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 (receivedfor review April 27, 1993) ABSTRACT Many theories of memory polate that pro- facilitation emerges, we hoped not only to help to character- cessing of information outlasts the earning situation and ize the substances themselves but also to shed light on the involves several different physiological substrates. If such dynamics of discrete phases of memory. The strategy of physiologically distinct mecanism or stages of memory do in examining time dependence of memory effects has a well- fact exist, they should be differentially affected by particular established history for substances that interfere with memory experimental manipulations. Accordingly, a selective improve- retention but has hardly been adopted in studies of memory ment ofthe processes underlying short-term memory should be enhancement. Even with interference treatments, reports of detectable only while the information is encoded in the short- delayed effects [as with cerebral electroshock, for example term mode, and a selective influence on long-term memory (14, 15)] and protein synthesis inhibition (e.g., see refs. should be detectable only from the moment when memory is 16-20) are relatively sparse. Moreover, some memory- based on the long-term trace. Our comparative study of the modulating substances have a wide range of unspecific side time course of the effects of the cholinergic agonist arecoline, effects that complicate interpretation-i.e., direct drug- the -aminobutyric acid type B receptor antagonist CGP induced behavioral effects can interfere with the behavioral 36742, the angiotensin-converting enzyme inhibitor captopril, manifestation of the memory effects at the time of retest. In and the nootropic oxiracetam, four substances with completely the present study, we focused largely on the memory- different primary sites of action, show that the memory- enhancing effect of oxiracetam, with which the risks of enhancing effects consistently come into evidence no sooner interference are minimal given that it has no known acute than 16-24 h after the learning trial. On the one hand, this effects on spontaneous behavior at the dose used; but par- finding suggests that all these substances act by way ofthe same ticular parallel experiments were also carried out with arec- type of mechanism; on the other hand, it demonstrates that the oline, CGP 36742, and captopril. 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., DESIGN, METHODS, PROCEDURES before the effects of the substances are measurable-it can be General Design. With oxiracetam four questions were inferred that retention during this time is mediated by other addressed. The first was to determine precisely when the mechanisms that are not influenced by any of the substances. enhancement of retention becomes manifest. Retention was therefore assessed in separate groups of animals at various The group ofpreparations already found to display memory- intervals (1, 2, 4, 8, 16, and 24 h) after the learning trial. The enhancing effects in experimental learning situations is sur- second question was whether the timing of the injection in prisingly large. Even more surprising is the fact that the relation to the learning trial and to the retention test itselfhad primary pharmacological sites of action of these substances an influence on the time ofappearance ofenhanced retention. are very heterogeneous. Among others, the list includes For this purpose, oxiracetam was administered 1, 4, or 8 h cholinergic agonists at the muscarinic and nicotinic receptors before or immediately after the learning trial. The third (1, 2); cholinesterase inhibitors such as physostigmine (3); question was whether the time of day had an influence on calcium-channel blockers such as nimodipine (4); angioten- performance during retention. To this end, retention was sin-converting enzyme (ACE) inhibitors such as captopril (5); always measured simultaneously in a group that had already transmitters such as, e.g., norepinephrine (6); t-aminobu- been subjected to training and treatment 1 day beforehand. tyric acid type B (GABAB) receptor blockers such as CGP Since the corresponding controls were also tested at each 36742 (7); peptides such as vasopressin and corticotropin (8); interval, the experiments included an additional seven glucose (9); and, not least, the large group of so-called treated and seven control groups with retention intervals of nootropics including oxiracetam and piracetam (10, 11). 25, 26, 28, 32, 40, 44, and 48 h. The fourth question was how Despite their different primary sites of action, all of the long the enhanced retention could be seen to persist. This was above-mentioned substances have a similar effect in a simple assessed by using retention intervals of4, 8, and 16 days after one-trial passive avoidance task; if administered, e.g., 1 h the learning trial. before the learning trial, they improve the retention perfor- The generality of the delayed emergence of enhanced mance at retest 24 h later (12, 13). At the time of retention retention was then also studied with the muscarinic agonist testing, the changes induced by the substances have already arecoline, the GABAB receptor blocker CGP 36742, and the been expressed at a stage generally reckoned to be in ACE inhibitor captopril. With these three substances, only long-term memory, but it is not known when these changes immediate posttrial administration was used. To test the occurred. Indeed, it is often implicitly assumed that the possible influence of time of day, the same parallel control facilitatory effects occurred concomitantly with the initial measurements were made as in the experiments with oxirac- learning itself, demonstrable at short retention intervals. By etam. making a comparative study of the time at which memory For all substances, the optimal dosage was first determined in pilot experiments in which dose-response profiles for The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: ACE, angiotensin-converting enzyme; GABAB, in accordance with 18 U.S.C. §1734 solely to indicate this fact. y-aminobutyric acid type B receptor. 2041 Downloaded by guest on October 1, 2021 2042 Psychology: Mondadori et al. Proc. Natl. Acad Sci. USA 91 (1994) memory enhancement were obtained. Accordingly, the doses retest latencies permitted retests to be carried out within an used were 100 mg of oxiracetam per kg, 0.3 mg of arecoline acceptable time frame (in some cases, five groups had to be per kg, 10 mg of CGP 36742 per kg, and 3 mg of captopril per tested simultaneously); (ii) the conditions were such that only kg. a few animals were included in the analysis, with cutoff Animals. Male mice (MA 01 Tif; 20-22 g) from our own latencies of 150 sec, which enhanced the power of the animal breeding unit were used throughout the experiment. statistical evaluation; and (iii) it provided an optimal baseline They were housed in plastic cages in a room lit from 6 a.m. against which to detect enhancement of retention. to 6 p.m. and had free access to food and water at all times. In all experiments, the animals were randomly assigned to Apparatus and Procedures. The apparatus consisted of an the different groups (n = 25) receiving the drug or saline. All electrifiable grid (50 x 50 cm) of stainless-steel rods (4 mm in administration was i.p. Retests were performed blind. diameter; spaced 13 mm apart), enclosed by gray poly(vinyl Learning was assessed by comparing the drug and saline chloride) (PVC) walls 50 cm high. In the middle of the grid groups with no-shock controls. Drug effects were assessed in was a platform 12 mm high and 67 mm in diameter, which was relation to a group receiving saline injection but otherwise enclosed by a removable gray PVC tube (180 mm high; treated identically to the drug groups. The data deriving from 68-mm inner diameter). The mice were placed one by one on such experiments are comparable to "failure-time data" (21). the platform inside the tube, which was removed after 10 sec. The appropriate statistical procedure to analyze the outcome With a few exceptions, they stepped down from the platform of single sets of experiments is a generalized Wilcoxon test within 10 sec and received a footshock (1 mA; 1 sec). (22). 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") RESULTS up to a maximum cutoff time of 150 sec. The experimental Oxiracetam. Time ofemergence ofretention enhancement. parameters were adjusted to produce minimal indications of If the compound was administered 1 h before the learning learning. There were three reasons for doing so: (i) the short trial, its effect on memory emerged reliably only after a 150- 100- S 0 0 * * 0 ..' 0 10 .-N 0 1 2 4 8 16 20 24 hours 0) .0-..4-. 150- 0) 0 100- 0 *6I0 *6,; ~~~~~~~~~~m~~~~~~~~10: 0 25 26 28 32 40 44 48 hours FIG. 1. Step-down passive avoidance in mice. Effects of pretrial oxiracetam on retention performance at various retention intervals after training 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.