Physiology & Behavior, Vol. 19, pp. 569-572. Pergamon Press and Brain Research Publ., 1977. Printed in the U.S.A.
BRIEF COMMUNICATION Effect of Hippocampal Lesions on Rat Shuttle Responses in Four Different Behavioral Tests I LINI~U S. C A L D E R A Z Z O FILHO 2 , ANTONIS MOSCHOVAKIS 3 AND IV,/~N I ZQ U I ERD O
Disciplina de Neurofisiologia, Departamento de Biofisica e Fisiologia, Escola Paulista de Medicina, Rua Botucatu 862, 04023 Sito Paulo, SP, Brasil (Received 21 March 1977) CALDERAZZO FILHO, L. S., A. MOSCHOVAKIS AND I. IZQUIERDO. Effect of hippocampal lesions on rat shuttle responses in four different behavioral tests. PHYSIOL. BEHAV. 19(4) 569-572, 1977. - Rats with lesions in subareas CA1 - CA2 of the dorsal hippocampus made more shuttle responses to a buzzer than sham-operated or intact control animals in four different shuttle-box situations using buzzers and footshocks as stimuli. These differences were much more marked in two of the tests, in which the buzzer-shock interval was varied at random (a pseudoconditioning situation and an avoidance paradigm without stimulus pairing), than in the other two tests, in which the stimuli were paired on all trials (a Pavlovian paradigm and a typical two-way avoidance situation). These data agree closely with those of a previous report on the effect of hippocampal spreading depression on the same four tests. The results suggest that the hippocampus plays a role both in the inhibition of nonassociative effects of the footshocks and in the establishment of stimulus-stimulus relations in the brain. Hippocampus Drive inhibition Stimulus pairing Classical conditioning Instrumental conditioning
Avoidance contingency
A RAT IN a shuttle box normally does not perform shuttle responses to a buzzer (SBs) unless it receives footshocks during the same session [12]. In the past two years, this laboratory has studied intensively four different buzzershock situations in which SBs may be obtained [3, 12, 13, 14, 15, 16]: the D test (pseudoconditioning, [9, 10, 11, 12] ) in which buzzers and shocks are presented in random order and at random intervals; the DP test (classical or Pavlovian conditioning) in which buzzers and shocks are paired on all trialsregardless of the occurrence of SBs; the DC test (avoidance conditioning without stimulus pairing) in which the buzzer-shock interval is varied at random but in which every SB cancels the next scheduled shock; the DPC test (typical two-way avoidance) in which the two stimuli are paired on all trials, as in DP, but shocks are omitted whenever there is an SB, as in DC. In the D test, SBs develop as a consequence of nonassociative effects of shocks [6, 12, 15, 16], which may be grouped under the convenient, if somewhat vague term drive (D) [2, 12, 13]. In the DP test, the constant temporal association between buzzers and shocks (which may be defined as pairing, P) plays a role in addition to drive. In the DC test there is no pairing in the sense just defined, but the avoidance contingency (C) is a factor together with drive. Finally, in
Shuttle behavior
the DPC test, all three factors, D, P and C, are present [3, 6, 12, 13, 14, 15, 16]. The influence of a large number of variables on SB performance in the four tests was investigated. These variables include a wide variety of drug treatments [ 12, 13, 14, 16], maturation [15], and hippocampal and neocortical spreading depression [3]. These studies have led to the following conclusions: (a) in all four tests, D, P and C operate as separate factors with apparent additive properties. Thus, for any given group of rats, performance in a DP test minus performance in a D test is a measure of the P factor; and performance in a DC test minus D is a measure of the C factor [ 16] ; and, in consequence, D + (DP - D) + (DC - D) = DPC. So far, this simple equation has been found to hold in 16-different groups of rats, including untreated adult and weanling [ 15], saline- or drug-treated animals [16], and rats submitted to the implant of brain cannulae and/or to hippocampal or to neocortical spreading depression[3]. (b) D, P, and C may, therefore, be considered to be the three main factors in shuttle avoidance (DPC), since all SB performance in the DPC test can be explained by the sum of D + P + C [ 15,16]. (c) the three factors, D, P, and C, operate each through a distinct physiological mechanism of its own, since drugs [12, 13,
1Supported by an institutional grant from Financiadora de Projetos (FINEP) to the Escola Paulista de Medicina, and by research grants from Fundaq~io de Amparo ~ Pesquisa do Estado de S~io Paulo (FAPESP) and from Conselho Nacional de Desenvolvimento Cientffico e Tecnol6gico (CNPq) to Dr. Ivan Izquierdo, to whom reprint requests should be addressed. 2 Fellow from FAPESP, Brasil. 3IBRO fellow on leave from Department of Biochemistry, University of Athens, Greece 569
570
CALDERAZZO FILttO, MOSCtlOVAKIS AND IZQUII;RI)()
14, 16], maturation [15] and hippocampal spreading depression [3] have different effects on SB performance depending on the behavioral test (D, DP, DC, DPC) in which their influence is studied. Several of the studies mentioned above have provided valuable hints as to the possible nature of the physiological mechanism of D, P and C. In particular, a recent study on the effect of hippocampal spreading depression has suggested that the hippocampus may normally play an inhibitory role on D, and a stimulant or facilitatory role on P [3]. The present report is an attempt to replicate those findings with the use of chronic electrolytic lesions of subareas CA1-CA2 of the dorsal hippocampus of rats. METHOD
One hundred forty adult Wistar rats of both sexes ( 1 5 0 - 2 5 0 g) were used. They were divided into three major groups: animals with bilateral hippocampal lesions (n = 45), sham-operated animals (n = 48), and intact controls (n = 47). The number of animals within each group that were submitted to each of the four behavioral tests (see below) appears in Table 1. Hippocampal lesions were placed stereotaxically under deep ketamine (30 mg/kg, IM) plus pentobarbital ( 2 0 - 3 0 mg/kg, IP) anesthesia. A 0.125 mm steel electrode whose insulation was scratched off for 0.5 mm at the tip was positioned during surgery with the tip aimed at the dorsal hippocampus (coordinates A 3.4, L 3.0, V +3.0 of the atlas by DeGroot, [4] ). Current from a DC source (1 mA, 15 sec) was passed between the hippocampal electrode and a rectal cathode. The procedure was carried out bilaterally. Lesion placement was verified post-mortem in formalin-fixed brain sections. Figure 1 shows maximum and minimum extent of lesions. Sham-operated animals were submitted to the same general procedure, except that the electrode was lowered only to 1 mm above the hippocampus and left there for 15 sec without passing any current. Lesioned and sham-operated animals were allowed to rest for 7 - 1 0 days after surgery before they were submitted to behavioral testing.
Rats in each group were submitted to either i). ~r DP. ,~ DC, or DPC tests, carried out as follows: D test [3, 9, 10, 12, 15, 16]+ 50 buzzers tduration. 5 sec) were presented at intervals which varied randomly between 10 and 40 sec. Interspersed among the buzzers, 25 footshocks (1.5 mA) were given at randomly variable buzzer-shock intervals of 5 to 30 sec. Footshocks were delivered according to a predetermined schedule and regardless of whether the rats did or not perform SBs to the buzzers. D P test [3, 13, 14, 15, 16]. 50 buzzers were given at 1 0 - 4 0 sec intervals as above, but each was followed immediately after its offset by footshocks, irrespective of whether SBs were or not performed. D C test [3, 13, 14, 15, 16]. 50 buzzers were given as above, and each was followed by a footshock after an interval which varied randomly between 5 and 30 sec. The. scheduled shock was omitted every time that the animals performed an SB to the preceding buzzer (avoidance contingency). Responses occurring in the buzzer-shock interval were exceedingly rare [13] even in the hippocampal group; they did not serve to avoid shocks and were counted as intertrial shuttlings (see below). (In this test, animals show a marked tendency to freeze during the variable buzzer-shock interval, which differentiates this procedure from the more traditional trace routines. [13,141. D P C t e s t [3, 12, 13, 15, 16]. 50 buzzer-shock trials were made, as in DP, but omitting shocks every time there was an SB, as in DC. In the four behavioral situations, rats were placed in the shuttle box 5 - 7 min before the onset of the first trial. The shuttle box (50 x 25 x 25 cm) was made of wood painted gray except for the front wall which was of glass. An 8-W light bulb hung from the midline and a buzzer rested on the opposite side of the lid, also at the midline. The floor was a grid consisting of 2 mm bronze bars spaced 7 mm apart. At the midline on the grid a piece of wood (0.5 x 0.5 x 25 cm) constituted the only separation between the right and left side of the floor. All animals responded to all shocks with shuttling in less than 1 sec (escape response). SBs (shuttle responses during the buzzers) and intertrial crossings (shuttle responses made during buzzer-buzzer, buzzer-shock, or shock-buzzer periods) were counted. SB performance in the D test was taken as a measure of the D factor [3, 15, 16]. The P factor was measured by subtracting from each individual performance in the DP test, the mean performance of animals belonging to the same treatment group (hippocampals, sham-operated, intact controls) in the D test (DP - D). The C factor was measured by subtracying from each individual SB performance in the DC test the mean of that group in the D test (DC - D) [3]. Statistical comparisons were by way of a randomized-group ANOVA followed by a Duncan multiple-range test [12, 13, 14, 15, 161. RESULTS
FIG. 1. Minimum (darkened) and maximum (striped) extent of hippocampal lesions, and most medial and most lateral electrode tracks. Note that hippocampal lesions involvs subareas CAt and/or CA2 only, sparing other areas of the hippoeampus proper and the dentate gyrus as well. The brain section depicted here corresponds to approximately plane A 3.4 from the atlas by De Groot [4 ].
The data on SB performance appear in Table 1. Rats with hippocampal lesions made more SBs than any of the two control groups in the four tests. There were no differences in any of the tests between sham-operated and control animals. Differences between hippocampal rats and controls were larger for the D (+25.4 and +26.0) and DC tests (+25.8 and +28.0) than for the DP (+11.3 and +14.4)
HIPPOCAMPAL LESIONS AND SHUTTLE BEHAVIOR
571
TABLE 1
TABLE 2
PERCENT OF SHUFFLE RESPONSES TO A BUZZER PERFORMED BY RATS WITH HIPPOCAMPAL LESIONS, BY SHAM-OPERATED RATS, AND BY INTACT CONTROLS, IN FOUR DIFFERENT BEHAVIORAL SITUATIONS (D, DP, DC AND DPC)
NUMBER OF INTER-TRIAL (SPONTANEOUS) SI-IUT~LINGS MADE BY RATS WITH ItIPPOCAMPAL LESIONS, BY SHAM-OPERATED RATS, AND BY INTACT CONTROLS, IN FOUR DIFFERENT BEHAVIORAL SITUATIONS (D, DP, De AND DPC)
Behavioral paradigm
Hippocampal lesioned group
Sham-operated group
Intact controls
Behavioral paradigm
Hippocampal lesioned group
Sham-operated group
Intact controls
D
3 6 . 9 - 6.4t (16) 37.3 ± 5.6* (11) 51.6 -+ 6.6t (9) 53.8 ± 6.5* (9)
11.5--- 1.6 (16) 26.0 ± 2.5 (12) 23.6 ± 4.1 (10) 38.6 ± 4.8 (10)
10.9± 1.4 (16) 22.9 ± 1.4 (11) 25.8 ± 2.9 (10) 36.6 ± 3.2 (10)
D DP DC DPC
7.5±1.9t 5.5±1.3" 5.2±0.9* 6.1±1.3"
2.1±0.6 1.8±0.9 2.5±1.1 2.5±1.4
1.9±0.4 2.4±1.1 1.7±0.7 2.3±0.8
D+(DP-D) +(DC-D)
52.0
38.1
37.8
DP-I)) DC-I))
0.4 ±- 5.8* 14.7 ± 6.4
14.5 ± 2.3 12.1 - 4.1
12.0 -+ 2.4 14.9 --- 3.5
DP DC DPC
In this and in the following table, data are e x p r e s s e d as means ± SE. Number of animals is in parentheses. *significant difference from both control groups at 5% level in Duncan-multiple range test; tsame, at 1% level.
and DPC situations (+15.2 and +17.2). The P factor (DP D) was significantly reduced in the lesion group when c o m p a r e d to any of the two c o n t r o l groups. There was n o difference in the C factor (DC - D) a m o n g groups. In Table 2, it can be seen that h i p p o c a m p a l animals made m o r e inter-trial shuttlings in all tests than the animals in any of the t w o c o n t r o l groups. DISCUSSION These results are practically identical w i t h those previously r e p o r t e d for the effect of h i p p o c a m p a l spreading depression on the D, DP, DC and DPC situations [ 3 ] . As in that paper, the increased SB p e r f o r m a n c e of h i p p o c a m p a l animals in all tests m a y be a t t r i b u t e d to a release of the D factor f r o m a h i p p o c a m p u s - d e p e n d e n t inhibition. The n u m e r o u s data in the literature on increased DPC perf o r m a n c e by rats with h i p p o c a m p a l lesions [1, 5, 7, 11] may thus be explained by this e n h a n c e d o p e r a t i o n of D. The relatively l o w e r increase of SB p e r f o r m a n c e observed in h i p p o c a m p a l animals in those tests which include the P factor (DP and DPC) m a y be a t t r i b u t e d to an i m p a i r m e n t of the normal o p e r a t i o n of P [ 3 ] . C seems to have been u n a f f e c t e d by h i p p o c a m p a l injury : on one hand, there were
Number of animals per group per test, same as in Table 1. *significant difference from the two control groups at 5% level in Duncan-multiple range test; tDsame, at 1% level. no differences in D C - D values a m o n g groups; and, on the other, the increase of responding observed in the hippocampal group in the DC test was of about the same m a g n i t u d e as the one observed in the h i p p o c a m p a l group in the DC test was of a b o u t the same m a g n i t u d e as the one observed in the D test. The same had been found with hippocampal spreading depression [ 3 ] . H i p p o c a m p a l animals made m o r e inter-trial shuttlings in all tests than controls. Again, this was similar to what we had previously r e p o r t e d w i t h h i p p o c a m p a l spreading depression [ 3 ] , and may be explained either by a r e d u c t i o n of the natural t e n d e n c y of rats to freeze [2] during inter-trial periods, or by the recklessness or by the perseverative tendencies caused by the lesions [ 1,11]. It is possible that the effect of h i p p o c a m p a l lesions on inter-trial crossings, and the one on the D factor, were related. In s u m m a r y , b o t h the present data and those previously r e p o r t e d on spreading depression [ 3 ] , suggest that the h i p p o c a m p u s plays a dual role in shuttle behavior. One would be an inhibitory influence on D, and the o t h e r a facilitatory one on P. This suggestion fits with previous h y p o t h e s e s on a role of the h i p p o c a m p u s in behavioral inhibition [ 1,5], and on one in the processing o f stimulusstimulus-related i n f o r m a t i o n [3, 8, 1 1, 17]. S o l o m o n [ 17] recently p r o p o s e d that the h i p p o c a m p u s would be important in the tuning out of stimuli which do not lead to responses appropriate to particular behavioral situations. Even t h o u g h S o l o m o n ' s hypothesis was derived from observations on behavioral paradigms different from ours, it might be a basis for the unification of the dual role of the h i p p o c a m p u s suggested by the present results. Our data, in fact, suggest that bottl h i p p o c a m p a l functions (D inhibition, P facilitation) are probably carried out by the same subareas (CA 1-CA2).
REFERENCES
1. Altman, J., R. L. Brunner and S. Bayer. The hippocampus and behavioral maturation. Behav. Biol. 8: 557-596, 1973. 2. Anisman, H. and T. G. Waller. Effects of inescapable shock on subsequent avoidance performance: role of response repertoire changes. Behav. Biol. 9 : 3 3 1 - 3 5 5 , 1973. 3. Cavalheiro, E. A. and I. lzquierdo. The effect of hippocampal and of neocortical spreading depression on rat shuttle behavior in four different experimental paradigms. Physiol. Behav., in press, 1977. 4. De Groot, J. The rat forebrain in stereotaxic coordinates. Trans. Royal Neth. Acad. ScL 52:(40 pp.), 1959.
5. Douglas, R. J. The hippocampus and behavior. Psychol. Bull. 67: 416-422. 6. Frontali, M., L. Amorico, L. De Acetis and G. Bignami. A pharmacological analysis of processes underlying differential responding: a review and further experiments with scopolamine, amphetamine, lysergic acid diethylamide (LSD-25), chlordiazepoxide, physostigmine, and chlorpromazine. Behav. Biol. 18: 1-74, 1976. 7. Isaacson, R. L., R. J. Douglas and R. Y. Moore. The effect of radical hippocampal ablation on acquisition of an avoidance response. J. comp. physiol. Psychol. 5 4 : 6 2 5 - 6 2 8 , 1 9 6 1 .
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11. 12.
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lzquierdo, 1. Regulation of information processing in the h i p p o c a m p u s by K+ release and (K÷)o accumulation. In: Neurohumoral Coding of Brain Function, edited by R. D. Myers and R. R. Drucker-Colin. New York: Plenum Press, 1974, pp. 1 5 1 - 1 6 6 . Izquierdo, I. Possible peripheral adrenergic and cholinergic m e c h a n i s m s in pseudoeonditioning. Psychopharmacolo~ia 35: 1 8 9 - 1 9 3 , 1974. Izquierdo, I. Effect on pseudoconditioning of drugs with k n o w n central nervous activity. Psychopharmacolo~gia 38: 259--266, 1974. Izquierdo, I. The h i p p o c a m p u s and learning. Prog. Neurobiol. 5: 3 7 - 7 5 , 1975. Izquierdo, I. Relations between orienting, conditioned and pseudoconditioned responses in the shuttle-box - a pharmacological analysis by m e a n s of LSD and dibenamine. Behav. Biol. 15" 1 9 3 - 2 0 5 , 1975.
13.
14.
15.
16.
17.
FILHO,
MOSCJtOVAKIS
,.\NI~ [ Z O t I! R!;~
Izquierdo, I. A pharmacological separation t,t ~Iunuius paJnng and of the response-reinforcement contingency as tactors ]n the elicitation of shuttle responses to a buzzer m ,-s~,s.B~,/t,,,~. Biol. 18: 7 5 . 8 7 , 1976. Izquierdo, 1. Stimulus pairing and the response-reinforcement contingency as separate factors in the elicitation of shuttle responses. Ciencia e Cult. (5'00 Paulo) 28:1334 1336, 1976. Izquierdo, I. and E. A. Cavalheiro. The influence of stimulus pairing and of the shuttle-shock contingency o11 lhe performance of shuttle responses lo a buzzer by weanling rats. Behav. Biol. 17: 119- 122, 1976. Izquierdo, 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. Psychopharmacolo£y 4 9 : 1 4 5 --157, 1976. Solomon, P. R. Role of the h i p p o c a m p u s in blocking and conditioned inhibition of the rabbit's nictitating m e m b r a n e response. J. comp. physiol. Psychol. 9 1 : 4 0 7 .-417, 1977.
BRIEF COMMUNICATION Effect of Hippocampal Lesions on Rat Shuttle Responses in Four Different Behavioral Tests I LINI~U S. C A L D E R A Z Z O FILHO 2 , ANTONIS MOSCHOVAKIS 3 AND IV,/~N I ZQ U I ERD O
Disciplina de Neurofisiologia, Departamento de Biofisica e Fisiologia, Escola Paulista de Medicina, Rua Botucatu 862, 04023 Sito Paulo, SP, Brasil (Received 21 March 1977) CALDERAZZO FILHO, L. S., A. MOSCHOVAKIS AND I. IZQUIERDO. Effect of hippocampal lesions on rat shuttle responses in four different behavioral tests. PHYSIOL. BEHAV. 19(4) 569-572, 1977. - Rats with lesions in subareas CA1 - CA2 of the dorsal hippocampus made more shuttle responses to a buzzer than sham-operated or intact control animals in four different shuttle-box situations using buzzers and footshocks as stimuli. These differences were much more marked in two of the tests, in which the buzzer-shock interval was varied at random (a pseudoconditioning situation and an avoidance paradigm without stimulus pairing), than in the other two tests, in which the stimuli were paired on all trials (a Pavlovian paradigm and a typical two-way avoidance situation). These data agree closely with those of a previous report on the effect of hippocampal spreading depression on the same four tests. The results suggest that the hippocampus plays a role both in the inhibition of nonassociative effects of the footshocks and in the establishment of stimulus-stimulus relations in the brain. Hippocampus Drive inhibition Stimulus pairing Classical conditioning Instrumental conditioning
Avoidance contingency
A RAT IN a shuttle box normally does not perform shuttle responses to a buzzer (SBs) unless it receives footshocks during the same session [12]. In the past two years, this laboratory has studied intensively four different buzzershock situations in which SBs may be obtained [3, 12, 13, 14, 15, 16]: the D test (pseudoconditioning, [9, 10, 11, 12] ) in which buzzers and shocks are presented in random order and at random intervals; the DP test (classical or Pavlovian conditioning) in which buzzers and shocks are paired on all trialsregardless of the occurrence of SBs; the DC test (avoidance conditioning without stimulus pairing) in which the buzzer-shock interval is varied at random but in which every SB cancels the next scheduled shock; the DPC test (typical two-way avoidance) in which the two stimuli are paired on all trials, as in DP, but shocks are omitted whenever there is an SB, as in DC. In the D test, SBs develop as a consequence of nonassociative effects of shocks [6, 12, 15, 16], which may be grouped under the convenient, if somewhat vague term drive (D) [2, 12, 13]. In the DP test, the constant temporal association between buzzers and shocks (which may be defined as pairing, P) plays a role in addition to drive. In the DC test there is no pairing in the sense just defined, but the avoidance contingency (C) is a factor together with drive. Finally, in
Shuttle behavior
the DPC test, all three factors, D, P and C, are present [3, 6, 12, 13, 14, 15, 16]. The influence of a large number of variables on SB performance in the four tests was investigated. These variables include a wide variety of drug treatments [ 12, 13, 14, 16], maturation [15], and hippocampal and neocortical spreading depression [3]. These studies have led to the following conclusions: (a) in all four tests, D, P and C operate as separate factors with apparent additive properties. Thus, for any given group of rats, performance in a DP test minus performance in a D test is a measure of the P factor; and performance in a DC test minus D is a measure of the C factor [ 16] ; and, in consequence, D + (DP - D) + (DC - D) = DPC. So far, this simple equation has been found to hold in 16-different groups of rats, including untreated adult and weanling [ 15], saline- or drug-treated animals [16], and rats submitted to the implant of brain cannulae and/or to hippocampal or to neocortical spreading depression[3]. (b) D, P, and C may, therefore, be considered to be the three main factors in shuttle avoidance (DPC), since all SB performance in the DPC test can be explained by the sum of D + P + C [ 15,16]. (c) the three factors, D, P, and C, operate each through a distinct physiological mechanism of its own, since drugs [12, 13,
1Supported by an institutional grant from Financiadora de Projetos (FINEP) to the Escola Paulista de Medicina, and by research grants from Fundaq~io de Amparo ~ Pesquisa do Estado de S~io Paulo (FAPESP) and from Conselho Nacional de Desenvolvimento Cientffico e Tecnol6gico (CNPq) to Dr. Ivan Izquierdo, to whom reprint requests should be addressed. 2 Fellow from FAPESP, Brasil. 3IBRO fellow on leave from Department of Biochemistry, University of Athens, Greece 569
570
CALDERAZZO FILttO, MOSCtlOVAKIS AND IZQUII;RI)()
14, 16], maturation [15] and hippocampal spreading depression [3] have different effects on SB performance depending on the behavioral test (D, DP, DC, DPC) in which their influence is studied. Several of the studies mentioned above have provided valuable hints as to the possible nature of the physiological mechanism of D, P and C. In particular, a recent study on the effect of hippocampal spreading depression has suggested that the hippocampus may normally play an inhibitory role on D, and a stimulant or facilitatory role on P [3]. The present report is an attempt to replicate those findings with the use of chronic electrolytic lesions of subareas CA1-CA2 of the dorsal hippocampus of rats. METHOD
One hundred forty adult Wistar rats of both sexes ( 1 5 0 - 2 5 0 g) were used. They were divided into three major groups: animals with bilateral hippocampal lesions (n = 45), sham-operated animals (n = 48), and intact controls (n = 47). The number of animals within each group that were submitted to each of the four behavioral tests (see below) appears in Table 1. Hippocampal lesions were placed stereotaxically under deep ketamine (30 mg/kg, IM) plus pentobarbital ( 2 0 - 3 0 mg/kg, IP) anesthesia. A 0.125 mm steel electrode whose insulation was scratched off for 0.5 mm at the tip was positioned during surgery with the tip aimed at the dorsal hippocampus (coordinates A 3.4, L 3.0, V +3.0 of the atlas by DeGroot, [4] ). Current from a DC source (1 mA, 15 sec) was passed between the hippocampal electrode and a rectal cathode. The procedure was carried out bilaterally. Lesion placement was verified post-mortem in formalin-fixed brain sections. Figure 1 shows maximum and minimum extent of lesions. Sham-operated animals were submitted to the same general procedure, except that the electrode was lowered only to 1 mm above the hippocampus and left there for 15 sec without passing any current. Lesioned and sham-operated animals were allowed to rest for 7 - 1 0 days after surgery before they were submitted to behavioral testing.
Rats in each group were submitted to either i). ~r DP. ,~ DC, or DPC tests, carried out as follows: D test [3, 9, 10, 12, 15, 16]+ 50 buzzers tduration. 5 sec) were presented at intervals which varied randomly between 10 and 40 sec. Interspersed among the buzzers, 25 footshocks (1.5 mA) were given at randomly variable buzzer-shock intervals of 5 to 30 sec. Footshocks were delivered according to a predetermined schedule and regardless of whether the rats did or not perform SBs to the buzzers. D P test [3, 13, 14, 15, 16]. 50 buzzers were given at 1 0 - 4 0 sec intervals as above, but each was followed immediately after its offset by footshocks, irrespective of whether SBs were or not performed. D C test [3, 13, 14, 15, 16]. 50 buzzers were given as above, and each was followed by a footshock after an interval which varied randomly between 5 and 30 sec. The. scheduled shock was omitted every time that the animals performed an SB to the preceding buzzer (avoidance contingency). Responses occurring in the buzzer-shock interval were exceedingly rare [13] even in the hippocampal group; they did not serve to avoid shocks and were counted as intertrial shuttlings (see below). (In this test, animals show a marked tendency to freeze during the variable buzzer-shock interval, which differentiates this procedure from the more traditional trace routines. [13,141. D P C t e s t [3, 12, 13, 15, 16]. 50 buzzer-shock trials were made, as in DP, but omitting shocks every time there was an SB, as in DC. In the four behavioral situations, rats were placed in the shuttle box 5 - 7 min before the onset of the first trial. The shuttle box (50 x 25 x 25 cm) was made of wood painted gray except for the front wall which was of glass. An 8-W light bulb hung from the midline and a buzzer rested on the opposite side of the lid, also at the midline. The floor was a grid consisting of 2 mm bronze bars spaced 7 mm apart. At the midline on the grid a piece of wood (0.5 x 0.5 x 25 cm) constituted the only separation between the right and left side of the floor. All animals responded to all shocks with shuttling in less than 1 sec (escape response). SBs (shuttle responses during the buzzers) and intertrial crossings (shuttle responses made during buzzer-buzzer, buzzer-shock, or shock-buzzer periods) were counted. SB performance in the D test was taken as a measure of the D factor [3, 15, 16]. The P factor was measured by subtracting from each individual performance in the DP test, the mean performance of animals belonging to the same treatment group (hippocampals, sham-operated, intact controls) in the D test (DP - D). The C factor was measured by subtracying from each individual SB performance in the DC test the mean of that group in the D test (DC - D) [3]. Statistical comparisons were by way of a randomized-group ANOVA followed by a Duncan multiple-range test [12, 13, 14, 15, 161. RESULTS
FIG. 1. Minimum (darkened) and maximum (striped) extent of hippocampal lesions, and most medial and most lateral electrode tracks. Note that hippocampal lesions involvs subareas CAt and/or CA2 only, sparing other areas of the hippoeampus proper and the dentate gyrus as well. The brain section depicted here corresponds to approximately plane A 3.4 from the atlas by De Groot [4 ].
The data on SB performance appear in Table 1. Rats with hippocampal lesions made more SBs than any of the two control groups in the four tests. There were no differences in any of the tests between sham-operated and control animals. Differences between hippocampal rats and controls were larger for the D (+25.4 and +26.0) and DC tests (+25.8 and +28.0) than for the DP (+11.3 and +14.4)
HIPPOCAMPAL LESIONS AND SHUTTLE BEHAVIOR
571
TABLE 1
TABLE 2
PERCENT OF SHUFFLE RESPONSES TO A BUZZER PERFORMED BY RATS WITH HIPPOCAMPAL LESIONS, BY SHAM-OPERATED RATS, AND BY INTACT CONTROLS, IN FOUR DIFFERENT BEHAVIORAL SITUATIONS (D, DP, DC AND DPC)
NUMBER OF INTER-TRIAL (SPONTANEOUS) SI-IUT~LINGS MADE BY RATS WITH ItIPPOCAMPAL LESIONS, BY SHAM-OPERATED RATS, AND BY INTACT CONTROLS, IN FOUR DIFFERENT BEHAVIORAL SITUATIONS (D, DP, De AND DPC)
Behavioral paradigm
Hippocampal lesioned group
Sham-operated group
Intact controls
Behavioral paradigm
Hippocampal lesioned group
Sham-operated group
Intact controls
D
3 6 . 9 - 6.4t (16) 37.3 ± 5.6* (11) 51.6 -+ 6.6t (9) 53.8 ± 6.5* (9)
11.5--- 1.6 (16) 26.0 ± 2.5 (12) 23.6 ± 4.1 (10) 38.6 ± 4.8 (10)
10.9± 1.4 (16) 22.9 ± 1.4 (11) 25.8 ± 2.9 (10) 36.6 ± 3.2 (10)
D DP DC DPC
7.5±1.9t 5.5±1.3" 5.2±0.9* 6.1±1.3"
2.1±0.6 1.8±0.9 2.5±1.1 2.5±1.4
1.9±0.4 2.4±1.1 1.7±0.7 2.3±0.8
D+(DP-D) +(DC-D)
52.0
38.1
37.8
DP-I)) DC-I))
0.4 ±- 5.8* 14.7 ± 6.4
14.5 ± 2.3 12.1 - 4.1
12.0 -+ 2.4 14.9 --- 3.5
DP DC DPC
In this and in the following table, data are e x p r e s s e d as means ± SE. Number of animals is in parentheses. *significant difference from both control groups at 5% level in Duncan-multiple range test; tsame, at 1% level.
and DPC situations (+15.2 and +17.2). The P factor (DP D) was significantly reduced in the lesion group when c o m p a r e d to any of the two c o n t r o l groups. There was n o difference in the C factor (DC - D) a m o n g groups. In Table 2, it can be seen that h i p p o c a m p a l animals made m o r e inter-trial shuttlings in all tests than the animals in any of the t w o c o n t r o l groups. DISCUSSION These results are practically identical w i t h those previously r e p o r t e d for the effect of h i p p o c a m p a l spreading depression on the D, DP, DC and DPC situations [ 3 ] . As in that paper, the increased SB p e r f o r m a n c e of h i p p o c a m p a l animals in all tests m a y be a t t r i b u t e d to a release of the D factor f r o m a h i p p o c a m p u s - d e p e n d e n t inhibition. The n u m e r o u s data in the literature on increased DPC perf o r m a n c e by rats with h i p p o c a m p a l lesions [1, 5, 7, 11] may thus be explained by this e n h a n c e d o p e r a t i o n of D. The relatively l o w e r increase of SB p e r f o r m a n c e observed in h i p p o c a m p a l animals in those tests which include the P factor (DP and DPC) m a y be a t t r i b u t e d to an i m p a i r m e n t of the normal o p e r a t i o n of P [ 3 ] . C seems to have been u n a f f e c t e d by h i p p o c a m p a l injury : on one hand, there were
Number of animals per group per test, same as in Table 1. *significant difference from the two control groups at 5% level in Duncan-multiple range test; tDsame, at 1% level. no differences in D C - D values a m o n g groups; and, on the other, the increase of responding observed in the hippocampal group in the DC test was of about the same m a g n i t u d e as the one observed in the h i p p o c a m p a l group in the DC test was of a b o u t the same m a g n i t u d e as the one observed in the D test. The same had been found with hippocampal spreading depression [ 3 ] . H i p p o c a m p a l animals made m o r e inter-trial shuttlings in all tests than controls. Again, this was similar to what we had previously r e p o r t e d w i t h h i p p o c a m p a l spreading depression [ 3 ] , and may be explained either by a r e d u c t i o n of the natural t e n d e n c y of rats to freeze [2] during inter-trial periods, or by the recklessness or by the perseverative tendencies caused by the lesions [ 1,11]. It is possible that the effect of h i p p o c a m p a l lesions on inter-trial crossings, and the one on the D factor, were related. In s u m m a r y , b o t h the present data and those previously r e p o r t e d on spreading depression [ 3 ] , suggest that the h i p p o c a m p u s plays a dual role in shuttle behavior. One would be an inhibitory influence on D, and the o t h e r a facilitatory one on P. This suggestion fits with previous h y p o t h e s e s on a role of the h i p p o c a m p u s in behavioral inhibition [ 1,5], and on one in the processing o f stimulusstimulus-related i n f o r m a t i o n [3, 8, 1 1, 17]. S o l o m o n [ 17] recently p r o p o s e d that the h i p p o c a m p u s would be important in the tuning out of stimuli which do not lead to responses appropriate to particular behavioral situations. Even t h o u g h S o l o m o n ' s hypothesis was derived from observations on behavioral paradigms different from ours, it might be a basis for the unification of the dual role of the h i p p o c a m p u s suggested by the present results. Our data, in fact, suggest that bottl h i p p o c a m p a l functions (D inhibition, P facilitation) are probably carried out by the same subareas (CA 1-CA2).
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