BEHAVIOURAL BRAIN RESEARCH

ELSEVIER
Behavioural Brain Research 83 (1997) 71-74

Research report

Learning/memory processes under stress conditions

Hiroshi Kaneto *

Department of Pharmacology, Faculty of Pharmaceutical Sciences, Nagasaki University, Nagasaki 852, Japan
Received 8 July 1995; revised 14 November 1995; accepted 22 November 1995

Abstract Using mice, changes in the learning/memory processes under various stress conditions were investigated in one-trial step-through type passive avoidance learning task. Pre-, post-training and pre-test foot shock (FS)-stress induced long-lasting, at least 96 h, facilitation of test trial latencies. Pre-training psychological (PSY)-stress induces facilitation and pre-test swimming (SW)-stress provokes impairment of test trial latencies. These effects of FS-, PSY- and SW-stress are all dependent on the timing of their exposure and due to their acute effect. Intraperitoneal administration of 1 mg/kg scopolamine 30 min pre-training caused impairment of test latencies in naive and pre-test FS-stressed animals but failed to affect both pre- and post-training FS-induced enhancement. Taken all these data together, it seems that cholinergic mechanism is partly involved in the FS-stress induced facilitation of test latencies, though the research on the changes in ACh levels at the action site in brain after FS-stress exposure is necessary for a definite conclusion.

Keywords: Learning/memory; Passive avoidance learning; Mouse; Stress; Cholinergic mechanism; Scopolamine

1. Introduction

experiments were carried out between 9 : 0 0 a.m. and 3 : 00 p.m. to minimize diurnal variation. 2.2. Exposure to stress Foot shock (FS)-stress: The communication box (30 x 30 x 30 crn) which was divided into 9 compartments (10 x 10 x 30 crn) with transparent plastic walls was used. Inescapable and unsignaled electric foot shock (FS, 2 mA, 1 s duration, 0.2 Hz) duration) was delivered for 5 min through the floor grid. Psychological (PSY)-stress: The same box was used. Plastic plates were placed on the grids of 5 compartments to prevent the animals from receiving direct shock, but they were exposed to PSYstress by watching and hearing the struggle, jumping and vocalization of the animal receiving FS in the adjacent compartments, for 5 min. Swimming (SW)stress: Mice were forced to swim in a water bath, 40(L) x 35(W) x 20(H) cm, 15 cm of water depth at 20C for 5 rain. After removing the animal from water returned to the home cage before subsequent testing. 2.3. Step-through type passive avoidance learning test The apparatus consisted of an illuminated and a dark compartment (each 4 x 13 x 19 crn) adjoining each other

We have demonstrated that foot-shock (FS)-, psychological (PSY)- and forced swimming (SW)-stress exposure can modulate the learning/memory processes, acquisition, consolidation, retention and retrieval in a different way, and suggested the involvement of both opioid and non-opioid mechanisms in the processes depending on the type of stress [4]. On the other hand, an important role of cholinergic system in cognitive function is well recognized [1,5]. Thus, in this experiment, the changes of learning/memory processes under various stress conditions were investigated in relation to the involvement of cholinergic mechanisms.
2. Materials and methods

2.1. Animal and experimental conditions Male mice of the ddY strain (Ohtsubo Exp. Animals, Nagasaki, Japan) weighing 18-20 g were purchased and housed in 42 26 x 15 cm plastic cages with free access to food and water under a natural day/night cycle. All
* Corresponding author. Tel.: (+ 81 ) 958-4%1111, Ext. 2538; Fax: {+ 81 ) 958-44~4248.

0166-4328/97/$17.00 Copyright 1997 Elsevier Science B.V. All rights reserved PII SO166-4328 (96) 00096-4

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11. Kaneto/Behavioural Brain Research 83 (1997) 71-74 Control (non-stressed) 600

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through a small gate (3 cm in diameter) with a grid floor. On the training trial, the animal was placed in the illuminated compartment facing away from the dark compartment. When the animal entered into the dark compartment, an electric shock (ES; 0.6 mA) was delivered through the grid floor until the animal returned to the illuminated compartment. At the test trial, 24 h after the training session unless otherwise specified, the animal placed in the illuminated compartment and its latency to enter the dark compartment (maximum 600 s) was measured. The effect of stress exposure on learning/memory processes were tested at 3 different stages. (1) Pretraining: Animals were exposed to stress and ret,~.ned to their home cages until the training trial. (2j Posttraining: Mice were exposed to stress at various intervals after the training trial and returned to their home cages. (3) Pre-test: Stress was delivered at different time intervals before the test trial.

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2.4. Statistical analysis

Data are expressed as medians and interquartile ranges (s). All data were analyzed by the Kruskal-Wallis non-parametric one-way analysis of variance, and subsequently, post-hoc tests were performed with the twotailed Mann-Whitney U-test. In statistical evaluations, P<0.05 was used as the criterion for statistical significance.

96

Training- test interval (hr) Fig. 1. Duration of the enhancement of the test latencies induced by pre-, post-training and pre-test FS-stress. Test was performed at 24 (open column), 48 (dark grey column), 72 (light grey column) and 96 (hartched column) h after training trial. +P<0.05, + P<0.01, compared with the value at 24 h after training in non-stressed control group. **P<0.01, compared with the control group at corresponding time.

Pro-training PSY-stress

Post-training SW-stress

3. Results As shown in Fig. 1, non-stressed control animals subjetted to one trial in the passive avoidance learning paradigm showed an enhanced test latencies 24 h after training that decreased time-dependently in the following days and at 72 and 96 h after training the latency returned to the control level. In contrast, pre-, posttraining and pre-test FS-stress exposure resulted in enhanced test latencies compared to non-stressed control group, showing an incidence of maximum retention latencies of 600 s that lasted at least for 96 h. In all cases, training latencies were not affected by stress exposure (FS: 16, 11-24; PSY: 20, 11-25; SW: 20, 13-22). In contrast, as reported previously [41, pre-training PSY-stress induced enhancement and post-training SW-stress evoked impairment of the test trial latencies at 24 h after training trial, but post-training and pre-test PSY-stress and pre-training and pre-test SW-stress failed to affect the test trial latencies. These effects induced by PSY- and SW-stress were rapidly diminished and returned to the control level by 48 h (Fig. 2).

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Cont 24 48 72 96 Cont 24 48 72 96 Training- test interval (hr) Fig. 2. Duration of the enhancing effect of pre-training PSY-stress and impairing effect of post-training SW-stress. Test was performed at 24, 48, 72 and 96 h after training trial. *P<0.05, compared with the control group.

3.1. Time dependency of the stress-induced effect

FS-stress exposure 30 min pre-training produced a marked increase of retention latencies at the test trial that decreased gradually and disappeared when the interval between FS-stress exposure and training trial

11. Kaneto/Behavioural Brain Research 83 (1997) 71-74

73

was 1 and 2 h. Similar results were obtained by posttraining and pre-test exposure to FS-stress (Fig. 3). The effects of exposure to pre-training PSY-stress and post-training SW-stress on the test latencies determined at 24 h after training were also time-dependent. When the interval between stress and training trial was more than 1 h the enhancing and impairing effects were lost (Fig. 4).

induced amnesic effect was blocked by stress exposure. In contrast, pre-test FS-stress failed to block the amnesic effect of Scop (Fig. 5). Whereas FS-stress exposed 30 min pre-training blocked the amnesic effect of Scop, 1 and 2 h pre-training FS-stress did not affect the amnesic effect of Scop (Fig. 6).

4. Discussion 3.2. Effect of FS-stress on scopolamine-induced amnesic effect

Intraperitoneal injection of 1 mg/kg scopolamine (Scop) 30 min before training resulted in a profound decrease of test trial latencies in non-stressed control animals. However, facilitated test latencies from animals subjected to pre- and post-training FS-stress were not affected by pre-training Scop, indicating that Scop
Pretraining Posttraining

The present study has been carried out using mice, since mice trained only once establish a long-lasting memory that lasts at least 24 h in step-through type passive avoidance learning task. We confirmed our previous data 1-5] that test trial latencies at 24 h after training trial were significantly prolonged in mice exposed to
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Stress (FS) - training interval (hr) Fig. 3. Time dependency of the FS-stress induced facilitation of the test latencies on the interval of stress exposure and training trial. Mice were exposed to FS-stress 30 rain, 1 and 2 h pre-training, post-training and pre-test. Test was performed 24 h after training trial. **P<0.01, compared with the control (Cont.) group. Pre-training PSY-stress 600* Post-trnining SW-stress

Fig. 5. Effect of FS-stress on Scop-induced amnesic effect. Scop ( 1 mg/kg) was administered 30 rain before training. FS-stress was delivered 30 rain pre-, post-training and 30 rain pre-test. Test trial was done 24 h after training trial. Control (given saline, open columns) and Scoptreated (black columns) group. **P<0.01, compared with non-stressed control group. ~P<0.01, compared with the corresponding stressed group given saline.

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Fig. 4. Time dependency of the PSY-stress induced facilitation and the Sw-induced impairment of the test latencies on the interval of stress exposure and training trial. Mice were exposed to pre-training PSYstress and to post-training SW-stress at the time interval of 30 rain, 1 and 2 h between stress exposure and training trial, respectively. *P <0.05, compared with respective control (Cont.) group.

Fig. 6. Effect of pre-training exposure to FS-stress at different time intervals on the Scop-induced amnesic effect. Mice were exposed to FS-stress 30 min, 1 and 2 h before training, and Stop was given 30 rain pre-training. Test trial was measu "d 24 h after training. Control (given saline, open columns} and Scop-treated (black columns} group. *P<0.05, **P<0.O1, compared with non-stressed control group. ##P<O.01, compared with the corresponding stressed group given saline.

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1-1. Kaneto/Behavioural Brain Research 83 (1997) 71-74

FSostress, pre-training, post.training and pre-test, indicating that memory forming processes, acquisition, consolidation, retention and retrieval might be facilitated by stress exposure, However, the effects of stresses were different each other depending on their type, and pretraining PSY-stress exposure also enhanced the test latencies at 24 h after the training but failed to modify the test latencies by post-training or pre-test exposure. In contrast, SW-stress produced no effect by pre-training and pre-test exposure but impaired the test latencies by post-training exposure. The fact that pre-, post-training and pre-test FS-stress induced facilitation was long-lasting, at least 96 h, but the enhancement caused by pre-training PSY-stress and the impairment induced by post-training SW-stress at 24 h after the training trial disappeared in the following days may suggest the implication of the different mechanisms among 3 types of stresses for their production. On the other hand, the facilitation induced by FS-stress applied at 3 different stages of learning/memory processes and pre-training PSY-stress, and impairment caused by post-training SW-stress are due to their acute effect, suggesting that the interval between stress exposure and training trail are critical factor for their production. The involvement of cholinergic mechanism in the process of facilitation induced by FS-stress was investigated, since there have been many reports that give evidence of the impairing effects of cognition of systemically administered antimuscarinic agents, such as scopolamine [1,3], and, moreover, the essential role of cholinergic mechanism in a number of learning/memory pradigrns have been suggested [2,5-7]. In this experiment, we found that FS-stress induced learning enhancement lasted at least 96 h, indicating that ACh-induced changes of excitability can be long-lasting and causes a mechanism of central plasticity. It has been suggested that FS-stress may increase the ACh levels in the brain, and higher levels of ACh would increase synaptic effectiveness of the neural circuit controlling retention latencies presumably regulated through muscarinic receptors. This possibility is supported by systemic administration

of a single 1 mg/kg Stop causing a profound amnesic effect that completely disappeared by 30 min pre- and post-training FS-stress, suggesting that increased levels of ACh induced by FS-exposure would compete with exogenously administered Scop for muscarinic binding, showing a blocking of Scop-induced amnesic effect and a sustained enhancement of test latencies. However, exposure to FS-stress 30 min before test failed to enhance test latencies in animals given pre-training Scop, indicating that Scop antagonism of muscarinic receptors before training would block the formation of the central circuit responsible for the induction of facilitation of test latencies. One and 2 h pre-training FS-exposure did not elicit significant enhancement of test latencies compared with that of non-stressed control group. Failure to activate a long-term facilitation process in animals exposed 1 or 2 h before training may be as well due to the progressive decrease of ACh levels at the action sites, and blocking effect of pre-test Scop also suggests the possibility. Timing of stress exposure and ACh pharmacokinetics appear to be key elements in this process but further research on the problem is needed to reach a definite conclusion.

References
El'] Fibiger, H.C., Central cholinergic systems and memory. In Squire, L.R. and Lindenlaub, E. (Eds.) The Biology of Memory. Schattauer-Verlag, Stuttgart, New York, 1989, pp. 381-426. [2.] Glick, S.D. and Zimmembvrg, B., Amnesic effects of scopolamine. Behav. Biol., 7 (1972) 245-254. 1"3] Izquierdo, I., Mechanism of action of scopolamine as an amnesic. Trends PharmacoL Sci., l0 (1989) 175-177. r4.] Jodar, L., Takahashi, M. and Kaneto, H., Effects of foot-shock-, psychological- and swimming-stress on the learning and memory processes: Involvement of opioidergic pathways. Jpn. 3. Pharmacol., 67 (1995) 143-147. [51 Kameyama, T., Nabeshima, T. and Noda, Y., Cholinergic modulation of memory for step-down passive avoidance task in mice. Res. Commun. PsychoL Psychiat. Behav., 11 (1986) 193-215. ~6] Rush, D.K., Reversal of scopolamine-inducext amnesia of passive avoidance by pre- and post-training naloxone. Psychopharmacology, 100 (1986) 27-30. 17] Rush, D.K. and Streit, K., Memory modulation with peripherally acting cholinergic drugs. Psychopharmacology, 106 (199t) 375-382.