Memory channels in the rat: Effect of post-training application of potassium chloride on the hippocampus

BEHAVIORAL AND NEURAL BIOLOGY 27, 354-361 (1979)

BRIEF REPORT Memory Channels in the Rat: Effect of Post-Training Application of Potassium Chloride on the Hippocampus E L A I N E ELISABETSKY, DEUSA A . V E N D I T E , AND IVAN IZQUIERDO 1

Departamento de Bioqulmica, Instituto de Biociencias, UFRGS, 90000 Prrto Alegre, RS, Brasil Rats were trained to perform shuttle responses to a buzzer either in a Pavlovian paradigm (50 buzzer-shock pairings every 10-40 sec) or in an avoidance situation without stimulus pairing (50 buzzers every 10-40 sec, each followed by a footshock at a randomly variable 5- to 35-sec interval unless the animals shuttled to the buzzer). Seven days later, animals were tested either in the same paradigm in which they had been trained, or in the other one. There was good evidence for retention in all groups, involving both "direct" (Pavlovian-to-Pavlovian, avoidance-to-avoidance) and "transfer" memory (Pavlovian-to-avoidance, avoidance-to-Pavlovian). The two memory "transfers" appeared enhanced in animals who received an application of potassium chloride to the dorsal hippocampus immediately after training. Control groups were either intact animals or rats with bilateral hippocampal cannulas. The data support the concept, advanced in a previous paper, of separate and parallel memory channels in the rat brain.

The same behavior may be acquired in different ways. For example, rats may learn to run across a shuttlebox in response to a buzzer, either by repeatedly pairing the buzzer with a footshock (Pavlovian conditioning) (Katzev & Mills, 1974; Izquierdo & Cavalheiro, 1976; Izquierdo & Elisabetsky, 1979), or by presenting the two stimuli at randomly variable intervals but omitting the shock every time that the animals shuttle to the preceding buzzer (avoidance without stimulus pairing) (Izquierdo & Cavalheiro, 1976; Izquierdo & Elisabetsky, 1979). In the Pavlovian (P) procedure, learning occurs mainly as a result of the repeated contiguous presentation of the two stimuli (Katzev & Mills, 1974); in the avoidance (A) paradigm, learning results from the presence of a response1Supported by research grants from FAPESP and UFRGS, Brasil (to I.I.) and by a fellowship from FAPESP (to E.E.). 354 0163-1047/79/110354-08502.00/0 Copyright(~ 1979by AcademicPress, Inc. All rights of reproductionin any form reserved.

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reinforcement (shuttle-no shock) contingency (Izquierdo & Cavalheiro, 1976). In spite of the fact that these two training modes involve acquisition of the s a m e response (shuttling) to the s a m e stimulus (a buzzer), they represent two different forms of learning, each with physiologic, biochemical, and pharmacologic properties of its own (Izquierdo, 1976; Izquierdo & Cavalheiro, 1976; Izquierdo & Elisabetsky, 1978, 1979; Calderazzo, Moschovakis, & Izquierdo, 1977; Cavalheiro & Izquierdo, 1977; Schutz & Izquierdo, 1979; Schutz, Schutz, Orsingher & Izquierdo, 1979). The relation of these two paradigms to one another and to the more common type of shuttle avoidance (in which there is both buzzer-shock pairing and a shuttle-no shock contingency) is amply commented on by Anisman and Bignami, (1978). (See also, Izquierdo & Cavalheiro, 1976; Izquierdo & Elisabetsky, 1979.) Animals trained in the P or A condition may be asked to retrieve the response 1 or 7 days later, either in the same paradigm in which they were trained, or in the other one. Thus, both memory of what was learned in each situation, and the possibility of memory transfer from one to the other, may be investigated. If the same mnemonic process (storage plus retrieval plus modulatory influences thereon) is involved in all cases, then all four possible training-test combinations (P-P, P-A, A-A, and A-P) should have similar biological properties, such as a similar time-course for buildup and/or decay, and a similar susceptibility to post-training drug treatments. If, on the contrary, different memory processes were involved in each case, then it should be possible to demonstrate the existence of different memory "channels," either by the use of appropriate interventive procedures (Hine and Paolino, 1970; Schneider, 1975; Kety, 1976; Izquierdo & Elisabetsky, 1978), or by appropriately timed trainingtest schedules (Izquierdo, 1979; Izquierdo & Elisabetsky, 1978, 1979). (A memory channel may be operationally defined as a system for the storage and retrieval of learned information with biological properties of its own, such as its time-course for buildup or forgetting, and its susceptibility to drugs or other modulatory influences (Izquierdo, 1979; Izquierdo & Elisabetsky, 1978, 1979)). The concept of memory channels is not new. Hine and Paolino (1970) and Schneider (1975) have shown that two separate memories arise from a single step-through inhibitory avoidance experience: one for a classically conditioned bradycardiac response, and one for the instrumental response. The former is insensitive to, and the latter is suppressed by, post-trial electroconvulsive shock treatment. Kety (1976) has argued compellingly in favor of the idea of multiple memory channels, each subserved by a peculiar "consortium of neural and metabolic processes" of its own. For further references on the diversity, as opposed to the unity, of memory processes, see Izquierdo and Elisabetsky (1978). In a series of recent experiments, we have succeeded in demonstrating

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the existence of four different channels for the retention of shuttle responses to a buzzer or a tone acquired through the P or the A mode in rats. The channels may be designated (for the lack of a better nomenclature) by the tasks in which the animals were trained and by those in which they were tested: P-P, P-A, A-A, A-P. Each of these channels has a time-course of buildup or decay of its own: P - P is fully operative 1 day after training and does not change through Day 7; P - A builds up between Days 1 and 7; A - A declines between Days 1 and 7; and A - P does not seem to be functional in normal animals either on Day I or on Day 7 (Izquierdo & Elisabetsky, 1978, 1979). The immediate post-training ip administration of metrazol (10 mg/kg) enhances operation of channels P - A and A - A as measured on Day 7. A similar treatment with d-amphetamine sulfate (1 mg/kg) results in an enhancement only of the A - A channel. Nicotine (0.2 mg/kg) selectively fosters the normally "dormant" A - P channel. P-P is unaffected by any of these three drugs (Izquierdo & Elisabetsky, 1978). The four channels are enhanced by post-training naloxone, but they differ in sensitivity to this drug, whereas 0.8 mg/kg are needed in order to "unclog" the A - P channel, A - A is stimulated by as little as 0.2 mg/kg of the drug, and P - P and P - A are sensitive to 0.4 mg/kg of naloxone (Izquierdo, 1979). In the present paper, we report on the effect of immediate post-training potassium chloride (KCI) application to the dorsal hippocampus on the operation of the four channels, P-P, P-A, A-A, and A-P. The hippocampus is widely assumed to play a role in memory, particularly in the early phase of it in which consolidation is supposed to occur (Zornetzer, Gold, & Boast, 1977). Humans with bilateral hippocampal damage are usually unable to consolidate new memories (Milner, 1959). In laboratory animals, hippocampal lesions may (Zornetzer et al., 1977) or may not (see Isaacson, 1975) have a similar effect. Post-training hippocampal stimulation may either facilitate (Destrade & Cardo, t974; Landfield, Tusa, & McGaugh, 1973) or disrupt retention (Kesner & Wilburn, 1974). Several amnesic drugs are believed to act through interference with hippocampal electrical or chemical processes (see Nakajima, 1973). Surprisingly, there are very few studies on the effect on memory of post-training application of KC1 to the hippocampus. Avis and Carlton (1968) reported that such an application, 1 day after inhibitory avoidance training, interferes with retention of this task. Hughes (1969) found that the same effect could be obtained if KC1 was applied up to 21 days after training, and that the effect was partially reversible. Kapp and Schneider (1971) confirmed the finding of Hughes, and, in addition, observed that if KC1 was applied immediately after training the amnesic effect was not reversible. In these studies, t h e effect of KC1 application was monitored electrographically, and the amnesia was attributed to KCl-induced hippocampal spreading depression. It is difficult to interpret the data by Avis and Carlton, or by Hughes, in

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relation to the possible role of this structure in consolidation. First, KC1 was applied 1 or more days after training; second, there was recovery from amnesia. On the other hand, the data of Kapp and Schneider on immediate post-trial KCI application are directly relevant to the problem, and fit well with the observations of others on the amnesic effect of restricted hippocampal lesions (Zornetzer et al., 1977). Adult male and female Wistar rats (200 and 300 g) were used. As reported previously, there was no sex difference in the acquisition or retention of P or A learning (Izquierdo & Elisabetsky, 1979). Thirty-seven animals were used as intact controls, and 103 were implanted bilaterally with stainless-steel 27-gauge cannulas in the dorsal hippocampus (coordinates A 1.8, V +2.0, L 4.0 to 4.5 of the atlas by DeGroot, 1959). Surgery was under deep anesthesia (30 mg/kg ketamine, im, plus 20-30 mg/kg pentobarbital, ip). Atropine sulfate (2 mg/kg ip) was also given during surgery to reduce nasopharyngeal secretion. The cannulas were cemented to the skull with acrylic, and kept closed with a tight-fitting inner probe leveled off with the tips of the cannulas. Twenty-five of the implanted animals were discarded, either because of infection, or because lesions involving more than the alveus were detected in postmortem histological examination. The remaining 78 implanted animals were subdivided into two major groups: one (37 rats) in which fine KC1 crystals (Merck Darmstadt AG) were pushed through the cannula with the inner probe, on both sides, immediately after the training sessions; and one (38 rats) in which the probe was simply withdrawn and replaced. The procedure of applying KC1 to both dorsal hippocampi took less than 2 min. The amount of KC1 applied was estimated by weighing the probe before and after application, and varied between 10 and 16/xg, which is within the range reported by Kapp and Schneider (1971). The electrical effect of the KC1 application was monitored in 10 animals which had, in addition to the cannulas, recording electrodes implanted in the dorsal (A 3.4, V +2.5, L 2.0 and 3.0) and ventral (A 3.4, V -2.5, L 4.0 and 5.0) hippocampus. The electrodes were 0.125-mm steel wires insulated except at the tip and connected to a miniature plug also fixed with acrylic to the skull (Cavalheiro & Izquierdo, 1977). Electroencephalographic recordings were made with a Beckman dynograph machine. In all animals, KCI application was immediately followed by spiking and eventual full-fledged seizures. This excitatory phase lasted between 60 and 90 sec. After it, there was a flattening of the recordings which lasted for about 1 hr, and corresponds to the phase of spreading depression (Kapp & Schneider, 1971; Cavalheiro & Izquierdo, 1977). These observations are coincident with all literature in the field (see Cavalheiro & Izquierdo, 1977, for references) and need not be illustrated here. The correctness of electrode and cannula placements was verified in postmortem histological sections.

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In the implanted animals, behavioral observations were carried out 7 to 14 days after surgery. The apparatus used for P and A training and testing was a noncompartmentalized wooden shuttlebox, 50 x 25 × 25 cm, as described elsewhere in detail (Izquierdo & Cavalheiro, 1976; Schutz & Izquierdo, 1979). The conditioned stimulus was a buzzer placed on the lid of the box and set on for 5 sec every 10 to 40 sec. The unconditioned stimulus was a 1.5-mA, 60-Hz footshock applied during 1 sec. All animals shuttled to the shock in less than 1 sec (100% escape responses in all groups). The interval between the training and the test session was 7 days. The sessions were as follows. Pavlovian conditioning (P): 50 buzzers were presented, each followed immediately after its offset by a footshock (contiguity), irrespective of whatever response was made to the former (Katzev & Mills, 1974; Izquierdo, 1976; Izquierdo & Cavalheiro, 1976). In this paradigm, the buzzer-shock contiguity is the major learning factor, and avoidance contingencies are absent by definition (Anisman and Bignami, 1978; Izquierdo, 1976; Izquierdo & Cavalheiro, 1976; Izquierdo & Elisabetsky, 1979; Schutz & Izquierdo, 1979). Avoidance without stimulus pairing (A): 50 buzzers were presented every 10-40 sec as above, and each was followed by a footshock at an interval which was varied randomly between 5 and 35 sec, unless the animals shuttled to the buzzer, in which case the next scheduled shock was canceled. As mentioned elsewhere (Izquierdo, 1976; Schutz & Izquierdo, 1979) shuttlings between the buzzer and the shock were exceedingly rare (four in total in all groups in this series), which is a major distinguishing factor between the present technique and trace avoidance routines. The A paradigm bears some resemblance to the so-called "discriminated" Sidman procedure (Anisman and Bignami, 1978). In the A situation, the shuttle-no shock or avoidance contingency is the main learning factor, and consistent temporal relations between the two stimuli are deliberately eschewed (Izquierdo, 1976; Izquierdo & Cavalheiro, 1976; Izquierdo & Elisabetsky, 1978, 1979; Schutz & Izquierdo, 1979; Schutz et al., 1979). Differences in training or test performances among groups were evaluated by a randomized-group analysis of variance followed by a Duncan multiple range test (Bliss, 1967). Results are presented in Table 1. Performance in the test session of the P - A and A - P groups was higher in the animals which received KC1 applications to their hippocampus, than in the intact or cannulated control animals. No significant differences among the three treatment groups was observed in performance in the test session of the other training-test schedules (P-P and A-A), or among P or A training sessions. Therefore, the application of KC1 to the hippocam-

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TABLE 1 Performance of Shuttle Responses to a Buzzer in Rats Submitted to Training and Test Sessions in a Pavlovian (P) and in an Avoidance (A) Mode in a Shuttlebox a

n

Training paradigm

Percentage responses in training session

Intact Cannulated Hippocampal KC1

9 10 9

P P P

25.1 ± 3.5 25.8 +-_2.5 22.7 ± 3.0

P P P

37.3 __. 2.1 37.6 ± 3.7 32.4 ___4.4

Intact Cannulated Hippocampal KC1

9 8 8

P P P

26.1 ± 2.6 22.8 ± 2.1 25.4 _+ 4.1

A A A

35.4 ± 1.4 35.7 --_ 6.7 46.6 ± 5.2b

Intact Cannulated Hippocampal KC1

10 11 8

A A A

28.8 _+ 3.5 28.5 ~- 6.2 27.1 _ 7.8

A A A

39.0 ± 1.5 39.6 _+ 5.9 40.3 ± 4.6

Intact Cannulated Hippocampal KC1

9 12 12

A A A

28.8 ± 1.5 27.8 ± 2.5 22.7 _+ 2.4

P P P

26.5 ___2.4 31.3 _ 2.3 46.0 _ 3.8c

Treatment group

Percentage Test responses in paradigm test session

a Means -+ SE. b Different from test session performance of the two other treatment groups at 5% level in Duncan multiple range test (Bliss, 1967). e Same, at 1% level, p u s r e s u l t e d in a n i n c r e a s e d o p e r a t i o n o f the two " t r a n s f e r " c h a n n e l s , P - A a n d A - P , a n d h a d n o effect o n that of the two " d i r e c t " c h a n n e l s , P-P and A-A. I n p r e v i o u s p a p e r s , it was f o u n d that a c u t e h i p p o c a m p a l s p r e a d i n g d e p r e s s i o n ( C a v a l h e i r o & I z q u i e r d o , 1977), or h i p p o c a m p a l l e s i o n s (Cald e r a z z o et al., 1977), i n c r e a s e r e s p o n d i n g i n P a n d A training s e s s i o n s . T h e p r e s e n t findings o n p e r f o r m a n c e in test s e s s i o n s c a n n o t be a t t r i b u t e d to a n y s u c h p r o a c t i v e i n f l u e n c e of the h i p p o c a m p a l m a n i p u l a t i o n s , howe v e r , for v a r i o u s r e a s o n s . First, the e l e c t r i c a l effect of KC1 a p p l i c a t i o n w o r e off in a b o u t 1 hr, a n d the t r a i n i n g - t e s t i n t e r v a l w a s 7 d a y s . S e c o n d , n o l e s i o n s of a n y h i p p o c a m p a l cell l a y e r w e r e p r e s e n t in the a n i m a l s o f the p r e s e n t series. T h i r d , a p p l i c a t i o n o f KC1 to the h i p p o c a m p u s had a n effect o n l y o n test p e r f o r m a n c e o f the t w o " t r a n s f e r " g r o u p s a n d n o t o n t h a t o f the o t h e r two g r o u p s . T h e p r e s e n t r e s u l t s w e r e u n e x p e c t e d , s i n c e t h e y are at v a r i a n c e w i t h the e x i s t i n g l i t e r a t u r e (see a b o v e ) , p a r t i c u l a r l y with the r e p o r t b y K a p p a n d S c h n e i d e r (1971) o n the a m n e s i c effect of i m m e d i a t e p o s t - t r i a l KC1 a p p l i c a t i o n to the h i p p o c a m p u s . O n e p o s s i b i l i t y is that the d i f f e r e n c e in tasks a n d task r e q u i r e m e n t s (activity in o u r tests, p a s s i v i t y in the o n e e m p l o y e d b y K a p p a n d S c h n e i d e r ) m a y a c c o u n t for the d i s c r e p a n c y . A n o t h e r p o s s i b i l i t y is that the effects r e p o r t e d in t h e p r e s e n t p a p e r re-

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suited f r o m the early p h a s e o f h i p p o c a m p a l stimulation c a u s e d b y the KC1 (Cavalheiro & I z q u i e r d o , 1977), and not f r o m the ensuing and longerlasting p h a s e o f spreading depression. In that case, o u r data w o u l d agree with t h o s e w h o f o u n d that post-trial h i p p o c a m p a l stimulation e n h a n c e s m e m o r y (Landfield et al., 1973; D e s t r a d e & C a r d o , 1974). At first sight, it is difficult to see just w h y the early stimulant p h a s e w o u l d be p r e d o m i n a n t in our e x p e r i m e n t s , and the s u b s e q u e n t d e p r e s s i v e p h a s e w o u l d prevail in t h o s e b y K a p p and S c h n e i d e r (1971). It is c o n c e i v a b l e , h o w e v e r , that there m a y be a different interaction b e t w e e n e a c h o f t h o s e t w o p h a s e s o f h i p p o c a m p a l activity and the physiological state o f the animals following e a c h set o f tasks. Different tasks are f o l l o w e d b y different n e u r o h u m o r a l states (Gold & M c G a u g h , 1975; Z o r n e t z e r , 1978; I z q u i e r d o , 1979), w h i c h m a y indeed be w h a t actually defines the s u b s e q u e n t o p e r a t i o n o f the various m e m o r y c h a n n e l s (Izquierdo, 1979), and the basis o f o t h e r disc r e p a n c i e s in the literature on the nature o f the effect o f drugs and o t h e r post-training t r e a t m e n t s on m e m o r y (Gold & M c G a u g h , 1975; Z o r n e t z e r , 1978; I z q u i e r d o & E l i s a b e t s k y , 1978).

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