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Effect of lesions in hippocampal subareas on rat shuttle behavior
Physiology &Behavior, Vol. 23, pp. 989--993.PergamonPress and BrainResearch Publ., 1979. Printed in the U.S.A.
Effect of Lesions in Hippocampal Subareas on Rat Shuttle Behavior' L I N E U S. C A L D E R A Z Z O F I L H O " A N D E S P E R A. C A V A L H E I R O
Disciplina de Neurofisiologia, Departamento de Fisiologia, Escola Paulista de Medicina. Rua Botucatu, 862, 04023, $6o Paulo, SP. Brasil AND IVAN IZQUIERDO
Departamento de Bioquirnica, lnstituto de Bioci~ncias da Universidade Federal do Rio Grande do Sul, 90000 Porto Alegre, RS, Brasil R e c e i v e d 12 F e b r u a r y 1979 CALDERAZZO FILHO, L. S., E. A. CAVALHEIRO AND I. IZQUIERDO. Effect of lesions in hippocampal s,bareas on rat shuttle behavior. PHYSIOL. BEHAV. 23(6) 98%993, 1979.--Rats with lesions in subareas CA3 and CA4/dentate gyrus of the dorsal hippocampus were submitted to four different experimental situations in a shuttlebox. Animals with CA3 lesions made less shuttle responses to a buzzer than sham-operated or intact control animals in two of the tests, in which the buzzer was paired (i.e., given contiguous) to a footshock: a Pavlovian paradigm, and a typical two-way avoidance situation. Animals with lesions in CA4/dentate gyrus showed no difference in their performance of shuttle responses to the buzzer in any of the four tests. The results suggest that CA3 plays a role in the establishment of stimulus-stimulus relations in the brain, and that CA4 and the dentate gyrus play no major role in any of the aversive situations examined in this paper. Pseudoconditioning Classical conditioning Stimulus pairing Avoidance contingency
Avoidance conditioning Hippocampus
RATS may be trained in a shuttle-box to perform shuttle responses to a buzzer or a tone in at least four different ways: pseudoconditioning (footshocks randomly interspersed among the buzzers at randomly variable intervals, 5, 7, 14, 15, 24); classical conditioning (buzzers paired to footshocks on every trial, regardless of what responses are made to the former, 5, 6, 7, 13, 14, 15, 16); avoidance conditioning without stimulus pairing (the buzzer-shock interval is varied at random but the shock is contingent upon the non-omission of a shuttle response to the buzzer, 5, 6, 7, 13, 14, 15, 20, 29); and standard or typical two-way avoidance, in which the two stimuli are paired (contiguous) as in the Pavlovian procedure but the footshock is omitted on those trials in which the animal shuttles to the buzzer [13, 14, 15, 16]. In the pseudoconditioning paradigm, shuttling to the buzzer apparently results from the mere presence of footshocks in the experimental situation (non-associative effect of shocks, 2, 3, 4; drive in the sense used by Anisman and Waller; [2], or by Skinner, [19] or by our own group in several papers, [14, 15, 20]. Performance in a 50-buzzer pseudoconditioning test is the same whether 10, 25, or 50 shocks are given among the
Non-associative factors
buzzers [24]. In the other tests, this drive (D) component is also present, but other factors play a role in addition to it: the Pavlovian stimulus-stimulus pairing (P) in both the classical and the typical two-way avoidance paradigms; and the shuttle-no shock or avoidance contingency (C) in the two avoidance tests. In consequence, the pseudoconditioning procedure has been called a D test, the Pavlovian paradigm a DP test, the avoidance situation without stimulus pairing a DC test, and the typical two-way avoidance paradigm a DPC test [5, 6, 7, 13, 14, 15, 20, 21, 24]. Operation of these three factors D, P, and C, has recently been shown to be independent, and therefore additive, at least in appearance [14]. Thus, performance in a DPC test may be predicted, for any given groups studied, from that in the other three tests, D + (DP - D) + (DC - D)= D + P + C = D P C [5, 7, 14, 15, 20]. This equation has been found to hold in all the groups studied so far in these four paradigms, which now number at least 44 (thirty mentioned in a recent review, [15], plus others in three papers now in press, [20, 21, 24]. Admittedly, this linear model may be overly simplistic, and the additive property of D, P, and C may just be a felicitous coincidence
~Supported by an institutional grant from FINEP to the Escola Paulista de Medicina, and by Research Grants from FAPESP and from CNPq to Dr. Iv~inlzquierdo. 2Fellow from FAPESP, BRASIL.
C o p y r i g h t © 1978 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/79/120989-05502.00/0
990
CALDERAZZO FILHO E T A L .
")
('"
FIG. 1. Minimum (darkened) and maximum (striped) extent of CA3 lesions. The brain section depicted here corresponds to approximately plane A 3.4 from the atlas by Krnig and Klippel [18].
FIG. 2. Minimum (darkened) and maximum (striped) extent of CA4/denate gyrus lesions. The brain section depicted here corresponds to approximately plane A 3.4 from the atlas by Krnig and Klippel [18]
resulting from a number of complex factors [12,14]. However, the model allows for, at least, a descriptive analysis of some of the major components of rat shuttle behavior, and thus might be of heuristic value [15]. In the present paper, we report on the effect of lesions placed in the hippocampal subareas, CA3, and CA4/dentate gyrus on pseudoconditioning, classical conditioning, and on the two forms of avoidance mentioned above. The purpose of the present paper is to continue the exploration of the role of the hippocampal formation in the operation of D, P, and C. In previous papers it was shown that lesions of the CAI/CA2 subareas of the dorsal hippocampus [5], or acute hippocampal spreading depression [7], enhance operation of the D factor, depress the operation of P, and have no effect on that of C. In addition, subtle alterations of hippocampal theta activity have been observed in rats trained in each of the behavioral procedures mentioned above [6].
groups of sham-operated animals were also tested.In the sham groups, electrodes were placed at the same A and L coordinates as in the lesioned animals, but were lowered only 0.5 mm down from the cortical surface, and current was not passed. Lesion placements were verified in post-mortem formalin-fixed brain sections. Figures 1 and 2 illustrate maximum and minimum extent of the two types of lesion.
METHOD Animals and Surgery
240 Adult male rats (weight, 200-300 g; age, 3-4 months) from a Wistar stock maintained at the Escola Paulista de Medicina for 12 years, were used. The animals were kept on an approximately 12 hr light/dark cycle, and housed in groups of 4 before surgery, and individually after that. The surgical procedure was carried out under deep anesthesia (ketamine, 60 mg/Kg IM, plus pentobarbital, 20 mg/Kg IP). Atropine sulfate (2 mg/Kg, IP) was also given during surgery in order to reduce nasopharyngeal secretion. The animals were placed in a stereotaxic instrument and holes were drilled at suitable positions in their parietal bones with a dental burr. Stereotaxic coordinates were selected according to the atlas by Krnig and Klippel [16], as follows: CA3 (A 3.4, L 3.4, V - 1.2), and CA4/dentate gyrus (A 3.4, L 2.0, V -1.6). Steel electrodes (0.125 mm, insulated except for 0.2 mm at the tip) were lowered to the desired positions and 1 mA DC was passed between their tip and a rectal cathode for 15 sec, first on one side and then on the other. In addition to these lesioned groups, 2 groups of intact controls, and 2
Training
Behavioral tests were carried out in a 50×25×25 cm shuttle-box made of wood painted gray except for the frontal wall which was glass. At the midline on the lid there was a 6 W light bulb hanging from the inside and a buzzer fixed to the outside. The light was on throughout the duration of each test. The floor of the box consisted of 2 mm bronze bars spaced 7 mm apart. A fiat piece of wood, 5 mm wide, was the only mark between the right and left side of the grid. The behavioral tests were as follows: D test. (pseudoconditioning): Fifty 5-sec, 70 dB buzzers delivered at intervals which varied randomly between 5 and 40 sec. Twenty-five 60 Hz, 1.5 mA, 2-sec scrambled footshocks were randomly interspersed among the buzzers at buzzer-shock intervals which also varied randomly between 5 and 30 sec. D P test. (classical conditioning): Fifty 5-sec buzzers were delivered at 5-40 sec intervals as above, but each was immediately followed by a footshock (stimulus contiguity). Footshocks were given on all trials, whether there was a shuttle response to the buzzer or not [13,16]. D C test. (avoidance without stimulus pairing): Fifty 5-sec buzzers were given at 5-40 sec intervals as above, but each was followed after a randomly variable 5-30 sec interval by a footshock, only in those trials in which the animals did not shuttle to the buzzer. Therefore, shuttling to the buzzer resulted in cancellation of the next scheduled shock (avoidance contingency). D P C test. (avoidance with stimulis pairing; typical or standard two-way avoidance): Fifty 5-sec buzzers were de-
HIPPOCAMPAL
LESIONS AND SHUTTLE
BEHAVIOR
991
TABLE 1 SHUTTLE RESPONSES TO A BUZZER PERFORMED BY RATS WITH CA3 HIPPOCAMPAL LESIONS, BY SHAM-OPERATED ANIMALS, AND BY INTACT CONTROLS, IN FOUR DIFFERENT BEHAVIORAL SITUATIONS Behavioral situation D (pseudoconditioning) DP (classical conditioning) DC (avoidance without stimulus pairing) DPC (avoidance with stimulus pairing) DP-[) DC-~) D + (DP-D) + (DC-D)
CA3 lesion group
Sham-operated group
Intact controls
4.8 _+ 2.0
5.5 _+ 1.6
4.9 + 2.6
6.2 _+ 2.6*
13.4 _+ 2.3
12.2 _+ 1.9
12.9 _+ 1.7
14.0 _+ 1.4
14.2 _+ 2.4
13.9 _+ 2.9* 1.4 _+ 1.7" 8.1 _+ 2.3 14.3
21.1 _+ 2.4 7.9 _+ 3.2 8.5 _+ 2.1 21.9
22.2 _+ 3.1 7.3 _+ 2.3 9.3 _+ 1.8 21.5
Data expressed as mean number of responses per test _+ SE. Ten animals per group. *: significant difference from the other two groups at 5% level in a Duncan multiple-range test.
TABLE 2 SHUTTLE RESPONSES TO A BUZZER PERFORMED BY RATS WITH CA4/DENTATE HIPPOCAMPAL LESIONS, BY SHAM-OPERATED ANIMALS, AND BY INTACT CONTROLS, IN FOUR DIFFERENT BEHAVIORAL SITUATIONS Behavioral situation D (pseudoconditioning) DP (classical conditioning) DC (avoidance without stimulus pairing) DPC (avoidance with stimulus pairing) DP-[) DC-D D + (DP-D) + (DC-D)
CA4/dentate lesion group
Sham-operated group
Intact controls
4.8 _+ 2.0
4.6 _+ 1.2
4.8 _+ 1.6
11.9 _+ 1.1
10.8 _+ 1.6
11.8 _+ 1.0
15.5 _+ 1.0
14,7 _+ 1.3
14.1 +_ 1.3
21.7 _+ 2.0 7.1 _+ 1.7 10.7 _+ 1.5 22.6
20,3 _+ !.9 6,2 _+ 2.1 10.1 _+ 2.2 20.9
22.7 _+ 1.8 7.0 _+ 1.5 9.3 + 1.8 21.1
Data expressed as mean number of responses per test -+ SE. Differences among groups for the same test not significant at 5% level in Duncan multiple-range test.
l i v e r e d at r a n d o m l y v a r i a b l e 5 - 4 0 sec i n t e r v a l s , as in the p r e c e d i n g tests; b u t e a c h w a s f o l l o w e d i m m e d i a t e l y b y a f o o t s h o c k (contiguity o r pairing, as in t h e D P test); u n l e s s t h e r e w a s a shuttle r e s p o n s e to t h e b u z z e r ( a v o i d a n c e cont i n g e n c y , as in t h e D C situation). F o r f u r t h e r details o n the a p p a r a t u s a n d o n t h e s e b e h a v ioral tests, see [5, 6, 7, 13, 14, 15, 20, 21, 24]. S h u t t l e r e s p o n s e s to t h e b u z z e r , a n d intertrial c r o s s i n g s (i.e., s h u t t l e r e s p o n s e s o c c u r r i n g in the b u z z e r - b u z z e r , b u z z e r - s h o c k , o r s h o c k - b u z z e r intervals) w e r e c o u n t e d . N o d i f f e r e n c e s in p e r f o r m a n c e o f intertrial c r o s s i n g s w e r e obs e r v e d a m o n g g r o u p s , a n d the d a t a o n t h e s e will n o t b e rep o r t e d h e r e . T h e n u m b e r o f s h u t t l e r e s p o n s e s to t h e b u z z e r in t h e D t e s t w a s t a k e n as a m e a s u r e of the D f a c t o r [5, 7, 14, 15, 20, 21]. T h e P f a c t o r w a s e s t i m a t e d b y s u b t r a c t i n g f r o m e a c h i n d i v i d u a l p e r f o r m a n c e in t h e D P t e s t , the m e a n perf o r m a n c e o f a n i m a l s in the s a m e t r e a t m e n t g r o u p (lesioned, s h a m - o p e r a t e d , i n t a c t c o n t r o l s ) in the D t e s t (DP - D). T h e
C f a c t o r w a s c a l c u l a t e d b y s u b t r a c t i n g f r o m e a c h individual p e r f o r m a n c e in the DC t e s t the m e a n for t h a t g r o u p in the D test ( D C - D) [5, 6, 7, 14, 15, 20, 24]. E l s e w h e r e , it w a s r e p o r t e d t h a t p e r f o r m a n c e in t h e D test is the s a m e regardless o f w h e t h e r 10, 25, o r 50 s h o c k s are g i v e n d u r i n g it [24]. T h e r e f o r e , it m a y b e a s s u m e d t h a t t h e r e l a t i v e c o n t r i b u t i o n o f the D f a c t o r w a s p r o b a b l y t h e s a m e in t h e DP, DC, or D C P s i t u a t i o n s , r e g a r d l e s s o f the fact t h a t different n u m b e r s o f f o o t s h o c k s w e r e g i v e n in e a c h o f t h o s e t e s t s [14, 15, 20, 24]. RESULTS P e r f o r m a n c e of s h u t t l e r e s p o n s e s to t h e b u z z e r in the D P a n d D P C s i t u a t i o n s was l o w e r in t h e CA3 lesion g r o u p t h a n in its c o r r e s p o n d i n g c o n t r o l s ( T a b l e 1). T h i s is a t t r i b u t a b l e to a d e p r e s s i o n o f the P f a c t o r ( D P - D), since D a n d C app e a r e d to n o r m a l in this group. T h e r e w e r e n o significant d i f f e r e n c e s in a n y o f the four tests, D, DP, DC, o r D P C , between sham-operated and intact controls.
992
CALDERAZZO FILHO ET AL.
As shown in Table 2, there was no significant difference in the number of shuttle responses to the buzzer in any of the four tests, among CA4/dentate-lesioned animals, its shamoperated group, and the intact controls. DISCUSSION These results, when put together with those previously reported on the effect of CAI-CA2 lesions [5], might, in appearance, provide a better understanding of the role of the hippocampus in rat shuttle behavior. Operation of the P factor would seem to depend on the integrity of CA1, CA2, and CA3. The subareas CAI and CA2 would, in addition, exert a tonic inhibitory influence on D. Finally, CA4 and the dentate gyrus would appear to be of little relevance to any of the three factors, D, P, or C. Vinogradovfi [25] suggested that Schaeffer collaterals, which originate from CA3 neurons and terminate upon dendritic fields of CA I-CA2 pyramidal cells, may have a regulatory influence on the latter. Thus, it is possible that the effect of CA3 lesions on the P component of shuttle behavior may result from the suppression of this regulatory influence of CA3 on CA1 and CA2. If this were true, since both CA3 and CAI-CA2 lesions [5] had a ceiling effect in depressing P, and since CA4/dentate lesions had no effect, one would expect that hippocampal spreading depression (which presumably affects all hippocampal subfields) would influence rat shuttle behavior in a manner indistinguishable from that of a CA1 or CA2 lesion. In fact, as shown in a previous report [7], this is exactly what happens. The role here proposed for C A I , CA2, and CA3, fits with hypotheses which suggest a role of the hippocampus in behavioral inhibition [1,8], as well as with those which suggest a role in the processing of paired sensory information [12,23]. Moreover, the apparent conflict between the two sets of hypotheses would be solved [5,12], and previous data by others on the effect of various types of hippocampal lesions on rat shuttle avoidance and related behaviors [8, 10,
11, 12[ would be satisfactorily explained. An even more straightforward interpretation of our previous [5,7] and present results may be found along the lines drawn by the more recent hypotheses of Hirsh [9], O 'K eef e and Nadel [18], and Osborne and Black [19]. Hirsh [9] suggested a general role of the hippocampus in the subtle processing of various kinds of information. O'Keefe and Nadel [18] consider it to be particularly related to spatial mapping. Accordingly, animals with hippocampal lesions would be predicted to have less flexible (in particular, stereotyped and/or repetitious) motivational responses [19], and to be defective in tasks which require subtle information processing leading to the elaboration of a spatial map (of which classical or instrumental learning in a shuttle-box are typical examples). The increased responding observed in the D test in rats with CAI-CA2 lesions [5] may thus be viewed as an expression of behavioral inflexibility in a situation in which such responding is essentially purposeless, and is due mainly, if not exclusively [13,14], to non-associative and/or motivational factors (i.e., what we chose to call drive) [13, 14, 15, 20]. The depressed operation of the P factor observed in the animals with CAICA2 [5] or CA3 lesions, may be viewed as a specific inability to elaborate adequate spatial maps with which to generate shuttle responses to the buzzer in the presence of a particular mode of sensory stimulation, namely, buzzer-shock contiguity. Mechanism through which the hippocampus could process paired sensory information have been suggested by several suthors [12,23]. For possible ways by which the hippocampus might translate such information into appropriate spatial maps, see O ' K e e f e and Nadel [18]. The apparent lack of effect of hippocampal lesions on the C factor may signify that the elaboration of spatial maps adequate for shuttle responding may proceed in the absence of an intact hippocampus, when a shuttle-no shock avoidance contingency is in operation; perhaps, as Hirsh [9], and Osborne and Black [19] have suggested, through the use of residual or vicarious systems.
REFERENCES
1. Airman, J., R. L. Brunner and S. Bayer. The hippocampus and behavioral maturation. Behav. Biol. 8: 557-596, 1973. 2. Anisman, H. Aversively motivated behavior as a tool in psychopharmacologic analysis. In: Psychopharmacology af A versively Motivated Behavior. edited by H. Anisman and G. Bignami. New York: Plenum Press, 1978, pp. 1-62. 3. Anisman, H. and T. G. Waller. Effects of inescapable shock upon subsequent avoidance performance: role of response repertoire changes. Behav. Biol. 9: 331-355, 1973. 4. Bignami, G. Nonassociative explanation of behavioral changes induced by central cholinergic drugs. Acta Neurobiol. Exp. 36: 5-90, 1976. 5. Calderazzo Filho, L. S., A. Moschovakis and 1. lzquierdo. Effect of hippocampal lesions on rat shuttle responses in four different behavioral tests. Physiol. Behav. 19: 96%972, 1977. 6. Cavalheiro, E. A. Role of 0 rhythm during rat shuttle behavior in four different experimental situations. Behav. Neural. Biol. 25: 20%220, 1979. 7. Cavalheiro, E. A. and 1.' lzquierdo. Effect of hippocampal and neocortical spreading depression on rat shuttle behavior in four different experimental paradigms. Physiol. Behav. 18: 10111016, 1977. 8. Douglas, R. J. The hippocampus and behavior. Psychol. Bull. 67: 416-422, 1967.
9. Hirsh, R. The hippocampus and contextual retrieval of information from memory: a theory. Behav. Biol. 12: 421-444, 1974. 10. lsaacson, R. L., R. J. Douglas and R. Y. Moore. The effect of radical hippocampal ablation on acquisition of an avoidance response. J. comp. physiol. Psyehol. 54: 626-628, 1961. 11. Isaacson, R. L. I7w Limbic System. New York: Plenum Press 1975.
12. lzquierdo, I. The hippocampus and" learning. Prog. Neurobiol. 5: 37-75, 1975. 13. Izquierdo, 1. A pharmacological separation of stimulus pairing and of the response-reinforcement contingency as factors in the elicitation of shuttle responses to a buzzer in rats. Behav. Biol. 18: 75-87, 1976. 14. lzquierdo, I. and E. A. Cavalheiro. Three main factors in rat shuttle behavior: their pharmacology and sequential entry in operation during a two-way avoidance session. Psyehopharmacology 49: 145-157. 1976. 15. Izquierdo, I. and E. Elisabetsky. Physiological and pharmacological dissection of the main factors in the acquisition and retention of shuttle behavior. In: Brain Mechanisms in Memory and Learning:from the Single Neuron to Man. edited by M. A. B. Brazier. IBRO Monograph Series, vol. 4, New York: Raven Press, 1979, pp. 227-248.
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16. Katzev, R. D. and S. K. Mills. Strain differences in avoidance conditioning as a function of the classical CS-UD contingency. J. comp. physiol. Psychol. 87: 661-671, 1974. 17. K6nig, J. F. R. and R. A. Klippel. The Rat Brain; a Stereotaxic Atlas. Krieger: Huntington, 1974. 18. O'Keefe, J. and L. Nadel. The Hippocampus as a Cognitive Map. London: Oxford University Press 1978. 19. Osborne, B. and A. H. Black. A detailed analysis of behavior during the transition from acquisition to extinction in rats with fornix lesions. Behav. Biol. 23: 271-290, 1978. 20. Schiitz, R. A. and I. Izquierdo. Effect of brain lesions on rat shuttle behavior in four different tests. Physiol. Behav. 23: 97-105, 1979. 21. SchCitz, R. A., M. T. B. Schiitz, O. A. Orsingher and 1. Izquierdo. Brain dopamine and noradrenaline levels in rats submitted to four different aversive behavioral tests. Psychopharmacology 63: 28%292, 1979,
22. Skinner, B. F. Science and Human Behavior. New York: MacMillan 1953. 23. Solomon, P. R. Role of the hippocampus in blocking and conditioned inhibition of the rabbit's nictitating membrane response. J. comp. physiol. Psychol. 91: 407-417, 1977. 24. Vendite, D. A., E. Elisabetsky, D. O. Souza and I. Izquierdo. Effect of the number and density of footshocks on pseudoconditioned responses in a shuttle-box. Acta physiol latinoam., in press, 1979. 25. Vinogradov~, O. S. Functional organization of the limbic system in the process of registration of information: facts and hypotheses. In: The Hippocampus, edited by R. L. Isaacson and K. H. Pribram. vol. 2, New York: Plenum Press, 1978, pp. 3-69.
Effect of Lesions in Hippocampal Subareas on Rat Shuttle Behavior' L I N E U S. C A L D E R A Z Z O F I L H O " A N D E S P E R A. C A V A L H E I R O
Disciplina de Neurofisiologia, Departamento de Fisiologia, Escola Paulista de Medicina. Rua Botucatu, 862, 04023, $6o Paulo, SP. Brasil AND IVAN IZQUIERDO
Departamento de Bioquirnica, lnstituto de Bioci~ncias da Universidade Federal do Rio Grande do Sul, 90000 Porto Alegre, RS, Brasil R e c e i v e d 12 F e b r u a r y 1979 CALDERAZZO FILHO, L. S., E. A. CAVALHEIRO AND I. IZQUIERDO. Effect of lesions in hippocampal s,bareas on rat shuttle behavior. PHYSIOL. BEHAV. 23(6) 98%993, 1979.--Rats with lesions in subareas CA3 and CA4/dentate gyrus of the dorsal hippocampus were submitted to four different experimental situations in a shuttlebox. Animals with CA3 lesions made less shuttle responses to a buzzer than sham-operated or intact control animals in two of the tests, in which the buzzer was paired (i.e., given contiguous) to a footshock: a Pavlovian paradigm, and a typical two-way avoidance situation. Animals with lesions in CA4/dentate gyrus showed no difference in their performance of shuttle responses to the buzzer in any of the four tests. The results suggest that CA3 plays a role in the establishment of stimulus-stimulus relations in the brain, and that CA4 and the dentate gyrus play no major role in any of the aversive situations examined in this paper. Pseudoconditioning Classical conditioning Stimulus pairing Avoidance contingency
Avoidance conditioning Hippocampus
RATS may be trained in a shuttle-box to perform shuttle responses to a buzzer or a tone in at least four different ways: pseudoconditioning (footshocks randomly interspersed among the buzzers at randomly variable intervals, 5, 7, 14, 15, 24); classical conditioning (buzzers paired to footshocks on every trial, regardless of what responses are made to the former, 5, 6, 7, 13, 14, 15, 16); avoidance conditioning without stimulus pairing (the buzzer-shock interval is varied at random but the shock is contingent upon the non-omission of a shuttle response to the buzzer, 5, 6, 7, 13, 14, 15, 20, 29); and standard or typical two-way avoidance, in which the two stimuli are paired (contiguous) as in the Pavlovian procedure but the footshock is omitted on those trials in which the animal shuttles to the buzzer [13, 14, 15, 16]. In the pseudoconditioning paradigm, shuttling to the buzzer apparently results from the mere presence of footshocks in the experimental situation (non-associative effect of shocks, 2, 3, 4; drive in the sense used by Anisman and Waller; [2], or by Skinner, [19] or by our own group in several papers, [14, 15, 20]. Performance in a 50-buzzer pseudoconditioning test is the same whether 10, 25, or 50 shocks are given among the
Non-associative factors
buzzers [24]. In the other tests, this drive (D) component is also present, but other factors play a role in addition to it: the Pavlovian stimulus-stimulus pairing (P) in both the classical and the typical two-way avoidance paradigms; and the shuttle-no shock or avoidance contingency (C) in the two avoidance tests. In consequence, the pseudoconditioning procedure has been called a D test, the Pavlovian paradigm a DP test, the avoidance situation without stimulus pairing a DC test, and the typical two-way avoidance paradigm a DPC test [5, 6, 7, 13, 14, 15, 20, 21, 24]. Operation of these three factors D, P, and C, has recently been shown to be independent, and therefore additive, at least in appearance [14]. Thus, performance in a DPC test may be predicted, for any given groups studied, from that in the other three tests, D + (DP - D) + (DC - D)= D + P + C = D P C [5, 7, 14, 15, 20]. This equation has been found to hold in all the groups studied so far in these four paradigms, which now number at least 44 (thirty mentioned in a recent review, [15], plus others in three papers now in press, [20, 21, 24]. Admittedly, this linear model may be overly simplistic, and the additive property of D, P, and C may just be a felicitous coincidence
~Supported by an institutional grant from FINEP to the Escola Paulista de Medicina, and by Research Grants from FAPESP and from CNPq to Dr. Iv~inlzquierdo. 2Fellow from FAPESP, BRASIL.
C o p y r i g h t © 1978 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/79/120989-05502.00/0
990
CALDERAZZO FILHO E T A L .
")
('"
FIG. 1. Minimum (darkened) and maximum (striped) extent of CA3 lesions. The brain section depicted here corresponds to approximately plane A 3.4 from the atlas by Krnig and Klippel [18].
FIG. 2. Minimum (darkened) and maximum (striped) extent of CA4/denate gyrus lesions. The brain section depicted here corresponds to approximately plane A 3.4 from the atlas by Krnig and Klippel [18]
resulting from a number of complex factors [12,14]. However, the model allows for, at least, a descriptive analysis of some of the major components of rat shuttle behavior, and thus might be of heuristic value [15]. In the present paper, we report on the effect of lesions placed in the hippocampal subareas, CA3, and CA4/dentate gyrus on pseudoconditioning, classical conditioning, and on the two forms of avoidance mentioned above. The purpose of the present paper is to continue the exploration of the role of the hippocampal formation in the operation of D, P, and C. In previous papers it was shown that lesions of the CAI/CA2 subareas of the dorsal hippocampus [5], or acute hippocampal spreading depression [7], enhance operation of the D factor, depress the operation of P, and have no effect on that of C. In addition, subtle alterations of hippocampal theta activity have been observed in rats trained in each of the behavioral procedures mentioned above [6].
groups of sham-operated animals were also tested.In the sham groups, electrodes were placed at the same A and L coordinates as in the lesioned animals, but were lowered only 0.5 mm down from the cortical surface, and current was not passed. Lesion placements were verified in post-mortem formalin-fixed brain sections. Figures 1 and 2 illustrate maximum and minimum extent of the two types of lesion.
METHOD Animals and Surgery
240 Adult male rats (weight, 200-300 g; age, 3-4 months) from a Wistar stock maintained at the Escola Paulista de Medicina for 12 years, were used. The animals were kept on an approximately 12 hr light/dark cycle, and housed in groups of 4 before surgery, and individually after that. The surgical procedure was carried out under deep anesthesia (ketamine, 60 mg/Kg IM, plus pentobarbital, 20 mg/Kg IP). Atropine sulfate (2 mg/Kg, IP) was also given during surgery in order to reduce nasopharyngeal secretion. The animals were placed in a stereotaxic instrument and holes were drilled at suitable positions in their parietal bones with a dental burr. Stereotaxic coordinates were selected according to the atlas by Krnig and Klippel [16], as follows: CA3 (A 3.4, L 3.4, V - 1.2), and CA4/dentate gyrus (A 3.4, L 2.0, V -1.6). Steel electrodes (0.125 mm, insulated except for 0.2 mm at the tip) were lowered to the desired positions and 1 mA DC was passed between their tip and a rectal cathode for 15 sec, first on one side and then on the other. In addition to these lesioned groups, 2 groups of intact controls, and 2
Training
Behavioral tests were carried out in a 50×25×25 cm shuttle-box made of wood painted gray except for the frontal wall which was glass. At the midline on the lid there was a 6 W light bulb hanging from the inside and a buzzer fixed to the outside. The light was on throughout the duration of each test. The floor of the box consisted of 2 mm bronze bars spaced 7 mm apart. A fiat piece of wood, 5 mm wide, was the only mark between the right and left side of the grid. The behavioral tests were as follows: D test. (pseudoconditioning): Fifty 5-sec, 70 dB buzzers delivered at intervals which varied randomly between 5 and 40 sec. Twenty-five 60 Hz, 1.5 mA, 2-sec scrambled footshocks were randomly interspersed among the buzzers at buzzer-shock intervals which also varied randomly between 5 and 30 sec. D P test. (classical conditioning): Fifty 5-sec buzzers were delivered at 5-40 sec intervals as above, but each was immediately followed by a footshock (stimulus contiguity). Footshocks were given on all trials, whether there was a shuttle response to the buzzer or not [13,16]. D C test. (avoidance without stimulus pairing): Fifty 5-sec buzzers were given at 5-40 sec intervals as above, but each was followed after a randomly variable 5-30 sec interval by a footshock, only in those trials in which the animals did not shuttle to the buzzer. Therefore, shuttling to the buzzer resulted in cancellation of the next scheduled shock (avoidance contingency). D P C test. (avoidance with stimulis pairing; typical or standard two-way avoidance): Fifty 5-sec buzzers were de-
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TABLE 1 SHUTTLE RESPONSES TO A BUZZER PERFORMED BY RATS WITH CA3 HIPPOCAMPAL LESIONS, BY SHAM-OPERATED ANIMALS, AND BY INTACT CONTROLS, IN FOUR DIFFERENT BEHAVIORAL SITUATIONS Behavioral situation D (pseudoconditioning) DP (classical conditioning) DC (avoidance without stimulus pairing) DPC (avoidance with stimulus pairing) DP-[) DC-~) D + (DP-D) + (DC-D)
CA3 lesion group
Sham-operated group
Intact controls
4.8 _+ 2.0
5.5 _+ 1.6
4.9 + 2.6
6.2 _+ 2.6*
13.4 _+ 2.3
12.2 _+ 1.9
12.9 _+ 1.7
14.0 _+ 1.4
14.2 _+ 2.4
13.9 _+ 2.9* 1.4 _+ 1.7" 8.1 _+ 2.3 14.3
21.1 _+ 2.4 7.9 _+ 3.2 8.5 _+ 2.1 21.9
22.2 _+ 3.1 7.3 _+ 2.3 9.3 _+ 1.8 21.5
Data expressed as mean number of responses per test _+ SE. Ten animals per group. *: significant difference from the other two groups at 5% level in a Duncan multiple-range test.
TABLE 2 SHUTTLE RESPONSES TO A BUZZER PERFORMED BY RATS WITH CA4/DENTATE HIPPOCAMPAL LESIONS, BY SHAM-OPERATED ANIMALS, AND BY INTACT CONTROLS, IN FOUR DIFFERENT BEHAVIORAL SITUATIONS Behavioral situation D (pseudoconditioning) DP (classical conditioning) DC (avoidance without stimulus pairing) DPC (avoidance with stimulus pairing) DP-[) DC-D D + (DP-D) + (DC-D)
CA4/dentate lesion group
Sham-operated group
Intact controls
4.8 _+ 2.0
4.6 _+ 1.2
4.8 _+ 1.6
11.9 _+ 1.1
10.8 _+ 1.6
11.8 _+ 1.0
15.5 _+ 1.0
14,7 _+ 1.3
14.1 +_ 1.3
21.7 _+ 2.0 7.1 _+ 1.7 10.7 _+ 1.5 22.6
20,3 _+ !.9 6,2 _+ 2.1 10.1 _+ 2.2 20.9
22.7 _+ 1.8 7.0 _+ 1.5 9.3 + 1.8 21.1
Data expressed as mean number of responses per test -+ SE. Differences among groups for the same test not significant at 5% level in Duncan multiple-range test.
l i v e r e d at r a n d o m l y v a r i a b l e 5 - 4 0 sec i n t e r v a l s , as in the p r e c e d i n g tests; b u t e a c h w a s f o l l o w e d i m m e d i a t e l y b y a f o o t s h o c k (contiguity o r pairing, as in t h e D P test); u n l e s s t h e r e w a s a shuttle r e s p o n s e to t h e b u z z e r ( a v o i d a n c e cont i n g e n c y , as in t h e D C situation). F o r f u r t h e r details o n the a p p a r a t u s a n d o n t h e s e b e h a v ioral tests, see [5, 6, 7, 13, 14, 15, 20, 21, 24]. S h u t t l e r e s p o n s e s to t h e b u z z e r , a n d intertrial c r o s s i n g s (i.e., s h u t t l e r e s p o n s e s o c c u r r i n g in the b u z z e r - b u z z e r , b u z z e r - s h o c k , o r s h o c k - b u z z e r intervals) w e r e c o u n t e d . N o d i f f e r e n c e s in p e r f o r m a n c e o f intertrial c r o s s i n g s w e r e obs e r v e d a m o n g g r o u p s , a n d the d a t a o n t h e s e will n o t b e rep o r t e d h e r e . T h e n u m b e r o f s h u t t l e r e s p o n s e s to t h e b u z z e r in t h e D t e s t w a s t a k e n as a m e a s u r e of the D f a c t o r [5, 7, 14, 15, 20, 21]. T h e P f a c t o r w a s e s t i m a t e d b y s u b t r a c t i n g f r o m e a c h i n d i v i d u a l p e r f o r m a n c e in t h e D P t e s t , the m e a n perf o r m a n c e o f a n i m a l s in the s a m e t r e a t m e n t g r o u p (lesioned, s h a m - o p e r a t e d , i n t a c t c o n t r o l s ) in the D t e s t (DP - D). T h e
C f a c t o r w a s c a l c u l a t e d b y s u b t r a c t i n g f r o m e a c h individual p e r f o r m a n c e in the DC t e s t the m e a n for t h a t g r o u p in the D test ( D C - D) [5, 6, 7, 14, 15, 20, 24]. E l s e w h e r e , it w a s r e p o r t e d t h a t p e r f o r m a n c e in t h e D test is the s a m e regardless o f w h e t h e r 10, 25, o r 50 s h o c k s are g i v e n d u r i n g it [24]. T h e r e f o r e , it m a y b e a s s u m e d t h a t t h e r e l a t i v e c o n t r i b u t i o n o f the D f a c t o r w a s p r o b a b l y t h e s a m e in t h e DP, DC, or D C P s i t u a t i o n s , r e g a r d l e s s o f the fact t h a t different n u m b e r s o f f o o t s h o c k s w e r e g i v e n in e a c h o f t h o s e t e s t s [14, 15, 20, 24]. RESULTS P e r f o r m a n c e of s h u t t l e r e s p o n s e s to t h e b u z z e r in the D P a n d D P C s i t u a t i o n s was l o w e r in t h e CA3 lesion g r o u p t h a n in its c o r r e s p o n d i n g c o n t r o l s ( T a b l e 1). T h i s is a t t r i b u t a b l e to a d e p r e s s i o n o f the P f a c t o r ( D P - D), since D a n d C app e a r e d to n o r m a l in this group. T h e r e w e r e n o significant d i f f e r e n c e s in a n y o f the four tests, D, DP, DC, o r D P C , between sham-operated and intact controls.
992
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As shown in Table 2, there was no significant difference in the number of shuttle responses to the buzzer in any of the four tests, among CA4/dentate-lesioned animals, its shamoperated group, and the intact controls. DISCUSSION These results, when put together with those previously reported on the effect of CAI-CA2 lesions [5], might, in appearance, provide a better understanding of the role of the hippocampus in rat shuttle behavior. Operation of the P factor would seem to depend on the integrity of CA1, CA2, and CA3. The subareas CAI and CA2 would, in addition, exert a tonic inhibitory influence on D. Finally, CA4 and the dentate gyrus would appear to be of little relevance to any of the three factors, D, P, or C. Vinogradovfi [25] suggested that Schaeffer collaterals, which originate from CA3 neurons and terminate upon dendritic fields of CA I-CA2 pyramidal cells, may have a regulatory influence on the latter. Thus, it is possible that the effect of CA3 lesions on the P component of shuttle behavior may result from the suppression of this regulatory influence of CA3 on CA1 and CA2. If this were true, since both CA3 and CAI-CA2 lesions [5] had a ceiling effect in depressing P, and since CA4/dentate lesions had no effect, one would expect that hippocampal spreading depression (which presumably affects all hippocampal subfields) would influence rat shuttle behavior in a manner indistinguishable from that of a CA1 or CA2 lesion. In fact, as shown in a previous report [7], this is exactly what happens. The role here proposed for C A I , CA2, and CA3, fits with hypotheses which suggest a role of the hippocampus in behavioral inhibition [1,8], as well as with those which suggest a role in the processing of paired sensory information [12,23]. Moreover, the apparent conflict between the two sets of hypotheses would be solved [5,12], and previous data by others on the effect of various types of hippocampal lesions on rat shuttle avoidance and related behaviors [8, 10,
11, 12[ would be satisfactorily explained. An even more straightforward interpretation of our previous [5,7] and present results may be found along the lines drawn by the more recent hypotheses of Hirsh [9], O 'K eef e and Nadel [18], and Osborne and Black [19]. Hirsh [9] suggested a general role of the hippocampus in the subtle processing of various kinds of information. O'Keefe and Nadel [18] consider it to be particularly related to spatial mapping. Accordingly, animals with hippocampal lesions would be predicted to have less flexible (in particular, stereotyped and/or repetitious) motivational responses [19], and to be defective in tasks which require subtle information processing leading to the elaboration of a spatial map (of which classical or instrumental learning in a shuttle-box are typical examples). The increased responding observed in the D test in rats with CAI-CA2 lesions [5] may thus be viewed as an expression of behavioral inflexibility in a situation in which such responding is essentially purposeless, and is due mainly, if not exclusively [13,14], to non-associative and/or motivational factors (i.e., what we chose to call drive) [13, 14, 15, 20]. The depressed operation of the P factor observed in the animals with CAICA2 [5] or CA3 lesions, may be viewed as a specific inability to elaborate adequate spatial maps with which to generate shuttle responses to the buzzer in the presence of a particular mode of sensory stimulation, namely, buzzer-shock contiguity. Mechanism through which the hippocampus could process paired sensory information have been suggested by several suthors [12,23]. For possible ways by which the hippocampus might translate such information into appropriate spatial maps, see O ' K e e f e and Nadel [18]. The apparent lack of effect of hippocampal lesions on the C factor may signify that the elaboration of spatial maps adequate for shuttle responding may proceed in the absence of an intact hippocampus, when a shuttle-no shock avoidance contingency is in operation; perhaps, as Hirsh [9], and Osborne and Black [19] have suggested, through the use of residual or vicarious systems.
REFERENCES
1. Airman, J., R. L. Brunner and S. Bayer. The hippocampus and behavioral maturation. Behav. Biol. 8: 557-596, 1973. 2. Anisman, H. Aversively motivated behavior as a tool in psychopharmacologic analysis. In: Psychopharmacology af A versively Motivated Behavior. edited by H. Anisman and G. Bignami. New York: Plenum Press, 1978, pp. 1-62. 3. Anisman, H. and T. G. Waller. Effects of inescapable shock upon subsequent avoidance performance: role of response repertoire changes. Behav. Biol. 9: 331-355, 1973. 4. Bignami, G. Nonassociative explanation of behavioral changes induced by central cholinergic drugs. Acta Neurobiol. Exp. 36: 5-90, 1976. 5. Calderazzo Filho, L. S., A. Moschovakis and 1. lzquierdo. Effect of hippocampal lesions on rat shuttle responses in four different behavioral tests. Physiol. Behav. 19: 96%972, 1977. 6. Cavalheiro, E. A. Role of 0 rhythm during rat shuttle behavior in four different experimental situations. Behav. Neural. Biol. 25: 20%220, 1979. 7. Cavalheiro, E. A. and 1.' lzquierdo. Effect of hippocampal and neocortical spreading depression on rat shuttle behavior in four different experimental paradigms. Physiol. Behav. 18: 10111016, 1977. 8. Douglas, R. J. The hippocampus and behavior. Psychol. Bull. 67: 416-422, 1967.
9. Hirsh, R. The hippocampus and contextual retrieval of information from memory: a theory. Behav. Biol. 12: 421-444, 1974. 10. lsaacson, R. L., R. J. Douglas and R. Y. Moore. The effect of radical hippocampal ablation on acquisition of an avoidance response. J. comp. physiol. Psyehol. 54: 626-628, 1961. 11. Isaacson, R. L. I7w Limbic System. New York: Plenum Press 1975.
12. lzquierdo, I. The hippocampus and" learning. Prog. Neurobiol. 5: 37-75, 1975. 13. Izquierdo, 1. A pharmacological separation of stimulus pairing and of the response-reinforcement contingency as factors in the elicitation of shuttle responses to a buzzer in rats. Behav. Biol. 18: 75-87, 1976. 14. lzquierdo, I. and E. A. Cavalheiro. Three main factors in rat shuttle behavior: their pharmacology and sequential entry in operation during a two-way avoidance session. Psyehopharmacology 49: 145-157. 1976. 15. Izquierdo, I. and E. Elisabetsky. Physiological and pharmacological dissection of the main factors in the acquisition and retention of shuttle behavior. In: Brain Mechanisms in Memory and Learning:from the Single Neuron to Man. edited by M. A. B. Brazier. IBRO Monograph Series, vol. 4, New York: Raven Press, 1979, pp. 227-248.
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16. Katzev, R. D. and S. K. Mills. Strain differences in avoidance conditioning as a function of the classical CS-UD contingency. J. comp. physiol. Psychol. 87: 661-671, 1974. 17. K6nig, J. F. R. and R. A. Klippel. The Rat Brain; a Stereotaxic Atlas. Krieger: Huntington, 1974. 18. O'Keefe, J. and L. Nadel. The Hippocampus as a Cognitive Map. London: Oxford University Press 1978. 19. Osborne, B. and A. H. Black. A detailed analysis of behavior during the transition from acquisition to extinction in rats with fornix lesions. Behav. Biol. 23: 271-290, 1978. 20. Schiitz, R. A. and I. Izquierdo. Effect of brain lesions on rat shuttle behavior in four different tests. Physiol. Behav. 23: 97-105, 1979. 21. SchCitz, R. A., M. T. B. Schiitz, O. A. Orsingher and 1. Izquierdo. Brain dopamine and noradrenaline levels in rats submitted to four different aversive behavioral tests. Psychopharmacology 63: 28%292, 1979,
22. Skinner, B. F. Science and Human Behavior. New York: MacMillan 1953. 23. Solomon, P. R. Role of the hippocampus in blocking and conditioned inhibition of the rabbit's nictitating membrane response. J. comp. physiol. Psychol. 91: 407-417, 1977. 24. Vendite, D. A., E. Elisabetsky, D. O. Souza and I. Izquierdo. Effect of the number and density of footshocks on pseudoconditioned responses in a shuttle-box. Acta physiol latinoam., in press, 1979. 25. Vinogradov~, O. S. Functional organization of the limbic system in the process of registration of information: facts and hypotheses. In: The Hippocampus, edited by R. L. Isaacson and K. H. Pribram. vol. 2, New York: Plenum Press, 1978, pp. 3-69.