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Effect of septal lesions on conditioned defensive burying
Physiology & Behavior, Vol. 27, pp. 1051-1056. Pergamon Press and Brain Research Publ., 1981. Printed in the U.S.A.
Effect of Septal Lesions on Conditioned Defensive Burying I D. S H A U N G R A Y , L O R I J. T E R L E C K I , D A L L A S T R E I T , A N D J O H N P. J. P I N E L z
Department of Psychology, University of British Columbia Vancouver, British Columbia, Canada, V6T 1W5 R e c e i v e d 27 N o v e m b e r 1980 GRAY, D. S., L. J. TERLECKI, D. TREIT AND J. P. J. PINEL. Effect of septal lesions on conditioned defensive burying. PHYSIOL. BEHAV. 27(6) 1051-1056, 1981.--Every control rat shocked by a wire-wrapped prod mounted on the wall of a test chamber buried the prod with bedding material from the floor of the chamber. Although this conditioned defensive burying was also observed in every rat with a lesion of the anterior septum (Experiment 2), no rat with a lesion of the entire septum (Experiment I) or a lesion limited to the posterior septum (Experiment 2) was observed to engage in this behavior. These results confirm previous reports of the involvement of the septal area in defensive behavior and provide the first demonstration of the utility of the conditioned defensive burying paradigm in assessing the behavioral effects of limbic lesions.
Aversive conditioning Septum
Burying
Conditioned defensive burying
BRADY and Nauta [3] were the first to report the heightened reactivity of rats with septal lesions to handling and other stimuli. Although this effect of septal lesions has been traditionally attributed to an increase in aggressiveness or irritability, Blanchard, Blanchard, Lee, and Nakamura [1] have recently argued that the septal hyperreactivity syndrome actually reflects an increase in defensive, rather than aggressive, behavior. They found that destruction of the anterior septum in rats enhanced defensive behaviors normally elicited by aggressive conspecifics (e.g., boxing, freezing, and ultrasonic vocalization). Consistent with this alternate view is the observation that septal lesions produce a decrease, rather than an increase, in attacks by dominant male rats and mice on conspecific subordinates [2, 8, 17]. The general purpose of the present experiments was to determine whether septal lesions enhance the defensive reactions of rats to threat in general or to conspecific threat per se. To answer this question, we studied the defensive burying response, a behavior that is reportedly used by rodents as a defense against predators and inanimate objects. Rats have been reported to respond to traps [18] or sources of noxious food [20] by burying them, and ground squirrels use the same response to drive off snakes [12] or to erect barriers to predators in their burrows [4]. Defensive burying behavior has been observed in both male and female rats, mice, and ground squirrels of a variety of strains and ages [4, 12, 19]. Pinel and Treit [14,15] have reported a particularly reliable conditioning technique for inducing defensive burying in
Defensive behavior
Septal lesions
laboratory rats. Rats shocked once by a wire-wrapped prod mounted on a wall of a test chamber buried the shock prod with bedding material from the floor of the chamber; whereas, unshocked control rats did not. Thus, the purpose of tile present experiments was to assess the effects of septal lesions on conditioned defensive burying in rats. EXPERIMENT 1 In Experiment 1, the conditioned defensive burying of rats with lesions of the entire septal area was compared with that of rats with sham lesions. METHOD
Subjects and Surgery Serving as subjects in Experiment 1 were 35 naive, male, 300 to 400 g, hooded Long-Evans rats obtained from the Canadian Breeding Farm and Laboratories (St. Constant, Quebec). Following sodium pentobarbital (Nembutal, 50 mg/kg, IP) anesthesia, bilateral septal lesions (n=18) were produced by passing a 2-mA, 15-sec anodal current through a stainless steel electrode (stainless steel insect pin, size '00', insulated with varathane except for 0.5 mm at the tip) positioned with its tip in the septal area (1.4 mm anterior and 0.7 mm lateral to bregma, and 4.5 mm ventral to the dura with the incisor bar elevated 5 mm above the interaural line). An anal cathode was used to complete the lesion circuit. Sham-
l-Portions of this research were presented at the 4lst Annual Meeting of the Canadian Psychological Association, 1980, Calgary, Alberta,
Canada. This research was supported by a Natural Sciences and Engineering Research Council of Canada grant awarded to J. P. J. Pinel. 2Address reprint requests to John Pinel, Department of Psychology, University of British Columbia, 2053 Main Mall, Vancouver, British Columbia, Canada V6T 1W5.
C o p y r i g h t © 1981 Brain R e s e a r c h Publications Inc.--0031-9384/81/121051-06502.00/0
1052 lesion controls (n= 17) were treated in the same way except that no current was passed through the electrode. Following surgery, all animals were individually housed in standard wire cages with food and water continuously available. At the conclusion of behavioral testing, the rats were sacrificed in a carbon dioxide chamber and perfused intracardially with saline (0.9%) and a 10% solution of Formalin. Frozen coronal sections (30 p.m) were the basis for determining the extent and location of each lesion.
GRAY E T A L .
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SHOCK
Following 17 days of post-surgical recovery, all 35 subjects were habituated to the test chamber on 4 consecutive days. On each of the 4 habituation days, each individual rat spent 15 min in the 44x30×40 cm Plexiglas test chamber, the floor of which was covered evenly with a 5 cm layer of bedding material (San-i-cel, Paxton Processing Co., Paxton, IL). Before the single conditioning trial on day 5, a wirewrapped wooden prod (6.5×0.5×0.5 cm) was mounted 2 cm above the level of the bedding material in the center of one of the end walls. Nine of the 18 rats with septal lesions and eight of the 17 rats with sham lesions received a single shock through this prod, whereas the remaining subjects in each group served as unshocked controls. Thus, the design was a 2 x 2 factorial. To begin each conditioning session, each rat was placed in the center of the test chamber facing away from the prod. When each of the subjects in the two shock groups touched the prod with a forepaw, the experimenter activated the shock circuit (an 800-V source in series with an 80 kI~ resistor) that delivered a shock to the subject between the two uninsulated wires wrapped around the prod. The shock was terminated by the withdrawal reflex of each subject. Shock duration and intensity were monitored with a storage oscilloscope (Techtronix Model DM64) connected in parallel with the shock circuit, and the behavioral reaction of the rat to the shock was scored on a five-point scale. The categories from 0 to 4 were: no discernible reaction, startle but no immediate withdrawal, startle and withdrawal to the far end of the chamber, jumping and/or squealing followed by rapid withdrawal, and finally, a reflexive j u m p to the far end of the chamber. The shock signalled the beginning of the 15-min test session during which the duration of approaches to within 3 cm of the prod, the number of contacts with the prod, and the duration of burying behavior were recorded. The burying behavior of rats is extremely stereotyped; rats orient directly toward the prod and spray bedding material at it with rapid pushing movements of the forepaws (cf. [14]). As has been the practice (cf. [15]), the duration of burying score was simply the cumulative duration of these bursts of directed forelimb spraying; movement of bedding by responses other than forelimb spraying or in directions other than at the prod was recorded separately but was rare. The behavior of the rats during conditioning and testing was monitored from a separate room via closed circuit television. Approaches to within 3 cm of the prod were determined by reference lines marked on the screen of the closed circuit television monitor. The behavioral measure of burying was confirmed at the end of each test session by measuring the height of the bedding material at the prod. The lesion and sham-lesion control subjects were tested in a similar fashion except that they received no shock. The 15-min test session commenced for each control rat when it
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FIG. I. Means and standard errors for height of bedding material at the prod (Panel A) and burying duration (panel B) in Experiment 1.
touched the prod with a forepaw. At the conclusion of behavioral testing, each rat was evaluated for septal hyperreactivity by scoring the rat's resistance to capture by a gloved experimenter according to the following five-point rating scale [16]: 0---no reaction; l - - s h i e d from the glove; 2--avoided the glove by running around box and/or struggled when captured; 3--jumped to avoid capture; 4 - - b i t when captured. RESULTS AND DISCUSSION The results of Experiment 1 are presented in Fig. 1. Readily apparent is the striking difference between the shocked and unshocked sham-lesion subjects. Although the sham-lesion rats that had received the single prod shock engaged in a substantial amount of burying, as indicated by both the height (Panel A) and duration (Panel B) measures, burying by the unshocked sham-lesion subjects was negligible. This result further illustrates the robustness of the conditioned defensive burying phenomenon (cf. [14]). Equally obvious in Fig. I is the fact that no subject with a septal lesion, shocked or unshocked, engaged in any burying behavior; septal lesions completely eliminated the defensive burying normally observed in rats following the single conditioning trial. Thus, the analysis of variance revealed that the rats with sham lesions buried significantly more than did those with septal lesions (burying duration, F(1,31)=18.79, p<0.001; height of bedding, F(1,31)=28.87, p<0.001) and that shocked rats buried significantly more than did unshocked rats (burying duration, F(1,31)=15.68, p<0.001; height of bedding, F(1,31)=27.75, p<0.001). The interaction effect was also significant for both the duration F(1,31)=18.69, p<0.001, and height, F(1,31)=33.02, p<0.001, measures. Post hoc analyses indicated that both shocked and unshocked subjects with septal lesions did not differ significantly from unshocked sham-lesion controls in terms of either measure of defensive burying (p>0.05). The fact that not a single subject with a septal lesion engaged in conditioned defensive burying established that septal lesions do not produce a general increase in defensive responding in the rat. Although anterior septal lesions were shown by Blanchard et al. [1] to enhance responses used by
SEPTAL LESIONS AND BURYING
1053
TABLE 1 MEAN SCORE AND STANDARD ERROR (PARENTHESES) FOR APPROACH, CONTACT, AND SHOCK-RESPONSE MEASURES IN SHOCKED AND NON-SHOCKED SEPTAL AND SHAM LESION GROUPS IN EXPERIMENT 1 Approach frequency*
Approach duration (sec)t
Contact frequencyt
Response to shock
Shock duration (msec)
Shock intensity (mA) 7.5 (0.39) 6.69(0.91)
Shock
Septal (n=9) Sham (n=8)
29.9(11.3) 16.2(3.9)
27.9(11.5) 20.8(3.9)
0.5(0.2) 0.3(0.2)
2.0(0.29) 2.0(0.37)
37.4(6.6) 26.6(5.1)
No Shock
Septal (n=9) Sham (n=8)
42.8(4.0) 35.5(3.3)
170.6(30.0) 156.1(20.6)
21.4(2.5) 13.8(2.9)
---
---
---
*Significant effect of shock vs no-shock (p<0.02). tSignificant effect of shock vs no-shock (p<0.001). All other comparisons were non-significant (p>0.05).
rats to defend themselves from conspecifics, lesions of the entire septum completely eliminated the conditioned defensive burying of an inanimate object. The failure o f the septal rats to bury the prod did not appear to result from a failure to be shocked, to f'md the shock aversive, or to learn the association between the shock and the prod. No significant differences were found in either the intensity, t(15)=0.818, p>0.05, or duration, t(15)= 1.29, p >0.05, of the shock received by the septal and sham-lesion rats, or in their responses to it, t(15)=0.0, p>0.05 (see Table 1). Moreover, the shock suppressed subsequent approaches to and contacts with the prod to the same extent in the septal and sham-lesion groups. Analysis of approach-frequency, approach-duration, and prodcontact scores (see Table 1) indicated only a main effect of shock, F(1,31)=6.06, p>0.05; F(1,31)=49.38, p > 0 . 0 5 ; F(1,31) = 74.78, p >0.05, respectively. Resistance to capture scores indicated that septallesioned rats were not significantly more reactive to handling than were the sham-lesioned control rats (septal mean=2.42, S.E.M. =0.392; sham mean= 1.77, S.E.M. =0.419; t(35)= 1.13, p>0.05). Thus, the differences observed in burying behavior did not appear to reflect a general increase in the reactivity to environmental stimuli on the part of the septal-lesioned rats. The maximal overall extent of the lesions and a typical lesion are presented in Fig. 2 (Panel A). The septal lesions were large and encompassed almost all of the of the medial and lateral septum. Damage extraneous to the septum was found to include portions of the fornix and caudate nucleus, and in some cases slight damage was found to extend below the septal area to include the stria terminalis, nucleus accumbens, and the anterior commissure. Both septal and sham-lesion rats were found to have some minor electrodeproduced damage limited to the dorsal surface of the neocortex. EXPERIMENT 2 Blanchard et al. [1] found that lesions of the anterior portions of the septum enhanced responses used for defense against conspecifics, whereas lesions restricted to the posterior septum had no effect. The purpose of Experiment 2 was to compare the effects of anterior and posterior lesions of the septal area on conditioned defensive burying in rats.
METHOD Serving as subjects were 40 naive, male, 300 to 400 g, hooded Long-Evans rats purchased and housed as in Experiment 1. Bilateral lesions of the anterior septum (1.5 mA, 15 sec) were produced in 12 of these subjects; whereas another 13 received bilateral lesions o f its posterior portions (1.5 mA, 15 sec). In both cases, the general surgical procedures were the same as those of Experiment 1, but the coordinates for both anterior (3 mm anterior and 0.5 mm lateral to bregma, and 5.5 mm ventral to the dura) and posterior (1 mm anterior and 0.5 mm lateral to bregma, and 4.5 mm ventral to the dura) lesions were those of Blanchard et al. [1]. The control subjects received sham lesions of the anterior (n=8) or posterior (n=7) septum, but because the placement of sham lesions within the septum did not affect the dependent measures, the data of these two groups of subjects were combined prior to statistical analysis. After 17 days of postsurgical recovery, all 40 subjects were habituated to the test chamber on 4 consecutive days as in Experiment 1. On day 5, all 40 rats received a single conditioning trial followed immediately by a 15-rain test session in accordance with the procedures of Experiment 1. Resistance to capture was again assessed following the conclusion of behavioral testing. RESULTS AND DISCUSSION The major results of Experiment 2 are summarized in Fig. 3. Whereas lesions of the anterior septum had little or no effect on the levels of conditioned defensive burying, posterior lesions completely suppressed the burying response. Accordingly, a one-way analysis of variance revealed a significant lesion effect in both the duration, F(2,37)=9.42,p<0.001, and height, F(2,37)= 15.65,p<0.001, scores. Subsequent post hoc analyses indicated that the sham-lesion group and the anterior-lesion group did not differ significantly from one another in terms of either measure (p >0.05), but that the posterior-lesion group differed significantly from both these two groups in terms of both measures (p<0.05). As in Experiment 1, the lesion-produced elimination of conditioned defensive burying did not appear to result from either a decrease in responsiveness to shock or to the failure of the lesioned subjects to learn the association between the
1054
GRAY ET AL.
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FIG. 2. Histological results for Experiments 1 (Panel A) and 2 (Panel B). In Panel A, septal lesions are illustrated in the first row whereas sham lesion cortical damage is illustrated in the second row. In Panel B, the first row illustrates posterior septal lesion damage and the second row illustrates anterior septai lesion damage. The shaded area of each section shows the overall maximal extent of all lesions in that group combined, whereas a lesion representative of the particular group is shown in black. The numbered sections correspond to coronal views anterior to bregma taken from Pellegrino and Cushman [13].
prod and the shock (see Table 2). Analysis of the shock parameters r e v e a l e d no significant differences in either the intensity, F(2,38)= 1.01, p >0.05, or duration, F(2,38)=0.392, p > 0 . 0 5 , o f the shocks r e c e i v e d by the three groups. And there w e r e no significant differences b e t w e e n the groups in
their reactivity to the shock, F(2,37)= 2.46, p >0.05, or in the frequency, F(2,37)=0.28, p > 0 . 0 5 , or duration, F(2,37) =2.25, p>0.05, of their approaches to the prod. Resistance to capture scores indicated that both anteriorand posterior-lesion rats were significantly more reactive to
SEPTAL L E S I O N S AND B U R Y I N G
1055 TABLE 2
MEAN SCORE AND STANDARD ERROR (PARENTHESES) FOR APPROACH AND SHOCK-RESPONSE MEASURES IN ANTERIOR SEPTAL, POSTERIOR SEPTAL, AND SHAM LESION GROUPS IN EXPERIMENT 2
Posterior Septal (n=13) Anterior Septal (n=12) Sham (n=15)
o
Approach frequency
Approach duration (sec)
Response to shock
22.3(4.3) 26.3(3.6) 23.1(5.0)
30.1(8.1) 49.4(12.1) 27.2(4.0)
1.86(0.25) 2.38(0.21) 1.73(0.18)
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37.85(5.55) 39.67(7.37) 44.33(3.74)
Shock intensity (mA) 9.46(0.58) 8.33(0.58) 8.73(0.50)
m
11
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"0
Shock duration (msec)
5 O POST
ANT
SHAM
POST
ANT
SHAM
FIG. 3. Means and standard errors for height of bedding material at the prod (Panel A) and burying duration (Panel B) in the anterior septal (ANT), posterior septal (POST) and sham lesion (SHAM) groups in Experiment 2.
handling than were their sham-lesion counterparts, F(2,38)=8.91, p<0.001. However, post-hoc analyses indicated that the anterior- and posterior-lesion groups did not differ significantly in this respect (anterior mean=2.54, S.E.M.=0.351; posterior mean=3.15, S.E.M.=0.320; sham mean=l.27, S.E.M.=0.316). The fact that anterior- and posterior-lesion groups did not differ in resistance to capture but did differ in burying behavior suggests that the enhanced reactivity contributed little to the deficits in burying behavior observed as a result of the posterior lesions. Although the increase in hyperreactivity produced by the septal lesions in Experiment 1 did not reach significance and the increase observed in Experiment 2 did, the two experiments did not differ substantially in this respect. Histological analyses (Fig. 2, Panel B) indicated that all lesions were placed in the appropriate areas of the septum. Whereas the posterior lesions were predominantly restricted to the medial and lateral septal nuclei, slight additional damage to the caudate nucleus, corpus callosum, or dorso-medial cortex was observable in all posterior subjects. Anterior septal lesions did not involve damage posterior to the level of the anterior commissure but did involve damage to adjacent forebrain areas.
G E N E R A L DISCUSSION In Experiment 1, lesions of the entire septum disrupted the conditioned defensive burying that was observed in rats with sham lesions. The results of the second experiment suggested that the area of the septum that is critical for conditioned defensive burying is located in its posterior portion. Lesions of the posterior septum disrupted conditioned defensive burying, whereas anterior lesions were without consequence. There are two features of the effect of posterior septal lesions on conditioned defensive burying that are worthy of emphasis: its reliability and selectivity. In both experiments, conditioned defensive burying was reliably observed in every shocked rat without damage to the posterior septum, but not a single subject with damage to this area was observed to engage in this behavior. Moreover, although posterior septal lesions completely suppressed conditioned defensive burying, they were without effect on any of the other dependent measures. Except for the fact that the rats with posterior septal lesions displayed no burying behavior, their behavior was indistinguishable from that of the shamlesion controls; they reacted to the shock in the same way and subsequently avoided it to the same degree. Thus, the deficit in conditioned burying cannot be attributed to an inability of the rats to perceive or fear the shock, or to associate it with the prod. The results of the present experiments confirm the view of Blanchard et al. [1] that the septum plays an important role in the regulation of defensive behavior and that anterior and posterior portions of the septal area exert differential control. However, in view of the fact that Blanchard et al. [1] found behaviors used by rats to defend themselves from conspecifics to be enhanced by anterior lesions but not by posterior lesions, the present results suggest that not all defensive behaviors are regulated by the septal area in the same way. In Experiment 2, posterior lesions completely eliminated conditioned defensive burying, whereas anterior lesions were ineffective. The conclusion that the posterior septum exerts control over conditioned defensive burying must be qualified in three important ways. First, although it is quite clear that some part of the posterior septal area mediates conditioned defensive burying, it is not clear whether it is the septal nuclei themselves or fibers passing through this portion of the septum that are of primary importance. For example, it is possible that the effects of septal lesions on defensive burying may have resulted from damage to the fornix, which includes afferent and efferent pathways of the hippocampus.
1056
GRAY E T A L .
Hippocampal disruption has been reported to result in a variety of behavioral deficits and some authors have interpreted these deficits to reflect a "spatial mapping" function of the hippocampus (e.g., [10,11]). However, a simple spatial-memory-deficit hypothesis cannot account for the present data. The rats with septal lesions were clearly able to localize and subsequently avoid contact with the source of the aversive stimulus. Second, the present results do not imply that the control of defensive behavior is the only function of the septal area; septal lesions have been shown to disrupt a variety of behaviors in rodents (see [6,7] for review). This has led to the view that the septum and other limbic structures such as the hippocampus may be involved in the sequencing of behavior in general rather than controlling one particular class of behaviors (cf. [5,9]). Third, it should be emphasized that the subjects in the present experiment were not tested until 17 days after surgery and were handled in the interim. Because some of the effects of septal lesions decline markedly with handling or just with the passage of time (e.g., hyperreactivity), the magnitude of the present effects on conditioned defensive burying are all the more striking. It should be noted that in contrast to the previous reports that septal lesions produce passive avoidance deficits [6,7], the septal lesions in the present experiments did not reduce
the tendency of the subjects to stay away from the conditoned stimulus. Although passive avoidance and conditioned defensive burying paradigms are similar in that the behavior of the subjects is changed following a single shock in both, they differ in several major respects. For example, in the conditioned defensive burying paradigm, the conditioned stimulus is a well-defined object, i.e., the prod; whereas, in the usual passive avoidance situations the shock is administered diffusely through the floor of the apparatus. Although it is interesting to speculate that conditioned passive avoidance may be resistant to disruption by septal lesions when the conditioned stimulus is a well-defined object, there are other methodological features of the present experiments (e.g., the availability of bedding or the relatively generous postsurgical recovery period) that could also account for the lack of a passive avoidance deficit in the lesioned subjects. The present experiments constitute the first demonstration of the utility of the conditioned defensive burying paradigm in assessing the effects of iimbic lesions. The reliability and robustness of the present data suggest that the conditioned defensive burying paradigm will prove to be an important addition to the behavioral paradigms available for assessing the behavioral effects of physiological manipulations.
REFERENCES I. Blanchard, D. C., R. J. Blanchard, E. M. C. Lee and S. Nakamura. Defensive behaviors in rats following septal and septal-amygdala lesions. J. comp. physiol. Psychol. 93: 378390, 1979. 2. Blanchard, D. C., R. J. Blanchard, L. Takahashi and T. Takahashi. Septal lesions and aggressive behavior. Behav. Biol. 21: 157-161, 1977. 3. Brady, J. V. and W. J. H. Nauta. Subcortical mechanisms in emotional behavior: Affective changes following septal forebrain lesions in the albino rat. J. comp. physiol. Psychol. 46: 339--346, 1953. 4. Coss, R. G. and D. H. Owings. Snake-directed behavior by snake naive and experienced California ground squirrels in a simulated burrow. Z. Tierpsychol. 48: 421--435, 1978. 5. Fleischer, S. and B. M. Slotnik, Disruption of maternal behavior in rats with lesions of the septal area. Physiol. Behav. 21: 189--200,1978. 6. Grossman, S. P. Behavioral functions of the septum: A reanalysis. In: The Septal Nuclei, edited by J. F. DeFrance. New York: Plenum Press, 1976. 7. Isaacson, R. L. The Limbic System. New York: Plenum Press, 1974. 8. Lau, P. and K. A. Miczek, Differential effects of septal lesions on attack and defensive-submission reaction during intraspecies aggression in rats. Physiol. Behav. 18: 479-485, 1977. 9. Michal, E. K. Effects of limbic lesions on behavior sequences and courtship behavior of male rats (rattus norvegicus). Behaviour 44: 264-285, 1973. 10. Nadel, L. and J. O'Keefe. The hippocampus in pieces and patches: An essay on modes of explanation in physiological psychology. In: Essays on the Nervous System. A Festschrift for Prof. J. Z. Young, edited by R. Bellairs and E. G. Gray. Oxford: Clarendon Press, 1974, pp. 367-390.
11. Olten, D. S., J. A. Walker and F. Grage. Hippocampal connections and spatial discrimination. Brain Res. 139: 255-308, 1978. 12. Owings, D. H. and R. G. Coss. Snake mobbing by California ground squirrels: Adaptive variation and ontogeny. Behaviour 62: 50-69, 1978. 13. Pellegrino, L. J. and A. J. Cushman. A Stereotaxic Atlas o f the Rat Brain. New York: Appleton-Century-Crofts, 1967. 14. Pinel, J. P. J. and D. Treit. Burying as a defensive response in rats. J. comp. physiol. Psychol. 92: 708-712, 1978. 15. Pinel, J. P. J. and D. Treit. Conditioned defensive burying in rats: Availability of burying materials. Anita. Learn. Behav. 7: 392-396, 1979. 16. Seggie, J. Effect of adrenalectomy or gonadectomy on affective behavior changes following septal lesions in rats. J. comp. physiol. Psychol. 74:11-19, 1974. 17. Slotnik, B. M. and M. F. McMullen. Intraspecific fighting in albino mice with septal forebrain lesions. Physiol. Behav. 8: 333-337, 1972. 18. Terlecki L. J., J. P. J. Pinel and D. Treit. Conditioned and unconditioned defensive burying in the rat. Learn. Motivat. 10: 337-350, 1979. 19. Treit, D., L. J. Terlecki and J. P. J. Pinel. Conditioned defensive burying in rodents: Organismic variables. Bull. Psychon. Soc. 16: 451-454, 1980. 20. Wilkie, D. M., A. J. MacLennan and J. P. J. Pinel. Rat defensive behavior: Burying noxious food. J. exp. Analysis Behav. 31: 299-306, 1979.
Effect of Septal Lesions on Conditioned Defensive Burying I D. S H A U N G R A Y , L O R I J. T E R L E C K I , D A L L A S T R E I T , A N D J O H N P. J. P I N E L z
Department of Psychology, University of British Columbia Vancouver, British Columbia, Canada, V6T 1W5 R e c e i v e d 27 N o v e m b e r 1980 GRAY, D. S., L. J. TERLECKI, D. TREIT AND J. P. J. PINEL. Effect of septal lesions on conditioned defensive burying. PHYSIOL. BEHAV. 27(6) 1051-1056, 1981.--Every control rat shocked by a wire-wrapped prod mounted on the wall of a test chamber buried the prod with bedding material from the floor of the chamber. Although this conditioned defensive burying was also observed in every rat with a lesion of the anterior septum (Experiment 2), no rat with a lesion of the entire septum (Experiment I) or a lesion limited to the posterior septum (Experiment 2) was observed to engage in this behavior. These results confirm previous reports of the involvement of the septal area in defensive behavior and provide the first demonstration of the utility of the conditioned defensive burying paradigm in assessing the behavioral effects of limbic lesions.
Aversive conditioning Septum
Burying
Conditioned defensive burying
BRADY and Nauta [3] were the first to report the heightened reactivity of rats with septal lesions to handling and other stimuli. Although this effect of septal lesions has been traditionally attributed to an increase in aggressiveness or irritability, Blanchard, Blanchard, Lee, and Nakamura [1] have recently argued that the septal hyperreactivity syndrome actually reflects an increase in defensive, rather than aggressive, behavior. They found that destruction of the anterior septum in rats enhanced defensive behaviors normally elicited by aggressive conspecifics (e.g., boxing, freezing, and ultrasonic vocalization). Consistent with this alternate view is the observation that septal lesions produce a decrease, rather than an increase, in attacks by dominant male rats and mice on conspecific subordinates [2, 8, 17]. The general purpose of the present experiments was to determine whether septal lesions enhance the defensive reactions of rats to threat in general or to conspecific threat per se. To answer this question, we studied the defensive burying response, a behavior that is reportedly used by rodents as a defense against predators and inanimate objects. Rats have been reported to respond to traps [18] or sources of noxious food [20] by burying them, and ground squirrels use the same response to drive off snakes [12] or to erect barriers to predators in their burrows [4]. Defensive burying behavior has been observed in both male and female rats, mice, and ground squirrels of a variety of strains and ages [4, 12, 19]. Pinel and Treit [14,15] have reported a particularly reliable conditioning technique for inducing defensive burying in
Defensive behavior
Septal lesions
laboratory rats. Rats shocked once by a wire-wrapped prod mounted on a wall of a test chamber buried the shock prod with bedding material from the floor of the chamber; whereas, unshocked control rats did not. Thus, the purpose of tile present experiments was to assess the effects of septal lesions on conditioned defensive burying in rats. EXPERIMENT 1 In Experiment 1, the conditioned defensive burying of rats with lesions of the entire septal area was compared with that of rats with sham lesions. METHOD
Subjects and Surgery Serving as subjects in Experiment 1 were 35 naive, male, 300 to 400 g, hooded Long-Evans rats obtained from the Canadian Breeding Farm and Laboratories (St. Constant, Quebec). Following sodium pentobarbital (Nembutal, 50 mg/kg, IP) anesthesia, bilateral septal lesions (n=18) were produced by passing a 2-mA, 15-sec anodal current through a stainless steel electrode (stainless steel insect pin, size '00', insulated with varathane except for 0.5 mm at the tip) positioned with its tip in the septal area (1.4 mm anterior and 0.7 mm lateral to bregma, and 4.5 mm ventral to the dura with the incisor bar elevated 5 mm above the interaural line). An anal cathode was used to complete the lesion circuit. Sham-
l-Portions of this research were presented at the 4lst Annual Meeting of the Canadian Psychological Association, 1980, Calgary, Alberta,
Canada. This research was supported by a Natural Sciences and Engineering Research Council of Canada grant awarded to J. P. J. Pinel. 2Address reprint requests to John Pinel, Department of Psychology, University of British Columbia, 2053 Main Mall, Vancouver, British Columbia, Canada V6T 1W5.
C o p y r i g h t © 1981 Brain R e s e a r c h Publications Inc.--0031-9384/81/121051-06502.00/0
1052 lesion controls (n= 17) were treated in the same way except that no current was passed through the electrode. Following surgery, all animals were individually housed in standard wire cages with food and water continuously available. At the conclusion of behavioral testing, the rats were sacrificed in a carbon dioxide chamber and perfused intracardially with saline (0.9%) and a 10% solution of Formalin. Frozen coronal sections (30 p.m) were the basis for determining the extent and location of each lesion.
GRAY E T A L .
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SHOCK
Following 17 days of post-surgical recovery, all 35 subjects were habituated to the test chamber on 4 consecutive days. On each of the 4 habituation days, each individual rat spent 15 min in the 44x30×40 cm Plexiglas test chamber, the floor of which was covered evenly with a 5 cm layer of bedding material (San-i-cel, Paxton Processing Co., Paxton, IL). Before the single conditioning trial on day 5, a wirewrapped wooden prod (6.5×0.5×0.5 cm) was mounted 2 cm above the level of the bedding material in the center of one of the end walls. Nine of the 18 rats with septal lesions and eight of the 17 rats with sham lesions received a single shock through this prod, whereas the remaining subjects in each group served as unshocked controls. Thus, the design was a 2 x 2 factorial. To begin each conditioning session, each rat was placed in the center of the test chamber facing away from the prod. When each of the subjects in the two shock groups touched the prod with a forepaw, the experimenter activated the shock circuit (an 800-V source in series with an 80 kI~ resistor) that delivered a shock to the subject between the two uninsulated wires wrapped around the prod. The shock was terminated by the withdrawal reflex of each subject. Shock duration and intensity were monitored with a storage oscilloscope (Techtronix Model DM64) connected in parallel with the shock circuit, and the behavioral reaction of the rat to the shock was scored on a five-point scale. The categories from 0 to 4 were: no discernible reaction, startle but no immediate withdrawal, startle and withdrawal to the far end of the chamber, jumping and/or squealing followed by rapid withdrawal, and finally, a reflexive j u m p to the far end of the chamber. The shock signalled the beginning of the 15-min test session during which the duration of approaches to within 3 cm of the prod, the number of contacts with the prod, and the duration of burying behavior were recorded. The burying behavior of rats is extremely stereotyped; rats orient directly toward the prod and spray bedding material at it with rapid pushing movements of the forepaws (cf. [14]). As has been the practice (cf. [15]), the duration of burying score was simply the cumulative duration of these bursts of directed forelimb spraying; movement of bedding by responses other than forelimb spraying or in directions other than at the prod was recorded separately but was rare. The behavior of the rats during conditioning and testing was monitored from a separate room via closed circuit television. Approaches to within 3 cm of the prod were determined by reference lines marked on the screen of the closed circuit television monitor. The behavioral measure of burying was confirmed at the end of each test session by measuring the height of the bedding material at the prod. The lesion and sham-lesion control subjects were tested in a similar fashion except that they received no shock. The 15-min test session commenced for each control rat when it
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FIG. I. Means and standard errors for height of bedding material at the prod (Panel A) and burying duration (panel B) in Experiment 1.
touched the prod with a forepaw. At the conclusion of behavioral testing, each rat was evaluated for septal hyperreactivity by scoring the rat's resistance to capture by a gloved experimenter according to the following five-point rating scale [16]: 0---no reaction; l - - s h i e d from the glove; 2--avoided the glove by running around box and/or struggled when captured; 3--jumped to avoid capture; 4 - - b i t when captured. RESULTS AND DISCUSSION The results of Experiment 1 are presented in Fig. 1. Readily apparent is the striking difference between the shocked and unshocked sham-lesion subjects. Although the sham-lesion rats that had received the single prod shock engaged in a substantial amount of burying, as indicated by both the height (Panel A) and duration (Panel B) measures, burying by the unshocked sham-lesion subjects was negligible. This result further illustrates the robustness of the conditioned defensive burying phenomenon (cf. [14]). Equally obvious in Fig. I is the fact that no subject with a septal lesion, shocked or unshocked, engaged in any burying behavior; septal lesions completely eliminated the defensive burying normally observed in rats following the single conditioning trial. Thus, the analysis of variance revealed that the rats with sham lesions buried significantly more than did those with septal lesions (burying duration, F(1,31)=18.79, p<0.001; height of bedding, F(1,31)=28.87, p<0.001) and that shocked rats buried significantly more than did unshocked rats (burying duration, F(1,31)=15.68, p<0.001; height of bedding, F(1,31)=27.75, p<0.001). The interaction effect was also significant for both the duration F(1,31)=18.69, p<0.001, and height, F(1,31)=33.02, p<0.001, measures. Post hoc analyses indicated that both shocked and unshocked subjects with septal lesions did not differ significantly from unshocked sham-lesion controls in terms of either measure of defensive burying (p>0.05). The fact that not a single subject with a septal lesion engaged in conditioned defensive burying established that septal lesions do not produce a general increase in defensive responding in the rat. Although anterior septal lesions were shown by Blanchard et al. [1] to enhance responses used by
SEPTAL LESIONS AND BURYING
1053
TABLE 1 MEAN SCORE AND STANDARD ERROR (PARENTHESES) FOR APPROACH, CONTACT, AND SHOCK-RESPONSE MEASURES IN SHOCKED AND NON-SHOCKED SEPTAL AND SHAM LESION GROUPS IN EXPERIMENT 1 Approach frequency*
Approach duration (sec)t
Contact frequencyt
Response to shock
Shock duration (msec)
Shock intensity (mA) 7.5 (0.39) 6.69(0.91)
Shock
Septal (n=9) Sham (n=8)
29.9(11.3) 16.2(3.9)
27.9(11.5) 20.8(3.9)
0.5(0.2) 0.3(0.2)
2.0(0.29) 2.0(0.37)
37.4(6.6) 26.6(5.1)
No Shock
Septal (n=9) Sham (n=8)
42.8(4.0) 35.5(3.3)
170.6(30.0) 156.1(20.6)
21.4(2.5) 13.8(2.9)
---
---
---
*Significant effect of shock vs no-shock (p<0.02). tSignificant effect of shock vs no-shock (p<0.001). All other comparisons were non-significant (p>0.05).
rats to defend themselves from conspecifics, lesions of the entire septum completely eliminated the conditioned defensive burying of an inanimate object. The failure o f the septal rats to bury the prod did not appear to result from a failure to be shocked, to f'md the shock aversive, or to learn the association between the shock and the prod. No significant differences were found in either the intensity, t(15)=0.818, p>0.05, or duration, t(15)= 1.29, p >0.05, of the shock received by the septal and sham-lesion rats, or in their responses to it, t(15)=0.0, p>0.05 (see Table 1). Moreover, the shock suppressed subsequent approaches to and contacts with the prod to the same extent in the septal and sham-lesion groups. Analysis of approach-frequency, approach-duration, and prodcontact scores (see Table 1) indicated only a main effect of shock, F(1,31)=6.06, p>0.05; F(1,31)=49.38, p > 0 . 0 5 ; F(1,31) = 74.78, p >0.05, respectively. Resistance to capture scores indicated that septallesioned rats were not significantly more reactive to handling than were the sham-lesioned control rats (septal mean=2.42, S.E.M. =0.392; sham mean= 1.77, S.E.M. =0.419; t(35)= 1.13, p>0.05). Thus, the differences observed in burying behavior did not appear to reflect a general increase in the reactivity to environmental stimuli on the part of the septal-lesioned rats. The maximal overall extent of the lesions and a typical lesion are presented in Fig. 2 (Panel A). The septal lesions were large and encompassed almost all of the of the medial and lateral septum. Damage extraneous to the septum was found to include portions of the fornix and caudate nucleus, and in some cases slight damage was found to extend below the septal area to include the stria terminalis, nucleus accumbens, and the anterior commissure. Both septal and sham-lesion rats were found to have some minor electrodeproduced damage limited to the dorsal surface of the neocortex. EXPERIMENT 2 Blanchard et al. [1] found that lesions of the anterior portions of the septum enhanced responses used for defense against conspecifics, whereas lesions restricted to the posterior septum had no effect. The purpose of Experiment 2 was to compare the effects of anterior and posterior lesions of the septal area on conditioned defensive burying in rats.
METHOD Serving as subjects were 40 naive, male, 300 to 400 g, hooded Long-Evans rats purchased and housed as in Experiment 1. Bilateral lesions of the anterior septum (1.5 mA, 15 sec) were produced in 12 of these subjects; whereas another 13 received bilateral lesions o f its posterior portions (1.5 mA, 15 sec). In both cases, the general surgical procedures were the same as those of Experiment 1, but the coordinates for both anterior (3 mm anterior and 0.5 mm lateral to bregma, and 5.5 mm ventral to the dura) and posterior (1 mm anterior and 0.5 mm lateral to bregma, and 4.5 mm ventral to the dura) lesions were those of Blanchard et al. [1]. The control subjects received sham lesions of the anterior (n=8) or posterior (n=7) septum, but because the placement of sham lesions within the septum did not affect the dependent measures, the data of these two groups of subjects were combined prior to statistical analysis. After 17 days of postsurgical recovery, all 40 subjects were habituated to the test chamber on 4 consecutive days as in Experiment 1. On day 5, all 40 rats received a single conditioning trial followed immediately by a 15-rain test session in accordance with the procedures of Experiment 1. Resistance to capture was again assessed following the conclusion of behavioral testing. RESULTS AND DISCUSSION The major results of Experiment 2 are summarized in Fig. 3. Whereas lesions of the anterior septum had little or no effect on the levels of conditioned defensive burying, posterior lesions completely suppressed the burying response. Accordingly, a one-way analysis of variance revealed a significant lesion effect in both the duration, F(2,37)=9.42,p<0.001, and height, F(2,37)= 15.65,p<0.001, scores. Subsequent post hoc analyses indicated that the sham-lesion group and the anterior-lesion group did not differ significantly from one another in terms of either measure (p >0.05), but that the posterior-lesion group differed significantly from both these two groups in terms of both measures (p<0.05). As in Experiment 1, the lesion-produced elimination of conditioned defensive burying did not appear to result from either a decrease in responsiveness to shock or to the failure of the lesioned subjects to learn the association between the
1054
GRAY ET AL.
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FIG. 2. Histological results for Experiments 1 (Panel A) and 2 (Panel B). In Panel A, septal lesions are illustrated in the first row whereas sham lesion cortical damage is illustrated in the second row. In Panel B, the first row illustrates posterior septal lesion damage and the second row illustrates anterior septai lesion damage. The shaded area of each section shows the overall maximal extent of all lesions in that group combined, whereas a lesion representative of the particular group is shown in black. The numbered sections correspond to coronal views anterior to bregma taken from Pellegrino and Cushman [13].
prod and the shock (see Table 2). Analysis of the shock parameters r e v e a l e d no significant differences in either the intensity, F(2,38)= 1.01, p >0.05, or duration, F(2,38)=0.392, p > 0 . 0 5 , o f the shocks r e c e i v e d by the three groups. And there w e r e no significant differences b e t w e e n the groups in
their reactivity to the shock, F(2,37)= 2.46, p >0.05, or in the frequency, F(2,37)=0.28, p > 0 . 0 5 , or duration, F(2,37) =2.25, p>0.05, of their approaches to the prod. Resistance to capture scores indicated that both anteriorand posterior-lesion rats were significantly more reactive to
SEPTAL L E S I O N S AND B U R Y I N G
1055 TABLE 2
MEAN SCORE AND STANDARD ERROR (PARENTHESES) FOR APPROACH AND SHOCK-RESPONSE MEASURES IN ANTERIOR SEPTAL, POSTERIOR SEPTAL, AND SHAM LESION GROUPS IN EXPERIMENT 2
Posterior Septal (n=13) Anterior Septal (n=12) Sham (n=15)
o
Approach frequency
Approach duration (sec)
Response to shock
22.3(4.3) 26.3(3.6) 23.1(5.0)
30.1(8.1) 49.4(12.1) 27.2(4.0)
1.86(0.25) 2.38(0.21) 1.73(0.18)
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7
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m
37.85(5.55) 39.67(7.37) 44.33(3.74)
Shock intensity (mA) 9.46(0.58) 8.33(0.58) 8.73(0.50)
m
11
..J
"0
Shock duration (msec)
5 O POST
ANT
SHAM
POST
ANT
SHAM
FIG. 3. Means and standard errors for height of bedding material at the prod (Panel A) and burying duration (Panel B) in the anterior septal (ANT), posterior septal (POST) and sham lesion (SHAM) groups in Experiment 2.
handling than were their sham-lesion counterparts, F(2,38)=8.91, p<0.001. However, post-hoc analyses indicated that the anterior- and posterior-lesion groups did not differ significantly in this respect (anterior mean=2.54, S.E.M.=0.351; posterior mean=3.15, S.E.M.=0.320; sham mean=l.27, S.E.M.=0.316). The fact that anterior- and posterior-lesion groups did not differ in resistance to capture but did differ in burying behavior suggests that the enhanced reactivity contributed little to the deficits in burying behavior observed as a result of the posterior lesions. Although the increase in hyperreactivity produced by the septal lesions in Experiment 1 did not reach significance and the increase observed in Experiment 2 did, the two experiments did not differ substantially in this respect. Histological analyses (Fig. 2, Panel B) indicated that all lesions were placed in the appropriate areas of the septum. Whereas the posterior lesions were predominantly restricted to the medial and lateral septal nuclei, slight additional damage to the caudate nucleus, corpus callosum, or dorso-medial cortex was observable in all posterior subjects. Anterior septal lesions did not involve damage posterior to the level of the anterior commissure but did involve damage to adjacent forebrain areas.
G E N E R A L DISCUSSION In Experiment 1, lesions of the entire septum disrupted the conditioned defensive burying that was observed in rats with sham lesions. The results of the second experiment suggested that the area of the septum that is critical for conditioned defensive burying is located in its posterior portion. Lesions of the posterior septum disrupted conditioned defensive burying, whereas anterior lesions were without consequence. There are two features of the effect of posterior septal lesions on conditioned defensive burying that are worthy of emphasis: its reliability and selectivity. In both experiments, conditioned defensive burying was reliably observed in every shocked rat without damage to the posterior septum, but not a single subject with damage to this area was observed to engage in this behavior. Moreover, although posterior septal lesions completely suppressed conditioned defensive burying, they were without effect on any of the other dependent measures. Except for the fact that the rats with posterior septal lesions displayed no burying behavior, their behavior was indistinguishable from that of the shamlesion controls; they reacted to the shock in the same way and subsequently avoided it to the same degree. Thus, the deficit in conditioned burying cannot be attributed to an inability of the rats to perceive or fear the shock, or to associate it with the prod. The results of the present experiments confirm the view of Blanchard et al. [1] that the septum plays an important role in the regulation of defensive behavior and that anterior and posterior portions of the septal area exert differential control. However, in view of the fact that Blanchard et al. [1] found behaviors used by rats to defend themselves from conspecifics to be enhanced by anterior lesions but not by posterior lesions, the present results suggest that not all defensive behaviors are regulated by the septal area in the same way. In Experiment 2, posterior lesions completely eliminated conditioned defensive burying, whereas anterior lesions were ineffective. The conclusion that the posterior septum exerts control over conditioned defensive burying must be qualified in three important ways. First, although it is quite clear that some part of the posterior septal area mediates conditioned defensive burying, it is not clear whether it is the septal nuclei themselves or fibers passing through this portion of the septum that are of primary importance. For example, it is possible that the effects of septal lesions on defensive burying may have resulted from damage to the fornix, which includes afferent and efferent pathways of the hippocampus.
1056
GRAY E T A L .
Hippocampal disruption has been reported to result in a variety of behavioral deficits and some authors have interpreted these deficits to reflect a "spatial mapping" function of the hippocampus (e.g., [10,11]). However, a simple spatial-memory-deficit hypothesis cannot account for the present data. The rats with septal lesions were clearly able to localize and subsequently avoid contact with the source of the aversive stimulus. Second, the present results do not imply that the control of defensive behavior is the only function of the septal area; septal lesions have been shown to disrupt a variety of behaviors in rodents (see [6,7] for review). This has led to the view that the septum and other limbic structures such as the hippocampus may be involved in the sequencing of behavior in general rather than controlling one particular class of behaviors (cf. [5,9]). Third, it should be emphasized that the subjects in the present experiment were not tested until 17 days after surgery and were handled in the interim. Because some of the effects of septal lesions decline markedly with handling or just with the passage of time (e.g., hyperreactivity), the magnitude of the present effects on conditioned defensive burying are all the more striking. It should be noted that in contrast to the previous reports that septal lesions produce passive avoidance deficits [6,7], the septal lesions in the present experiments did not reduce
the tendency of the subjects to stay away from the conditoned stimulus. Although passive avoidance and conditioned defensive burying paradigms are similar in that the behavior of the subjects is changed following a single shock in both, they differ in several major respects. For example, in the conditioned defensive burying paradigm, the conditioned stimulus is a well-defined object, i.e., the prod; whereas, in the usual passive avoidance situations the shock is administered diffusely through the floor of the apparatus. Although it is interesting to speculate that conditioned passive avoidance may be resistant to disruption by septal lesions when the conditioned stimulus is a well-defined object, there are other methodological features of the present experiments (e.g., the availability of bedding or the relatively generous postsurgical recovery period) that could also account for the lack of a passive avoidance deficit in the lesioned subjects. The present experiments constitute the first demonstration of the utility of the conditioned defensive burying paradigm in assessing the effects of iimbic lesions. The reliability and robustness of the present data suggest that the conditioned defensive burying paradigm will prove to be an important addition to the behavioral paradigms available for assessing the behavioral effects of physiological manipulations.
REFERENCES I. Blanchard, D. C., R. J. Blanchard, E. M. C. Lee and S. Nakamura. Defensive behaviors in rats following septal and septal-amygdala lesions. J. comp. physiol. Psychol. 93: 378390, 1979. 2. Blanchard, D. C., R. J. Blanchard, L. Takahashi and T. Takahashi. Septal lesions and aggressive behavior. Behav. Biol. 21: 157-161, 1977. 3. Brady, J. V. and W. J. H. Nauta. Subcortical mechanisms in emotional behavior: Affective changes following septal forebrain lesions in the albino rat. J. comp. physiol. Psychol. 46: 339--346, 1953. 4. Coss, R. G. and D. H. Owings. Snake-directed behavior by snake naive and experienced California ground squirrels in a simulated burrow. Z. Tierpsychol. 48: 421--435, 1978. 5. Fleischer, S. and B. M. Slotnik, Disruption of maternal behavior in rats with lesions of the septal area. Physiol. Behav. 21: 189--200,1978. 6. Grossman, S. P. Behavioral functions of the septum: A reanalysis. In: The Septal Nuclei, edited by J. F. DeFrance. New York: Plenum Press, 1976. 7. Isaacson, R. L. The Limbic System. New York: Plenum Press, 1974. 8. Lau, P. and K. A. Miczek, Differential effects of septal lesions on attack and defensive-submission reaction during intraspecies aggression in rats. Physiol. Behav. 18: 479-485, 1977. 9. Michal, E. K. Effects of limbic lesions on behavior sequences and courtship behavior of male rats (rattus norvegicus). Behaviour 44: 264-285, 1973. 10. Nadel, L. and J. O'Keefe. The hippocampus in pieces and patches: An essay on modes of explanation in physiological psychology. In: Essays on the Nervous System. A Festschrift for Prof. J. Z. Young, edited by R. Bellairs and E. G. Gray. Oxford: Clarendon Press, 1974, pp. 367-390.
11. Olten, D. S., J. A. Walker and F. Grage. Hippocampal connections and spatial discrimination. Brain Res. 139: 255-308, 1978. 12. Owings, D. H. and R. G. Coss. Snake mobbing by California ground squirrels: Adaptive variation and ontogeny. Behaviour 62: 50-69, 1978. 13. Pellegrino, L. J. and A. J. Cushman. A Stereotaxic Atlas o f the Rat Brain. New York: Appleton-Century-Crofts, 1967. 14. Pinel, J. P. J. and D. Treit. Burying as a defensive response in rats. J. comp. physiol. Psychol. 92: 708-712, 1978. 15. Pinel, J. P. J. and D. Treit. Conditioned defensive burying in rats: Availability of burying materials. Anita. Learn. Behav. 7: 392-396, 1979. 16. Seggie, J. Effect of adrenalectomy or gonadectomy on affective behavior changes following septal lesions in rats. J. comp. physiol. Psychol. 74:11-19, 1974. 17. Slotnik, B. M. and M. F. McMullen. Intraspecific fighting in albino mice with septal forebrain lesions. Physiol. Behav. 8: 333-337, 1972. 18. Terlecki L. J., J. P. J. Pinel and D. Treit. Conditioned and unconditioned defensive burying in the rat. Learn. Motivat. 10: 337-350, 1979. 19. Treit, D., L. J. Terlecki and J. P. J. Pinel. Conditioned defensive burying in rodents: Organismic variables. Bull. Psychon. Soc. 16: 451-454, 1980. 20. Wilkie, D. M., A. J. MacLennan and J. P. J. Pinel. Rat defensive behavior: Burying noxious food. J. exp. Analysis Behav. 31: 299-306, 1979.