Effect of Dietary Restriction on Learningand Memory Impairment and HistologicAlterations of Brain Stem inSenescence-Accelerated Mouse (SAM)P8 Strain

RYOYA TAKAHASHI, YUKARI KOMIYA, AND SATARO GOTO

Department of Biochemistry, Faculty of Pharmaceutical Sciences,Toho University 2-2-1 Miyama, Funabashi, Chiba, Japan

ABSTRACT: The age-associated spontaneous spongy degeneration in thebrain stem of senescence-accelerated mouse (SAM) P8 strain has beensuggested to be closely associated with the ability to learn and memorize.In this study, we investigated the effects of dietary restriction (DR) ini-tiated from weaning on learning and memory and histologic changes ofthe brain stem in P8 and control R1 mice. Although no effect of DR wasobserved in the retention of the passive-avoidance response in both theP8 and R1 mice, the acquisition of the task was significantly improved byDR in P8 mice. On the other hand, the total area and number of vacuolesin the brain stem was significantly higher in ad libitum-fed (AD)-P8 micethan in AD-R1 mice. However, no significant effect was observed on thevacuole formation in the brain stem of P8 mice by DR. These observa-tions suggest that the improvement of the acquisition of the task by DRin P8 mice is possibly due to changes in neuronal function rather thanhistologic alteration in brain stem.

KEYWORDS: dietary restriction; learning; memory; senescence-acceler-ated mouse; spongy degeneration; SAMP8

INTRODUCTION

Dietary restriction (DR) has been recognized to inhibit or delay (or both)the appearance and intensity of many late-life age-related cases of functionaldeterioration and other pathalogic changes, and to prolong mean and maximumlife span.1–4 In addition, DR is known to prevent or delay the occurrence of some

Address for correspondence: Dr. Ryoya Takahashi, Department of Biochemistry, Faculty of Phar-maceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan. Voice/Fax:+81-47-472-1562.

e-mail: takahasi@phar.toho-u.ac.jp

Ann. N.Y. Acad. Sci. 1067: 388–393 (2006). C© 2006 New York Academy of Sciences.doi: 10.1196/annals.1354.055

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genetic diseases, such as autoimmune diseases of B/W mice5 and hypertensionof SHR rats.6

The senescence-accelerated mouse (SAM) was established as an animalmodel of accelerated aging by Takeda et al.8 SAMP8, a substrain of SAM,has a much shorter life span (approximately 50% that of the control strainthe accelerated senescence-resistant strain, R1) and exhibits early deficits inlearning and memory in different tasks, such as passive-avoidance responsetests,8,9 active avoidance tasks,8–10 and spatial learning tasks (including theMorris water maze task).8,11 For example, the impairment of passive-avoidancebehavior in P8 mice began to occur at a much earlier age (about 2 months ofage) and increased with age.12 On the other hand, remarkable histopathologicchanges, such as vacuolization, have been found mainly in the brain stem of P8mice.13 Yagi et al.12 suggested that such pathologic change in the brain stemof P8 mice is closely associated with memory and the ability to learn.

In this study, therefore, we investigated the effects of DR initiated fromweaning on learning and memory and vacuolization in the brain stem in P8and control R1 mice.

MATERIALS AND METHODS

Animals and Dietary Restriction

SAMP8 and SAMR1 mice obtained from Dr. T. Takeda in 1988 were bredand maintained in our Laboratory Animal Center. The 50% survival of maleSAMP8/Toho and SAMR1/Toho was found at about 430 and 780 days of age,respectively.14

At the age of 21 days, all mice were housed individually and dietaryrestriction was started. Restricted mice were fed 60% of the amount of foodconsumed by the control animals.

Passive Avoidance Task

A two-compartment, step-through passive avoidance test was performed atthe age of 6 months according to the method described by Sasaki et al.15 Briefly,a Plexiglas two-compartment, step-through passive avoidance apparatus(Muromachi Kikai, Japan) was used for the test. In a pretraining trial, a mousewas placed in the illuminated chamber, the guillotine door was opened and themouse was adapted to the apparatus for 4 min and then returned to its homecage. In an acquisition trial 24 h after the pretraining trial, the mouse wasplaced in the illuminated chamber and the time (acquisition latency) beforeit entered the dark chamber was recorded. Immediately after the mouse hadentered the dark chamber, the door was closed and a scrambled foot shock(0.2 mA at 50 Hz) was applied to the floor grid for 3 s through a shock gen-erator (SGS-003, Muromachi Kagaku, Japan). Training was terminated when

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the mouse remained in the illuminated chamber for 300 consecutive seconds.The number of trials (entries into the dark chamber) and total training time(acquisition time) were recorded. Retention of avoidance behavior was tested24 h and more after the foot shock. The mouse was again placed in the illumi-nated chamber and latency to reenter the dark chamber was recorded up to amaximum of 300 s. If the mouse did not enter the dark chamber within 300 s,the retention test was terminated and a ceiling score of 300 s was assigned.

Spontaneous Activity

Locomotor activity in mice was quantified using an Animex activity meterType S (LKB, Farad, Sweden) at the same time of day as the passive avoidancetest. Spontaneous activity was expressed as signal counts automatically pro-duced by the movement of the animal placed on the top of the Animex activitymeter.

Study of Pathologic Changes

After completion of the behavior test, the mice were anesthetized by expo-sure to ethyl ether, and the brains were removed immediately and immersedin methacarn (methanol: chloroform: acetic acid = 6:3:1). The fixed brainswere embedded in paraffin and 3-�m-thick coronal sections were prepared.The sections of brain stem were stained with hematoxylin and eosin and pho-tographed with a light microscope. The number and area of the vacuoles in thebrain stem of P8 and R1 mice were measured using an image analyzer.

Statistical Analysis

All experimental results are given as the mean ± SEM. Analysis of variance,followed by Fisher’s protected least significant difference procedure for posthoc comparison, were used to verify significance between the two means.

RESULTS

Body Weight

Restriction of the diet was started at the age of 21 days and continued forabout 5 months. The body weight of DR mice fed 60% of the amount of foodconsumed by the control animals (fed ad libitums) was consistently lower thanthat of AD animals in both P8 and R1 mice. However, the general growthpattern was similar for the mice on the two dietary regimens; the body weightincreased rapidly during the first 2.5 months of age and increased at a lowerrate thereafter.

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TABLE 1. Effect of dietary restriction on the acquisition of the passive avoidance test

Strain Diet Acquisition time (s)

SAMP8 AD 432 ± 65a

DR 234 ± 34b

SAMR1 AD 95 ± 30DR 66 ± 20

Mean ± SE (n = 25).aSignificant difference between P8 and R1 in AD (P < 0.05).bSignificant difference between AD and DR (P < 0.05).

Learning and Memory Abilities

A two-compartment, step-through passive avoidance test was performed atthe age of 6 months. AD-P8 mice showed significant impairment in the acqui-sition of passive avoidance response (TABLE 1) and retention of the acquiredresponse assessed by repeated extinction trials compared with AD-R1 mice.

DR significantly improved the acquisition of the task in P8 mice (TABLE 1).However, no effect of DR was observed on the retention of the acquired re-sponse in both P8 and R1 mice.

It is conceivable that the improvement in the acquisition of passive avoid-ance response might be associated with altered motor activity by DR. There-fore, we next determined the spontaneous activities of AD and DR mice inboth strains. The spontaneous activity of individual mice was measured atthe same time of day as the passive avoidance test. No significant differ-ence was observed in the spontaneous activity between AD and DR mice inboth strains, suggesting that the improvement in the acquisition of the pas-sive avoidance task in P8 mice is not due to changes in the motor activityin response to DR.

Thus, DR improved the impaired acquisition of passive avoidance responsebut not the extinction of the acquired response in P8 mice.

Spongy Degeneration in Brain Stem

In SAMP8 mice, spongy degeneration (vacuole formation) in the brain stemis closely associated with deterioration of learning and memory.12 It is inter-esting how DR alters such pathologic changes of the brain stem in P8 mice.

We determined the total area and number of vacuoles in the brain stem ofAD and DR mice in both strains at the age of 6 months. The total area andthe number of vacuoles in the brain stem were significantly higher in AD-P8mice than in AD-R1 mice. However, no significant effect was observed on thevacuole formation in the brain stem of P8 mice by DR.

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DISCUSSION

The present study showed that DR from time of weaning significantly im-proved the acquisition but not the retention of the passive avoidance response inP8 mice. It has been demonstrated that P8 mice exhibit age-related deteriorationin learning and memory in passive avoidance response tests,16 especially in theacquisition stage.12 However, little is known about the mechanism underlyingthe acquisition impairment of the task in P8 mice. Yagi et al.12 suggested thatvacuolization in brain stem, especially the magnocellular reticular formation,might result in an age-related deterioration of learning and memory in P8 micebecause some neurons in the brain stem respond to tasks requiring learning andmemory. For example, destruction of pedunculo-pontine nucleus-parabrachialnucleus,17,18 or median raphe nucleus19 in the brain stem resulted in the dete-rioration of maze learning and of active avoidance and visual discriminationlearning. In addition, electrophysiological studies have also shown that neu-rons in the reticular formation of the brain stem have learning and memoryfunctions.20 Thus, neurons in the reticular formation of the brain stem playimportant roles in learning and memory. However, in the present study, wecould not observe any significant effect of DR on the vacuole formation in thebrain stem of P8 mice. These observations suggest that the improvement inthe acquisition of the task by DR in P8 mice is possibly due to changes in neu-ronal function,21 such as the amount of neurotransmitters and receptors, ratherthan a histologic alteration in the brain stem. However, possible changes inthe cerebellum must also be studied because they may be involved in learningand memory deficit in P8 mice.21–23 Further study will be required to clar-ify the effect of DR on the improvement in the acquisition of such tasks inP8 mice.

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