems of the limbic brain and behavioral inhibition. In R. A. Boakes and M. S. HaUiday (Eds.), "Inhibition and Learning," chap. 19. London: Academic Press. Isaacson, R. L. (1974). "The Limbic System," p. 163. New York/London: Plenum Press. Isaacson, R. L., Douglas, R. J., and Moore, R. V. (1961). The effect of radical hippocampai ablation on acquisition of avoidance response. J. Comp. Physiol. Psychol. 54, 625-628. Jarrard, L. E. (1968). Behavior of hippocampal lesioned rats in home cage and novel situations. Physiol. Behav. 3, 65-70. Jarrard, L. E. (1973). The hippocampus and motivation, Psychol. Bull. 79, 1-12. Kaada, B. R., Jansen, J., and Anderson, P. (1953). Stimulation of the hippocampus and medial cortical areas in unanesthetized cats. Neurology 3, 844-857. Kim, C., Choi, H., Kim, J. K., Chang, H. K., Park, R. S., and Kang, I. Y. (1970). General behavioral activity and its component patterns in hippocampectomized rats. Brain Res. 19, 379-394. Kimble, D. P. (1963). The effects of bilateral hippocampal lesions in rats. J. Comp. Physiol. Psychol. 56, 273-283. Kimble, D. P. (1968). Hippocampus and internal inhibition. Psychol. Bull. 70, 285-295. Kimble, D. P., and Coover, G. D. (1966). Effects of hippocampal lesions on food and water consumption in rats. Psychon. ScL 4, 91-92. Kimble, D., and Greene, E. (1968). Absence of latency learning in rats with hippoeampal lesions. Psychon. Sci. 11, 99-100. Kimura, D. (1958). Effects of selective hippocampal damage on avoidance behavior in the rat. Can. J. Psychol. 12, 213-218. Kluver, H., and Bucy, P. C. (1937). "Psychic blindness" and other symptons following bilateral lobectomy in Rhesus monkeys. Amer. J. PhysioL 119, 352-353. Kolb, B., and Nonneman, A. J. (1974). Frontolimbic lesions and social behavior in the rat. Physiol. Behavo 13, 637-643. Lubar, J. F., Herrmann, R. F., Moore, D. R., and Shouse, M. N. (1973). Effects of septal and frontal oblations on species-typical behavior in the rat. J. Comp. PhysioL Psychol. 83, 260-270. MacLean, P. D. (1949). Psychosomatic disease and the "Visceral Brain": Recent developments bearing on the Papez theory of emotion. Psychosom. Med. 11, 338 -353. MacLean, P. D. (1957). Chemical and electrical stimulation of hippocampus in unrestrained animals. II. Behavioral findings. Arch. Neurol. Psychiat. 78, 128-142. MacLean, P. D., and Delgado, J. M. R. (1953). Electrical and chemical stimulation of fronto-temporal portion of the limbic system in the waking animal. Electroencephalogr. Clin. NeurophysioL 5, 91-100. MacPhail, E. M. (1968). Effects of intracranial cholinergic stimulation in rats on drinking, EEG, and heart rate. J. Comp. Physiol. PsychoL 65, 42-49. McCleary, R. A. (1966). Response-modulating functions of the limbic systems: Initiation and suppression. In E. Stellar and H. M. Sprague (Eds.), "Progress in Physiological Psychology." New York: Academic Press.

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Nadel, L. (1968). Dorsal and ventral hippocampal lesions and behavior. Physiol. Behav. 3, 891-900. Nadel, L., and O'Keefe, J. O. (1974). The hippoeampus in pieces and patches: An essay on modes of explanation in physiological psychology. In R. Bellairs and E. G. Gray (Eds.), "Essays on the Nervous System." Oxford: Clarendon Press. Nonneman, A. J., and Kolb, B. E. (1974). Lesions of hippocampus or prefrontal cortex alter species-typical behaviors in the eat. Behav. Biol. 12, 41-54. O'Keefe, J., and Dostrovsky, J. (1971). The hippocampus as a spatial map. Preliminary evidence from unit activity in the freely-moving rat. Brain Res. 34, 171-175. Papez, J. W. (1937). A proposed mechanism of emotion. Arch. Neurol. Psychiat. 38, 725-743. Powell, E. W., and Hines, G. (1974). The limbic system: An interface. Behav. Biol. 12, 149-164. Pribram, K. H. (1960). A review of theory in physiological psychology. Annu. Rev. Psychol. 11, 1-40. Rosvold, H. E., Mirsky, A. F., and Pribram, K. H. (1954). Influence of amygdalectomy on social behavior in monkeys. J. Comp. Physiol. Psyehol. 47, 173-178. Samuels, I. (1972). Hippocampal lesions in the rat: Effects on spatial and visual habits. PhysioL Behav. 8, 1093-1098. Saporta, S., and Greene, E. (1974). orienting bias in the rat produced by hippocampal lesion. Behav. Biol. 11, 339-351. Seigel, D. and Leaf, R. C. Effects of septal and amygdoloid brain lesions in rat on mouse killing. Paper presented at the meeting of the Eastern Psychology Association, Philadelphia, April 1, 1969. Siegel, A., and Flynn, J. P. (1968). Differential effects of electrical stimulation and lesions of the hippocampus and adjacent regions upon attack behavior in cats. Brain Res. 7, 252-267. Teitelbaum, H., and Milner, P. M. (1963). Activity changes following partial hippocampal lesions in rats. J. Comp. Physiol. Psychol. 56, 284-289. Thomas, G. J., Hostetter, G., and Barker, D. J. (1968). Behavioral functions of the limbic system. In E. Stellar and H. M. Sprague (Eds.), "Progress in Physiological Psychology." New York: Academic Press. Vanderwolf, C. H. (1969). Hippocampal electrical activity and voluntary movement in the rat. Electroencephalogr. Clin. Neurophysiol. 26, 407-418. Whishaw, I. W. (1972). Hippocampal EEG activity in the Mongolian gerbil during natural behaviors and wheel running and in the rat during wheel running and conditioned immobility. Can. J. Psychol. 26,219-239.