Implications for the dynamics of long-term memory

Proc. Natl. Acad. Sci. USA

Vol. 91, pp. 2041-2045, March 1994 Psychology

Delayed emergence of effects of memory-enhancing drugs: Implications for the dynamics of long-term memory CESARE MONDADORI, BASTIAN HENGERER, THOMAS DUCRET, AND JUERGEN BORKOWSKI Pharmaceutical Research Division, CIBA-Geigy Limited, Basle, CH-4002, Switzerland

Communicated by L. Weiskrantz, November 22, 1993 (received for review April 27, 1993)

facilitation emerges, we hoped not only to help to characterize the substances themselves but also to shed light on the dynamics of discrete phases of memory. The strategy of examining time dependence of memory effects has a wellestablished history for substances that interfere with memory retention but has hardly been adopted in studies of memory enhancement. Even with interference treatments, reports of delayed effects [as with cerebral electroshock, for example (14, 15)] and protein synthesis inhibition (e.g., see refs. 16-20) are relatively sparse. Moreover, some memorymodulating substances have a wide range of unspecific side effects that complicate interpretation-i.e., direct druginduced behavioral effects can interfere with the behavioral manifestation of the memory effects at the time of retest. In the present study, we focused largely on the memoryenhancing effect of oxiracetam, with which the risks of interference are minimal given that it has no known acute effects on spontaneous behavior at the dose used; but particular parallel experiments were also carried out with arecoline, CGP 36742, and captopril.

ABSTRACT Many theories of memory polate that processing of information outlasts the earning situation and involves several different physiological substrates. If such physiologically distinct mecanism or stages of memory do in fact exist, they should be differentially affected by particular experimental manipulations. Accordingly, a selective improvement of the processes underlying short-term memory should be detectable only while the information is encoded in the shortterm mode, and a selective influence on long-term memory should be detectable only from the moment when memory is based on the long-term trace. Our comparative study of the time course of the effects of the cholinergic agonist arecoline, the -aminobutyric acid type B receptor antagonist CGP 36742, the angiotensin-converting enzyme inhibitor captopril, and the nootropic oxiracetam, four substances with completely different primary sites of action, show that the memoryenhancing effects consistently come into evidence no sooner than 16-24 h after the learning trial. On the one hand, this finding suggests that all these substances act by way of the same type of mechanism; on the other hand, it demonstrates that the substrate modulated by the compounds forms the basis of memory only after 16-24 h. From the observation that animals also show clear signs of retention during the first 16 h-i.e., before the effects of the substances are measurable-it can be inferred that retention during this time is mediated by other mechanisms that are not influenced by any of the substances.

DESIGN, METHODS, PROCEDURES General Design. With oxiracetam four questions were addressed. The first was to determine precisely when the enhancement of retention becomes manifest. Retention was therefore assessed in separate groups of animals at various intervals (1, 2, 4, 8, 16, and 24 h) after the learning trial. The second question was whether the timing of the injection in relation to the learning trial and to the retention test itself had an influence on the time of appearance of enhanced retention. For this purpose, oxiracetam was administered 1, 4, or 8 h before or immediately after the learning trial. The third question was whether the time of day had an influence on performance during retention. To this end, retention was always measured simultaneously in a group that had already been subjected to training and treatment 1 day beforehand. Since the corresponding controls were also tested at each interval, the experiments included an additional seven treated and seven control groups with retention intervals of 25, 26, 28, 32, 40, 44, and 48 h. The fourth question was how long the enhanced retention could be seen to persist. This was assessed by using retention intervals of 4, 8, and 16 days after the learning trial. The generality of the delayed emergence of enhanced retention was then also studied with the muscarinic agonist arecoline, the GABAB receptor blocker CGP 36742, and the ACE inhibitor captopril. With these three substances, only immediate posttrial administration was used. To test the possible influence of time of day, the same parallel control measurements were made as in the experiments with oxirac-

The group of preparations already found to display memoryenhancing effects in experimental learning situations is surprisingly large. Even more surprising is the fact that the primary pharmacological sites of action of these substances are very heterogeneous. Among others, the list includes cholinergic agonists at the muscarinic and nicotinic receptors (1, 2); cholinesterase inhibitors such as physostigmine (3); calcium-channel blockers such as nimodipine (4); angiotensin-converting enzyme (ACE) inhibitors such as captopril (5); transmitters such as, e.g., norepinephrine (6); t-aminobutyric acid type B (GABAB) receptor blockers such as CGP 36742 (7); peptides such as vasopressin and corticotropin (8); glucose (9); and, not least, the large group of so-called nootropics including oxiracetam and piracetam (10, 11). Despite their different primary sites of action, all of the above-mentioned substances have a similar effect in a simple one-trial passive avoidance task; if administered, e.g., 1 h before the learning trial, they improve the retention performance at retest 24 h later (12, 13). At the time of retention testing, the changes induced by the substances have already been expressed at a stage generally reckoned to be in long-term memory, but it is not known when these changes occurred. Indeed, it is often implicitly assumed that the facilitatory effects occurred concomitantly with the initial learning itself, demonstrable at short retention intervals. By making a comparative study of the time at which memory

etam. For all substances, the optimal dosage was first determined in pilot experiments in which dose-response profiles for

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Abbreviations: ACE, angiotensin-converting y-aminobutyric acid type B receptor. 2041

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Proc. Natl. Acad Sci. USA 91

Psychology: Mondadori et al.

(1994)

retest latencies permitted retests to be carried out within an acceptable time frame (in some cases, five groups had to be tested simultaneously); (ii) the conditions were such that only a few animals were included in the analysis, with cutoff latencies of 150 sec, which enhanced the power of the statistical evaluation; and (iii) it provided an optimal baseline against which to detect enhancement of retention. In all experiments, the animals were randomly assigned to the different groups (n = 25) receiving the drug or saline. All administration was i.p. Retests were performed blind. Learning was assessed by comparing the drug and saline groups with no-shock controls. Drug effects were assessed in relation to a group receiving saline injection but otherwise treated identically to the drug groups. The data deriving from such experiments are comparable to "failure-time data" (21). The appropriate statistical procedure to analyze the outcome of single sets of experiments is a generalized Wilcoxon test (22).

memory enhancement were obtained. Accordingly, the doses used were 100 mg of oxiracetam per kg, 0.3 mg of arecoline per kg, 10 mg of CGP 36742 per kg, and 3 mg of captopril per kg. Animals. Male mice (MA 01 Tif; 20-22 g) from our own animal breeding unit were used throughout the experiment. They were housed in plastic cages in a room lit from 6 a.m. to 6 p.m. and had free access to food and water at all times. Apparatus and Procedures. The apparatus consisted of an electrifiable grid (50 x 50 cm) of stainless-steel rods (4 mm in diameter; spaced 13 mm apart), enclosed by gray poly(vinyl chloride) (PVC) walls 50 cm high. In the middle of the grid was a platform 12 mm high and 67 mm in diameter, which was enclosed by a removable gray PVC tube (180 mm high; 68-mm inner diameter). The mice were placed one by one on the platform inside the tube, which was removed after 10 sec. With a few exceptions, they stepped down from the platform within 10 sec and received a footshock (1 mA; 1 sec). No-shock controls were run in parallel. At a specified interval after training, each animal was again placed on the platform and the step-down latency was recorded ("retest latency") up to a maximum cutoff time of 150 sec. The experimental parameters were adjusted to produce minimal indications of learning. There were three reasons for doing so: (i) the short

RESULTS

Oxiracetam. Time of emergence ofretention enhancement. If the compound was administered 1 h before the learning trial, its effect on memory emerged reliably only after a

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FIG. 1. Step-down passive avoidance in mice. Effects of pretrial oxiracetam on retention performance at various retention intervals after and treatment. Oxiracetam was always administered i.p. 1 h before the learning trial. Boxplots represent the retest latencies, in seconds, of the various experimental and control groups. Hatched box, saline control; open box, oxiracetam (100 mg/kg, i.p.); stippled box, no-shock controls. Prolongation of the latencies by comparison with the saline-treated controls indicates drug-induced memory facilitation. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by generalized Wilcoxon test. Corresponding retest latencies (Upper, 1- to 24-h interval; Lower, 25- to 48-h interval) were measured in parallel. To depict a distribution, a boxplot (23) consists of a box and tails, with outliers (if present) for each group. Horizontal line through the box depicts the median, and the top and bottom of the box mark the upper and lower quartiles. Thus, the box itself shows the range for exactly that half of the data points in the group that cluster around the median. Tails mark the actual range of 90% of the data in the group. Outliers are represented by small circles. training

Psychology: Mondadori et al.

Proc. Natl. Acad. Sci. USA 91 (1994)

retention interval of 20 h. At all shorter retention intervals, the animals showed the usual signs of having learned (i.e., their step-down latencies were longer than those of the no-shock controls), but there were no facilitatory drug effects. The results are shown in Fig. 1 (Upper). Time of day for retention testing. The parallel control experiments clearly indicate that the absence of any enhancement effect with retention intervals shorter than 20 h could not be due to circadian influences; at all intervals when no facilitation by oxiracetam could be detected, highly significant effects of the drug were found in the animals already trained and treated 1 day earlier (Fig. 1 Lower). These experiments also show that the effect that emerged after 16 h persisted at all intervals up to 48 h. Time of injection in relation to learning and retention tests. Regardless of whether the drug was administered 1 or 4 h before or immediately after the learning trial, the emergence of the memory-facilitation effect was the same; it did not become evident until an interval of 16-24 h had elapsed between training and retest. At each of the tests after longer intervals, the animals treated with oxiracetam showed a significant improvement of retention performance by comparison with the controls. Again, at all shorter intervals the effect of trainingper se was present (the retest latencies of the saline- and oxiracetam-treated animals were significantly longer than those of the no-shock controls), but there were no perceptible differences between the drug and saline groups. For the sake of clarity, in Fig. 2, the latencies are expressed as percentages of the (saline) controls. The data after 1-h pretrial administration are identical to those shown in Fig. 1. If injected 8 h before the learning test, oxiracetam had no facilitating effects on memory; the retest latencies of the controls and the treated animals were about the same (mean ± SEM: vehicle, 19.1 ± 3.8; oxiracetam, 14.3 ± 2.0; median: vehicle, 11.5; oxiracetam, 11.0). Stability of the memory-facilitating effect. The memoryenhancing effect of oxiracetam (posttrial treatment) was still clearly perceptible even after intervals as long as 16 days (Fig. 3), the longest time that was tested. It remains to be seen what the maximum interval might be and whether it extends beyond the course of natural forgetting of the initial learning experience.

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FIG. 3. Step-down passive avoidance in mice. Effects of posttrial oxiracetam on retention performance at various intervals after training and treatment. Oxiracetam (100 mg/kg) was always administered i.p. immediately after the learning trial. For details see legend to Fig. 1.

Arecoline, Captopril, and CGP 36742. Results comparable to those with oxiracetam were found for the emergence of memory enhancement; with intervals of