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Food restriction affects Y-maze spatial recognition memory in developing mice
Accepted Manuscript Title: Food restriction affects Y-maze spatial recognition memory in developing mice Author: Yu Fu Yanmei Chen Liane Li Yumei Wang Xiangyang Kong Jianhong Wang PII: DOI: Reference:
S0736-5748(17)30017-5 http://dx.doi.org/doi:10.1016/j.ijdevneu.2017.03.010 DN 2180
To appear in:
Int. J. Devl Neuroscience
Received date: Revised date: Accepted date:
19-1-2017 5-3-2017 21-3-2017
Please cite this article as: Fu, Y., Chen, Y., Li, L., Wang, Y., Kong, X., Wang, J.,Food restriction affects Y-maze spatial recognition memory in developing mice, International Journal of Developmental Neuroscience (2017), http://dx.doi.org/10.1016/j.ijdevneu.2017.03.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Food restriction affects Y-maze spatial recognition memory in developing mice
Yu Fua^#, Yanmei Chena^, Liane Lia, Yumei Wanga, Xiangyang Konga, Jianhong Wangb# (^: these authors contributed equally to this work) Medical Faculty, Kunming University of Science & Technology, Kunming, Yunnan 650500, PR China
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Kunming Primates Research Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan
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650223, PR China
Correspondence and reprint requests:
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Yu Fu (# corresponding author) Medical Faculty, Kunming University of Science & Technology
727 Jing Ming Nan Road, Chenggong County, Kunming, Yunnan Province, 650500, PR China
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E-mail: [email protected]
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Jianhong Wang (*corresponding author)
Kunming Institute of Zoology
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Chinese Academy of Sciences
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Key Laboratory of Animal Models and Human Disease Mechanisms
#32 Kunming Jiao Chang Dong Lu,Kunming, Yunnan, 650223, P.R. China; E-mail: [email protected]
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ABSTRACT The ambiguous effects of food restriction (FR) on cognition in rodents have been mostly explored in the aged brain by a variety of paradigms, in which either rewards or punishments are involved. This study aims to examine the effects of chronic and acute FR with varying intensities on spatial recognition memory in developing mice. We have used a Y-maze task that is based on the innate tendency of rodents to explore
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novel environments. In chronic FR, mice had 70%-30% chow of control for seven weeks. In acute FR, mice were food restricted for 12-48 h before the tests. We found that chronic FR had no effect on the preference
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of mice for novelty in the Y-maze, but severe FR (50%-30% of control) caused impairment on spatial recognition memory. The impairment significantly correlated with the slow weight growth induced by FR.
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Acute FR also did not affect the novelty preference of mice, but either improved or impaired the memory retention. These data suggest chronic FR impairs Y-maze spatial recognition memory in developing mice
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depending on FR intensity and individual tolerability of the FR. Moreover, acute FR exerts diverse effects on the memory, either positive or negative. Our findings have revealed new insights on the effects of FR on
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spatial recognition memory in developing animals.
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Keywords: food restriction; developing mice; spatial recognition memory; Y-maze, chronic; acute
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1.
Introduction Food restriction (FR; or dietary restriction / DR and caloric restriction / CR) has emerged as one of the
healthy dieting concepts, partially because of the increasing global burden of obesity. Obesity started from teenagers in recent years has inspired parents and health care to consider the regulation of food or diet to combat this disease. Accumulating evidence indicates the beneficial effects of FR or associated diet control
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on humans and animals. It not only reduces body weight, extends lifespan, reduces neuronal damage and rescues cognitive function, but also decreases incidence of age-related diseases (Mattson, 2010; Murphy et
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al., 2014; Pani, 2015).
It has been reported that FR attenuated age-related deficits in learning and memory in rodents (Yang et
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al., 2014) and improved cognitive performance in models of neurodegenerative diseases (Brownlow et al., 2014). However, other studies reported neutral or negative effects of FR on cognitive function in different
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animal models (Khabour et al., 2010; Oliveira-Silva et al., 2007; Rajab et al., 2014; Tucci et al., 2006; Yanai et al., 2004). No significant effect by FR was observed on the disruptive memory caused by aging process in the water maze in rats (Oliveira-Silva et al., 2007). FR reduces the exploration of a new environment in an
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open field test in mice (Tucci et al., 2006). These disparities in outcome are likely attributed to experimental variables, in particular the dosage (intensity and duration) of FR (Hashimoto and Watanabe, 2005). In
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apparent agreement, the effects of CR on lifespan of mice are closely related to the levels of restriction
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(Speakman and Mitchell, 2011). A mild CR results in lifespan enhancement; a severe CR leads to a diversity of response including life extension, no impact and life shortening; and a very severe CR induces life
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shortening .
Y-maze task is a simple two-trial recognition test for measuring spatial recognition memory (Conrad et al., 1997; Dellu et al., 2000; Dellu et al., 1992; Martin et al., 2003). It does not require learning of rules, and can avoid the effects of rewards and punishments that may have nonspecific effects. In this task, during the first trial, the animal is allowed to explore only two arms of the maze, with the third arm being blocked with a door. During the second trial, the door is opened and all three arms are accessible. Discrimination of novelty vs. familiarity can be analyzed by comparing animal’s exploration of all three arms. Since the task is based on the rodents’ natural tendency to explore novelty, novelty preference and memory can be assessed by varying the inter-trial interval (ITI) between the two trials (Dellu et al., 2000). Given the fact that the level of FR is probably detrimental to its ultimate effects on health, this study aims to investigate the effects of different levels of FR on spatial recognition memory in developing mice. We have used the Y-maze task to assess the effects of acute and chronic FR in mice younger than 3 months, 3
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which approximates to humans younger than 20 years of age (Harrison, 2011). Mice experienced chronic FR after weaning and experienced acute FR after sexual maturity. All behavioral tests were carried out on animals before being mature adults.
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Material and methods
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2.1. Animals and FR protocols Male ICR mice from breeding colonies at the Kunming Medical College were used in the experiment.
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They were housed under standard conditions (a 12-h light/12-h dark cycle with light on from 07:00 to 19:00 h) and reared one group (10-13 mice) per cage (50 cm × 35 cm × 20 cm; length × width × height). We put
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each group in a larger cage than the usual type for ease of implementing FR procedures. All experimental and animal care procedures were carried out in accordance with the guidelines for the National Care and Use
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of Animals and approved by the National Animal Research Authority.
In chronic FR (Fig. 1A), 50 mice (postnatal day / P25) were randomly separated into 4 groups: the control group was allowed to feed freely (n = 12; Ctrl); and three FR groups separately had 70% (n = 13;
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FR30), 50% (n = 12; FR50) and 30% (n = 13; FR70) of chow consumed by the control in the same number of mice. The FR mice were fed once a day at 8:30 in the morning, and all the chow had been consumed
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basically (the FR30 group had occasionally a little food pellets left at the beginning days of FR, which had
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been discarded). Here, 70% of control mainly referred to that “eating is only to seventy percent of the full” as the old saying goes in China. In acute FR (Fig. 1B), 50 mice (P42) were randomly divided into 4 groups:
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Group 1 was the control that was allowed to feed freely (n = 10; Ctrl); and Groups 2-5 were food restricted for 12 h (n = 10; FR12), 24 h (n = 10; FR24), 36 h (n =10; FR36) and 48h (n =10; FR48) before the Y-maze test, respectively. Mice had no access to food within the acute FR periods. Herein, 12-h fasting mainly referred to our routine diet: usually people do not eat during night (e.g., 7:00 pm -7:00 am). In addition, 24-h and 48-h fasting are the most frequently used procedures. All mice used in both experiments had free access to water.
Figure 1
2.2. Y-maze procedures 2.2.1. Effects of chronic food restriction on memory Chronic FR was performed on mice from P25 to P74 (Fig. 1A). Y-maze tests were repeated three times 4
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separately on P60, P67 and P74. There was a 1-week interval between each test. Previous studies have indicated that the performance in the Y-maze can be assessed several times in the same animals, such as 1 week later, since the retention memory dose not last longer than a few hours (Dellu et al., 2000). As described previously (Fu et al., 2008), 4 Y-mazes were made of grey Plexiglas or wood, covered with black paper, and consisted of three arms with an angle of 120º between adjacent arms (Fig. 2). Each arm was
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8 cm × 30 cm × 15 cm (width × length × height). The three identical arms were randomly designated: (1) start arm, in which the mouse started to explore (always open); (2) novel arm, which was blocked during the
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first trial, but open during the second trial, and (3) other arm (always open).Y-maze tests were performed in another room with a light. The floor of the maze was covered with sawdust, which was mixed after each
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individual trial in order to equate differential olfactory stimuli. Differential visual cues were placed on the walls of the mazes.
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The Y-maze test consisted of 2 trials (Fig. 2). The first trial (training) had 10-min duration and allowed the mouse to explore only two arms (start arm and other arm) of the maze, with the third arm (novel arm) blocked. After an ITI of 2 min, 1 h or 4 h, the second trial (testing / retention) was conducted, during which
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all three arms were accessible and novelty vs. familiarity was analyzed by comparing behavior in all three arms. For the second trial, the mouse was placed back in the maze in the same starting arm, with free access
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to all three arms for 5 min. By using a ceiling-mounted CCD camera, all trials were recorded on a VCR.
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Video recordings were later analyzed and the duration, distance and number of visits in each arm were
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analyzed.
Figure 2
2.2.2. Effects of acute food restriction on memory The same Y-maze procedure was applied in this experiment (Fig. 1B). Y-maze tests were also repeated three times, separately on P49, P63 and P77. There was a 2-week interval between each test. Longer interval was adopted here, compared with that used in the chronic experiment, mainly for avoiding the influence of the last FR on the next FR.
2.3. Statistical analysis Changes of body weights of mice were expressed as: (1) weight growth index in the chronic FR; and (2) weight loss index in the acute FR. Both indexes were calculated as the ratio of the weights of mice on the 5
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day when the behavioral task was carried out to their initial weights before the FR experiment. For example, a mouse weighed 20 g on P25 before the chronic FR, and weighed 32 g on the day of P60 when 2-min ITI Y-maze task was performed, so its weight growth index was calculated as 32/20, namely 1.6. Data in the Y-maze were expressed as: (1) Duration of arm visits, which is the time spent in each arm; (2) Number of arm visits, the number of entries in each arm; (3) Distance of arm visits, the ambulation distances
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of mice in each arm; and (4) the total number of arm visits and ambulation in the Y-maze. The (1)-(3) were as spatial recognition memory indices and the (4) as a locomotor activity index, all of which were in the 5
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min retention test.
Differences between groups were assessed with two-way analysis of variance with repeated measures
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(ANOVA-R), one-way ANOVA (ANOVA-1) where appropriate, using the SPSS statistical software package (version 10). Treatment (chronic: Ctrl, FR30, FR50 and FR70; acute: Ctrl, FR12, FR24, FR36 and FR48)
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was a between-group factor, whereas Time (hours or days of FR) and Arm (duration, distance and number of visits in each arm) was a within-group factor. Pearson correlation analysis and linear regression analysis
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Results
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significant and P <0.01 as highly significant.
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were also performed as needed. Data were expressed as mean ± SEM. A level of P < 0.05 was considered as
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3.1. Chronic FR delayed weight growth and acute FR caused weight loss Combined ANOVA-R showed that chronic FR led to a significant main effect of time (F(2, 90) =
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102.434, P < 0.001) and group (F(3, 45) = 32.670, P < 0.001), and a significant interaction of time × group (F(6, 90) = 46.340, P < 0.001) on the body weights of mice (Fig. 3). All chronic FR groups had significantly lower weights than the control group from P32 to P74 after FR (P < 0.01 for all). The weight growth index showed significant group difference before each onset of the three Y-maze tasks (2-min ITI: F(3, 49)=23.86, P < 0.001; 1-h ITI: F(3, 49)=38.09, P < 0.001; 4-h ITI: F(3, 48)=36.69, P < 0.001) (Table 1).
Figure 3 Table 1
Similarly, acute FR resulted in significant main effect of time (F(2, 90) = 13.445, P < 0.001) and group (F(4, 45) = 238.759, P < 0.001), and a significant interaction of time × group (F(8, 90) = 5.216, P < 0.001) on the body weight of mice. The weight of acute FR mice was significantly decreased before each Y-maze 6
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onset, compared with that of the control mice (2-min ITI: F(4, 49)=56.29, P < 0.001; 1-h ITI: F(4, 49)=99.34, P < 0.001; 4-h ITI: F(4, 49)=149.68, P < 0.001) (Table 2).
Table 2
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3.2. Severe chronic FR impaired memory retention The percentages of time, visits and ambulation spent in the novel arm during the retention test were
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evaluated as an index of novelty exploration and compared with chance level (33.3%). After 2-min ITI, the control group and all three chronic FR groups had scores significantly higher than chance level for the three
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indices (P < 0.01 for all, Fig. 4A-C left). In contrast, after 1-h ITI, only the control and FR30 groups showed significantly higher scores than chance level (P < 0.05 for all), and no significant difference was observed
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for both FR50 and FR70 groups (P > 0.05 for all) (Fig. 4A-C middle). In addition, after 4-h ITI, the control group and both FR30 and FR50 groups had either one or two indices with significantly higher scores than chance level (P < 0.05 for all), but FR70 group did not have (P > 0.05 for all) (Fig. 4A-C right).
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Combined ANOVA-R displayed significant differences in percentage time, number of arm visits and ambulation in the three arms after 2-min, 1-h, and 4-h ITI (P < 0.001 for all, data not shown), except for the
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percentage time after 4-h ITI, which showed a trend towards significant (P = 0.060). After 1-h ITI, a
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significant group × arm interaction was also found in the ambulation distance (P < 0.042). By ANOVA-1, there was a significant main effect of ‘group’ in the percentage of ambulation spent in the novel arm (F(3,
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49)=2.837, P < 0.05). Post-hoc LSD analysis revealed that the FR50 group had a significantly lower score than the control group (P < 0.05; Fig. 4C middle). These results have demonstrated that chronic FR could impair Y-maze spatial recognition memory. Figure 4
We next analyzed the relationship between weight changes of mice and their testing score in the Y maze. Fig. 5 showed the combined results of correlation analysis for the FR groups. The mean scores in novel arm ( i.e., the average percentage of time, visits and ambulation spent in the novel arm during the retention test) increased with the increasing index of the weight of mice, which could be found after 1-h ITI (Fig. 5A) and 4-h ITI (Fig. 5B). There were positive correlations between mean score in novel arm and weight growth index for both 1-h ITI (r = 0.346, P < 0.05) and 4-h ITI (r = 0.353, P < 0.05). The linear regression function of 1-h-ITI for FR groups was: y=13.24x+23.03; and that of 4-h ITI was: y=10.10x+25.21. For these FR mice, 7
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no correlation could be found for the 2-min ITI (r = 0.030, P = 0.859). In addition, there was no correlation for the control group for either 2-min (r = 0.124, P = 0.700), 1-h (r = -0.021, P = 0.948) or 4-h ITIs (r = -0.016, P = 0.961).
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Figure 5
3.3. Acute FR diversely affected memory retention
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Acute FR led to significant differences in percentage time, number of arm visits and ambulation in the three arms with 2-min and 1-h ITIs (P < 0.001 for all). After 4-h ITI, the percentages of distance and number
and the percentage of time was close to significance (P = 0.059).
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of arm visits also showed significant difference among the three arms (distance, P < 0.05; number, P < 0.01),
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As shown in Fig. 6, the percentages of the three indices spent in the novel arm during testing were first compared with 33.3%. The control group and all FR groups had scores significantly higher than chance level
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for 1-3 indices after 2-min ITI (P < 0.05 for all, Fig. 6A-C left). After 1-h ITI, all except FR24 groups still showed higher scores than chance level for 1-3 indices (P < 0.05 for all, Fig. 6A-C middle). After 4-h ITI,
Figure 6
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novel arm (P < 0.05, Fig. 6A-C right).
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only the FR12 group showed significantly higher score than chance level on the percentage of number in the
In addition, correlation analysis was performed between mean score in novel arm of Y-maze and weight loss index for acute FR groups. No significant correlation could be found either after 2-min (r = -0.008, P = 0.963), 1-h (r = 0.171, P = 0.290) or 4-h ITI (r = 0.281, P = 0.079). For the control group, there were also no correlations between mean scores in novel arm and their weight changes (2-min ITI: r = 0.092, P = 0.800; 1-h ITI: r = -0.296, P = 0.406; 4-h ITI: r = 0.328, P = 0.355).
3.4. Acute but not chronic FR decreased locomotor activity Both chronic and acute FR had no effects on the total number of arm visits in the Y-maze (chronic FR: F(3, 49)=0.692, P = 0.561; acute FR: F(4, 49)=0.999, P = 0.418) (Fig. 7A-B). On the other side, the total ambulation distance was a direct index of the locomotor activity. Chronic FR has no significant effect on this index (F(3, 49)=0.838, P = 0.480). In contrast, acute FR decreased the ambulation of the mice (F(4, 8
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49)=6.293, P <) (Fig. 7C-D). Post-hoc LSD analysis revealed that all acute FR groups had significantly lower ambulation distances than the control group (P < 0.05; Fig. 7D). Figure 7
Discussion
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4.
In this study, we mainly found that: (1) both chronic and acute FR had no effect on the preference of
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mice for novelty in Y-maze; (2) severe chronic FR impaired the Y-maze performance, depending on FR intensity and individual tolerability of the FR, and acute FR affected the performance to some extent, either
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improving or impairing the retention scores; (3) acute FR reduced the locomotor activity in mice. The two-trial Y-maze task is a specific and sensitive test of spatial recognition memory in rodents (Dellu
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et al., 2000). We now explore the ambulation as an index for measuring memory of mice in the Y-maze. Compared to the other two indices, it displayed scores greater than 45% during test after 2-min and 1-h ITI, such as for the control mice (chronic FR: 59% for 2-min ITI and 48% for 1-h ITI; acute FR: 50% for 2-min
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ITI and 46% for 1-h ITI). In addition, in acute FR experiment, after 2-min ITI the FR36 group and after 1-h ITI the FR48 group showed higher percentages of ambulation than chance level, which were not observed in
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Y-maze task.
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the other two indices. Thus, the ambulation is a good supplement to the other two indices for this kind of
All chronic and acute FR groups exhibited a preferable level of novelty exploration. The result was
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consistent with the previous open field test revealing no difference in exploratory activity between the CR (e.g., 20%-40% CR) and the control (Cardoso et al., 2016; Wu et al., 2003) . These data showed that FR did not affect the preference of mice for novelty in Y-maze. Severe chronic FR impaired the retention of mice in the two-trial Y-maze tests. Significant correlation was found between the impairment and the slow weight growth induced by FR. The data suggest that chronic FR impairs spatial recognition memory, which is not only related to the FR dosage but also to the individual tolerability of FR. There are limited studies assessing the effects of FR in spatial tasks unrelated to rewards and punishments (Benau et al., 2014; Conrad, 2010). In one study, FR or shock as an unpredictable stress lasting for 4 weeks caused an impairment of spatial memory in rats (Orsetti et al., 2007). In addition, previous studies indicated impairment of chronic stress (e.g., chronic restraint up to 21 d) on spatial memory in rats (Conrad, 2010). Thus, severe chronic FR may serve as a significant stressor, which over time can affect memory of individuals. 9
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Acute FR led to diverse effects on the retention of the Y-maze test in mice. After 4-h ITI, the retention was kept only in FR for 12 h group, but not in the control and other FR groups. Studies with subtle FR, DR or CR have exhibited the enhancement effects (Ko et al., 2015). For example, 2 h of 40% DR for 5 consecutive days increased learning and memory in water maze in adult mice (Rajab et al., 2014). After 1-h ITI, however, acute FR for 24 h impaired the retention. This result is paradoxical because the negative effect
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was not observed on acute FR for longer durations (36-48 h). An important consideration was that the effect of acute FR is dynamic and reversible, which has been raised on changes in the hippocampus caused by
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many chronic stress paradigms (Conrad, 2010). In addition, 12-h FR difference among acute FR groups was involved with natural circadian rhythm. It has reported that the rodents have the ability to integrate spatial
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and temporal information (Lukoyanov et al., 2002), and the effects of FR on learning and memory could be photoperiod-dependent (Steinman et al., 2011). Taken together, acute FR has positive or negative effect on
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spatial recognition memory in mice, which may be dynamic and reversible, or circadian-rhythm-dependent. All mice used in this study were with ages less than 3 months old, which is at an early stage in life. Up to now, few studies were performed in developing rodents to investigate the effects of FR on cognition.
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These limited data, however, exhibit inconsistent results. For example, the male but not female mice at an age of 1 month were fed by 20%-35% DR for 3 months (Wu et al., 2002), or after weaning experienced DR
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for 6 months (Wu et al., 2003), displaying increased learning but not retention in a Y-maze. Herein, the
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Y-maze task is very different from ours, which has foot shocks in start and error arms. However, in female mice after weaning, 40% CR for 4 weeks improved cognitive performance in water maze (Kuhla et al.,
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2013). In the same paradigm, a recent study has reported that the male rats aged 4 weeks were 40% FR for 2 months, and their performance was impaired for spatial learning but not for memory retention (Cardoso et al., 2016) . The divergence may be due to different paradigms and some experimental variables, such as FR intensity and duration. Moreover, the effects of FR may be sex-dependent in developing mice. It will be very interesting in the future to investigate those effects of FR in both male and female animals. On the other side, for severe FR, the rats at an age of 30 days have received 75%-80% less chow than the control rats, and when tested in novel object recognition test after 4 and 14 weeks, their short- and/or long-term memory were improved (Molz et al., 2016). In our chronic study, the mice after weaning experienced 50%-70% FR for 6-7 weeks, displaying impaired recognition memory in the Y-maze test. Both novel object recognition task in that report and Y-maze in our study can test recognition memory basing on natural tendency of rodents to explore novelty, but the underlying neural mechanism may be different. Because it has been reported that object novelty recognition could be performed without an intact 10
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hippocampus, while in this case the Y-maze performance was impaired (Conrad, 2010). This may also explain that FR had no effects on preference of mice for novelty but had influenced their spatial recognition memory in our study. In rodents, the hippocampus is involved in spatial cognition processes (Morris et al., 1982). Previous studies have indicated that hippocampus is one of the brain regions that are more sensitive to the nutritional
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deficits (Cardoso et al., 2013; Hipolito-Reis et al., 2013). A number of previous studies suggest that FR (DR/CR) may lead to alterations of hippocampus (Benau et al., 2014; Conrad, 2010), which further
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contribute to changes of memory (Partadiredja and Bedi, 2011). For example, FR altered hippocampal CB1R expression and function in rats (Talani et al., 2016), and CR enhanced hippocampal neurogenesis and
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remote contextual fear memory in adult mice (Hornsby et al., 2016). Regarding the developing phase, it was published recently that young-onset CR could result in alterations of neurogenesis in the dentate gyrus,
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which may explain some impairment of spatial cognition (Cardoso et al., 2016). These findings also provide an explanation for the current results.
In the present study, the age of the mice approximates to humans less than 20 years old (Harrison, 2011).
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Previous studies have found that the volume of hippocampus has an inverted-U shape relationship with increasing age, which peaks before 20 years old (Krogsrud et al., 2014; Wierenga et al., 2014). At the
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corresponding age phase, we found that both chronic and acute FR treatment affected spatial recognition
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memory in mice, suggesting that FR may affect the key developmental stage of hippocampus since the brain region is essential to spatial cognition. Thus, our data indicate that adolescent animals are sensitive or
issue.
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vulnerable to FR treatment (Cardoso et al., 2016) and the FR routine in this age group should consider this
The ambulation was affected by the acute FR in this study, indicating the decreased locomotor activity. The decreased activity could not explain changes of the retention in the Y-maze, because the total number of arm visits showed no significant difference from the control. On the other hand, previous studies have found that food deprivation for 0-48 h had no effect on the distance traveled in an open field test (Dietze et al., 2016; Pierre et al., 2001). It was assumed that the duration of fasting positively correlates the response rate of animals (Moscarello et al., 2009). Therefore, the decrease of locomotion seems to be an inadequate response at the time when animals are suffering from hunger, which may be detrimental to their survival. However, previous data suggest that exploratory behavior is separable from seeking food and acquiring information associated with environmental aspects (Pierre et al., 2001). In addition, food deprivation for up to 48 h seemed not to alter anxiety or depression behavior (Dietze et al., 2016; Hendriksen et al., 2015). 11
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Thus, the underlying mechanism and function of the influence of acute FR on locomotor activity require further assessment. In conclusion, chronic FR impaired Y-maze spatial recognition memory in developing mice depending on FR intensity and individual tolerability of the FR, and acute FR positively or negatively affected the retention of the memory. Our data give new insights on the effects of FR on spatial cognition in developing
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Funding This work was supported by the National Science Foundation of China (81560221, 81560234, 31571109, 31560317, 61502214, and 81460007), the Research Foundation of Kunming University of Science and Technology (KKZ3201560006), and the Natural Science Foundation of Yunnan Province of
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China (2016FB107).
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Acknowledgements We thank Prof. Yuanye Ma (Kunming Institute of Zoology, Chinese Academy of Sciences) for supervision at this project and thank Dr. Junhong Su (Medical Faculty, Kunming University of Science & Technology) for his help with this work. We are especially grateful to Dr. Qiuwei Pan (Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center) for his constructive revising of
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Hipolito-Reis, J., Pereira, P.A., Andrade, J.P., Cardoso, A. (2013) Prolonged protein deprivation differentially affects calretinin-
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and parvalbumin-containing interneurons in the hippocampal dentate gyrus of adult rats. Neurosci Lett 555, 154-158. Hornsby, A.K., Redhead, Y.T., Rees, D.J., Ratcliff, M.S., Reichenbach, A., Wells, T., Francis, L., Amstalden, K., Andrews, Z.B., Davies, J.S. (2016) Short-term calorie restriction enhances adult hippocampal neurogenesis and remote fear memory in a Ghsr-dependent manner. Psychoneuroendocrinology 63, 198-207. Khabour, O.F., Alzoubi, K.H., Alomari, M.A., Alzubi, M.A. (2010) Changes in spatial memory and BDNF expression to concurrent dietary restriction and voluntary exercise. Hippocampus 20, 637-645. Ko, K.I., Root, C.M., Lindsay, S.A., Zaninovich, O.A., Shepherd, A.K., Wasserman, S.A., Kim, S.M., Wang, J.W. (2015) Starvation promotes concerted modulation of appetitive olfactory behavior via parallel neuromodulatory circuits. Elife 4. Krogsrud, S.K., Tamnes, C.K., Fjell, A.M., Amlien, I., Grydeland, H., Sulutvedt, U., Due-Tonnessen, P., Bjornerud, A., Solsnes, A.E., Haberg, A.K., Skrane, J., Walhovd, K.B. (2014) Development of hippocampal subfield volumes from 4 to 22 years. Hum Brain Mapp 35, 5646-5657. Kuhla, A., Lange, S., Holzmann, C., Maass, F., Petersen, J., Vollmar, B., Wree, A. (2013) Lifelong caloric restriction increases working memory in mice. Plos One 8, e68778. Lukoyanov, N.V., Pereira, P.A., Mesquita, R.M., Andrade, J.P. (2002) Restricted feeding facilitates time-place learning in adult rats. Behav Brain Res 134, 283-290. Martin, S., Jones, M., Simpson, E., van den Buuse, M. (2003) Impaired spatial reference memory in aromatase-deficient (ArKO) mice. Neuroreport 14, 1979-1982. Mattson, M.P. (2010) The impact of dietary energy intake on cognitive aging. Front Aging Neurosci 2, 5. 15
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Molz, P., Ellwanger, J.H., Zenkner, F.F., Campos, D., Pra, D., Putzke, M.T., Franke, S.I. (2016) Recognition memory and DNA damage in undernourished young rats. An Acad Bras Cienc 88, 1863-1873. Morris, R.G., Garrud, P., Rawlins, J.N., O'Keefe, J. (1982) Place navigation impaired in rats with hippocampal lesions. Nature 297, 681-683. Moscarello, J.M., Ben-Shahar, O., Ettenberg, A. (2009) Effects of food deprivation on goal-directed behavior, spontaneous locomotion, and c-Fos immunoreactivity in the amygdala. Behav Brain Res 197, 9-15. Murphy, T., Dias, G.P., Thuret, S. (2014) Effects of diet on brain plasticity in animal and human studies: mind the gap. Neural Plast 2014, 563160.
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Pani, G. (2015) Neuroprotective effects of dietary restriction: Evidence and mechanisms. Semin Cell Dev Biol 40, 106-114. Partadiredja, G., Bedi, K.S. (2011) Mice undernourished before, but not after, weaning perform better in motor coordination and spatial learning tasks than well-fed controls. Nutr Neurosci 14, 129-137.
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Rajab, E., Alqanbar, B., Naiser, M.J., Abdulla, H.A., Al-Momen, M.M., Kamal, A. (2014) Sex differences in learning and memory
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Speakman, J.R., Mitchell, S.E. (2011) Caloric restriction. Mol Aspects Med 32, 159-221. Steinman, M.Q., Crean, K.K., Trainor, B.C. (2011) Photoperiod interacts with food restriction in performance in the Barnes maze
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Sanna, E. (2016) Enhanced Glutamatergic Synaptic Plasticity in the Hippocampal CA1 Field of Food-Restricted Rats: Involvement of CB1 Receptors. Neuropsychopharmacology 41, 1308-1318. Tucci, V., Hardy, A., Nolan, P.M. (2006) A comparison of physiological and behavioural parameters in C57BL/6J mice undergoing
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food or water restriction regimes. Behav Brain Res 173, 22-29. Wierenga, L., Langen, M., Ambrosino, S., van Dijk, S., Oranje, B., Durston, S. (2014) Typical development of basal ganglia, hippocampus, amygdala and cerebellum from age 7 to 24. Neuroimage 96, 67-72. Wu, A., Sun, X., Liu, Y. (2003) Effects of caloric restriction on cognition and behavior in developing mice. Neurosci Lett 339, 166-168.
Wu, A., Wan, F., Sun, X., Liu, Y. (2002) Effects of dietary restriction on growth, neurobehavior, and reproduction in developing Kunmin mice. Toxicol Sci 70, 238-244.
Yanai, S., Okaichi, Y., Okaichi, H. (2004) Long-term dietary restriction causes negative effects on cognitive functions in rats. Neurobiol Aging 25, 325-332.
Yang, F., Chu, X., Yin, M., Liu, X., Yuan, H., Niu, Y., Fu, L. (2014) mTOR and autophagy in normal brain aging and caloric restriction ameliorating age-related cognition deficits. Behav Brain Res 264, 82-90.
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Figure captions Fig. 1. Experimental workflow. (A) In chronic experiment, mice experienced food restriction (FR) from P25 (postnatal day 25) to P74, and three ITIs Y-maze tests were performed on P60, P67, P74, individually. (B) In acute experiment, mice experienced acute FR separately before the three ITIs Y-maze tests, which were performed on P49, P63 and P77. Please refer to the methods for details.
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Fig. 2. Schematic illustration of the 2-trial Y-maze tests. During the first trial (Training), the animal is allowed to explore only two arms of the maze, with the third arm being blocked by a door. During the second trial (Testing), the door is opened and all three arms are accessible.
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Fig. 3. Changes of body weights with time during chronic FR. All FR groups showed signicantly difference from the control group on P32 - P74 (P < 0.01 for all).
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Fig. 4. Differences from chronic FR groups in exploration of novelty after a 2-min (left), 1-h (middle) and 4-h (right) ITIs. Percentages of time (A), visits (B) and ambulation (C) made to the novel arm during the 5 min of the retrieval trial. Comparisons with chance (dotted line, 33.33%): ***P < 0.001; **P < 0.01; *P < 0.05. Comparisons between groups: #P < 0.05.
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Fig. 5. Relationships between changes on weights of mice after chronic FR and their retention performance in the novel arm after 1-h (A) and 4-h (B) ITIs. Weight growth index was calculated as the ratio of weights before and after FR. Mean score in the novel arm (%) was expressed as the average percentage of time, visits and ambulation spent in the novel arm during the retention test. The horizontal dotted lines represent the criteria of 40%, which is a common index of evaluating if mice exhibit a significant exploration of novelty in the two-trial Y-maze, and the vertical dotted lines represent the corresponding weight index from the linear regression functions. Pearson correlation analysis: *P < 0.05.
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Fig. 6. Differences from acute FR groups in exploration of novelty after a 2-min (left), 1-h (middle) and 4-h (right) ITIs. Percentages of time (A), visits (B) and ambulation (C) made to the novel arm during the 5 min of the retrieval trial. Comparisons with chance (dotted line, 33.33%): ***P < 0.001; **P < 0.01; *P < 0.05.
Fig. 7. Effects of chronic (A, C) and acute (B, dD) FRs on locomotor activity. (A) and (B) Total number of arm visits in the Y-maze during the retrieval trial; (C) and (D) Total ambulation in the Y-maze during the retrieval trial. Comparison with the control group: ###P < 0.001; ##P < 0.01; #P < 0.05.
Tables Table 1 Descriptive statistics of weight growth index after chronic FR in mice. Weight Ctrl FR30 FR50 growth index Mean SD N Mean SD N Mean SD
FR70 N
Mean
SD
N
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2-min ITI 1.64 0.19 12 1.53 0.20 13 1.37** 0.18 12 1-h ITI 1.96 0.23 12 1.60*** 0.22 13 1.37*** 0.18 12 4-h ITI 1.92 0.23 12 1.68* 0.24 13 1.37*** 0.18 11 * ** *** P ≤ 0.05, P ≤ 0.01 and P ≤ 0.001; comparisons with the control. P-values were calculated by Post hoc least significant difference (LSD) using ANOVA-1.
1.02*** 0.22 1.07*** 0.22 1.02*** 0.25
13 13 13
Table 2
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0.93*** 0.03 0.86*** 0.03 0.87*** 0.02
N 10 10 10
Mean
SD
N
0.83*** 0.07 0.86*** 0.02 0.84*** 0.02
10 10 10
FR48 Mean
SD
N
0.83*** 0.04 0.82*** 0.03 0.83*** 0.01
10 10 10
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2-min ITI 1.05 0.03 10 0.98*** 0.02 10 1-h ITI 1.03 0.02 10 0.91*** 0.03 10 4-h ITI 1.02 0.02 10 0.92*** 0.02 10 *** P ≤ 0.001; comparisons with the control. P-values were calculated by post-hoc LSD using ANOVA-1.
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Descriptive statistics of weight loss index after acute FR in mice. Weight Ctrl FR12 FR24 loss index Mean SD N Mean SD N Mean SD
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Highlights ：
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We used a Y-maze task avoiding reward or punishment to test in developing mice. The levels of chronic and acute food restrictions were systematically varied.
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Severe chronic food restriction caused impairment on spatial recognition memory.
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The impairment significantly correlated with slow weight growth.
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Acute restriction diversely affected memory retention, positively or negatively.
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S0736-5748(17)30017-5 http://dx.doi.org/doi:10.1016/j.ijdevneu.2017.03.010 DN 2180
To appear in:
Int. J. Devl Neuroscience
Received date: Revised date: Accepted date:
19-1-2017 5-3-2017 21-3-2017
Please cite this article as: Fu, Y., Chen, Y., Li, L., Wang, Y., Kong, X., Wang, J.,Food restriction affects Y-maze spatial recognition memory in developing mice, International Journal of Developmental Neuroscience (2017), http://dx.doi.org/10.1016/j.ijdevneu.2017.03.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Food restriction affects Y-maze spatial recognition memory in developing mice
Yu Fua^#, Yanmei Chena^, Liane Lia, Yumei Wanga, Xiangyang Konga, Jianhong Wangb# (^: these authors contributed equally to this work) Medical Faculty, Kunming University of Science & Technology, Kunming, Yunnan 650500, PR China
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Kunming Primates Research Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan
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a
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650223, PR China
Correspondence and reprint requests:
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Yu Fu (# corresponding author) Medical Faculty, Kunming University of Science & Technology
727 Jing Ming Nan Road, Chenggong County, Kunming, Yunnan Province, 650500, PR China
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E-mail: [email protected]
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Jianhong Wang (*corresponding author)
Kunming Institute of Zoology
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Chinese Academy of Sciences
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Key Laboratory of Animal Models and Human Disease Mechanisms
#32 Kunming Jiao Chang Dong Lu,Kunming, Yunnan, 650223, P.R. China; E-mail: [email protected]
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ABSTRACT The ambiguous effects of food restriction (FR) on cognition in rodents have been mostly explored in the aged brain by a variety of paradigms, in which either rewards or punishments are involved. This study aims to examine the effects of chronic and acute FR with varying intensities on spatial recognition memory in developing mice. We have used a Y-maze task that is based on the innate tendency of rodents to explore
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novel environments. In chronic FR, mice had 70%-30% chow of control for seven weeks. In acute FR, mice were food restricted for 12-48 h before the tests. We found that chronic FR had no effect on the preference
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of mice for novelty in the Y-maze, but severe FR (50%-30% of control) caused impairment on spatial recognition memory. The impairment significantly correlated with the slow weight growth induced by FR.
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Acute FR also did not affect the novelty preference of mice, but either improved or impaired the memory retention. These data suggest chronic FR impairs Y-maze spatial recognition memory in developing mice
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depending on FR intensity and individual tolerability of the FR. Moreover, acute FR exerts diverse effects on the memory, either positive or negative. Our findings have revealed new insights on the effects of FR on
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spatial recognition memory in developing animals.
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Keywords: food restriction; developing mice; spatial recognition memory; Y-maze, chronic; acute
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1.
Introduction Food restriction (FR; or dietary restriction / DR and caloric restriction / CR) has emerged as one of the
healthy dieting concepts, partially because of the increasing global burden of obesity. Obesity started from teenagers in recent years has inspired parents and health care to consider the regulation of food or diet to combat this disease. Accumulating evidence indicates the beneficial effects of FR or associated diet control
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on humans and animals. It not only reduces body weight, extends lifespan, reduces neuronal damage and rescues cognitive function, but also decreases incidence of age-related diseases (Mattson, 2010; Murphy et
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al., 2014; Pani, 2015).
It has been reported that FR attenuated age-related deficits in learning and memory in rodents (Yang et
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al., 2014) and improved cognitive performance in models of neurodegenerative diseases (Brownlow et al., 2014). However, other studies reported neutral or negative effects of FR on cognitive function in different
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animal models (Khabour et al., 2010; Oliveira-Silva et al., 2007; Rajab et al., 2014; Tucci et al., 2006; Yanai et al., 2004). No significant effect by FR was observed on the disruptive memory caused by aging process in the water maze in rats (Oliveira-Silva et al., 2007). FR reduces the exploration of a new environment in an
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open field test in mice (Tucci et al., 2006). These disparities in outcome are likely attributed to experimental variables, in particular the dosage (intensity and duration) of FR (Hashimoto and Watanabe, 2005). In
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apparent agreement, the effects of CR on lifespan of mice are closely related to the levels of restriction
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(Speakman and Mitchell, 2011). A mild CR results in lifespan enhancement; a severe CR leads to a diversity of response including life extension, no impact and life shortening; and a very severe CR induces life
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shortening .
Y-maze task is a simple two-trial recognition test for measuring spatial recognition memory (Conrad et al., 1997; Dellu et al., 2000; Dellu et al., 1992; Martin et al., 2003). It does not require learning of rules, and can avoid the effects of rewards and punishments that may have nonspecific effects. In this task, during the first trial, the animal is allowed to explore only two arms of the maze, with the third arm being blocked with a door. During the second trial, the door is opened and all three arms are accessible. Discrimination of novelty vs. familiarity can be analyzed by comparing animal’s exploration of all three arms. Since the task is based on the rodents’ natural tendency to explore novelty, novelty preference and memory can be assessed by varying the inter-trial interval (ITI) between the two trials (Dellu et al., 2000). Given the fact that the level of FR is probably detrimental to its ultimate effects on health, this study aims to investigate the effects of different levels of FR on spatial recognition memory in developing mice. We have used the Y-maze task to assess the effects of acute and chronic FR in mice younger than 3 months, 3
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which approximates to humans younger than 20 years of age (Harrison, 2011). Mice experienced chronic FR after weaning and experienced acute FR after sexual maturity. All behavioral tests were carried out on animals before being mature adults.
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Material and methods
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2.1. Animals and FR protocols Male ICR mice from breeding colonies at the Kunming Medical College were used in the experiment.
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They were housed under standard conditions (a 12-h light/12-h dark cycle with light on from 07:00 to 19:00 h) and reared one group (10-13 mice) per cage (50 cm × 35 cm × 20 cm; length × width × height). We put
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each group in a larger cage than the usual type for ease of implementing FR procedures. All experimental and animal care procedures were carried out in accordance with the guidelines for the National Care and Use
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of Animals and approved by the National Animal Research Authority.
In chronic FR (Fig. 1A), 50 mice (postnatal day / P25) were randomly separated into 4 groups: the control group was allowed to feed freely (n = 12; Ctrl); and three FR groups separately had 70% (n = 13;
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FR30), 50% (n = 12; FR50) and 30% (n = 13; FR70) of chow consumed by the control in the same number of mice. The FR mice were fed once a day at 8:30 in the morning, and all the chow had been consumed
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basically (the FR30 group had occasionally a little food pellets left at the beginning days of FR, which had
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been discarded). Here, 70% of control mainly referred to that “eating is only to seventy percent of the full” as the old saying goes in China. In acute FR (Fig. 1B), 50 mice (P42) were randomly divided into 4 groups:
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Group 1 was the control that was allowed to feed freely (n = 10; Ctrl); and Groups 2-5 were food restricted for 12 h (n = 10; FR12), 24 h (n = 10; FR24), 36 h (n =10; FR36) and 48h (n =10; FR48) before the Y-maze test, respectively. Mice had no access to food within the acute FR periods. Herein, 12-h fasting mainly referred to our routine diet: usually people do not eat during night (e.g., 7:00 pm -7:00 am). In addition, 24-h and 48-h fasting are the most frequently used procedures. All mice used in both experiments had free access to water.
Figure 1
2.2. Y-maze procedures 2.2.1. Effects of chronic food restriction on memory Chronic FR was performed on mice from P25 to P74 (Fig. 1A). Y-maze tests were repeated three times 4
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separately on P60, P67 and P74. There was a 1-week interval between each test. Previous studies have indicated that the performance in the Y-maze can be assessed several times in the same animals, such as 1 week later, since the retention memory dose not last longer than a few hours (Dellu et al., 2000). As described previously (Fu et al., 2008), 4 Y-mazes were made of grey Plexiglas or wood, covered with black paper, and consisted of three arms with an angle of 120º between adjacent arms (Fig. 2). Each arm was
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8 cm × 30 cm × 15 cm (width × length × height). The three identical arms were randomly designated: (1) start arm, in which the mouse started to explore (always open); (2) novel arm, which was blocked during the
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first trial, but open during the second trial, and (3) other arm (always open).Y-maze tests were performed in another room with a light. The floor of the maze was covered with sawdust, which was mixed after each
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individual trial in order to equate differential olfactory stimuli. Differential visual cues were placed on the walls of the mazes.
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The Y-maze test consisted of 2 trials (Fig. 2). The first trial (training) had 10-min duration and allowed the mouse to explore only two arms (start arm and other arm) of the maze, with the third arm (novel arm) blocked. After an ITI of 2 min, 1 h or 4 h, the second trial (testing / retention) was conducted, during which
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all three arms were accessible and novelty vs. familiarity was analyzed by comparing behavior in all three arms. For the second trial, the mouse was placed back in the maze in the same starting arm, with free access
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to all three arms for 5 min. By using a ceiling-mounted CCD camera, all trials were recorded on a VCR.
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Video recordings were later analyzed and the duration, distance and number of visits in each arm were
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analyzed.
Figure 2
2.2.2. Effects of acute food restriction on memory The same Y-maze procedure was applied in this experiment (Fig. 1B). Y-maze tests were also repeated three times, separately on P49, P63 and P77. There was a 2-week interval between each test. Longer interval was adopted here, compared with that used in the chronic experiment, mainly for avoiding the influence of the last FR on the next FR.
2.3. Statistical analysis Changes of body weights of mice were expressed as: (1) weight growth index in the chronic FR; and (2) weight loss index in the acute FR. Both indexes were calculated as the ratio of the weights of mice on the 5
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day when the behavioral task was carried out to their initial weights before the FR experiment. For example, a mouse weighed 20 g on P25 before the chronic FR, and weighed 32 g on the day of P60 when 2-min ITI Y-maze task was performed, so its weight growth index was calculated as 32/20, namely 1.6. Data in the Y-maze were expressed as: (1) Duration of arm visits, which is the time spent in each arm; (2) Number of arm visits, the number of entries in each arm; (3) Distance of arm visits, the ambulation distances
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of mice in each arm; and (4) the total number of arm visits and ambulation in the Y-maze. The (1)-(3) were as spatial recognition memory indices and the (4) as a locomotor activity index, all of which were in the 5
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min retention test.
Differences between groups were assessed with two-way analysis of variance with repeated measures
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(ANOVA-R), one-way ANOVA (ANOVA-1) where appropriate, using the SPSS statistical software package (version 10). Treatment (chronic: Ctrl, FR30, FR50 and FR70; acute: Ctrl, FR12, FR24, FR36 and FR48)
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was a between-group factor, whereas Time (hours or days of FR) and Arm (duration, distance and number of visits in each arm) was a within-group factor. Pearson correlation analysis and linear regression analysis
3.
Results
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significant and P <0.01 as highly significant.
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were also performed as needed. Data were expressed as mean ± SEM. A level of P < 0.05 was considered as
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3.1. Chronic FR delayed weight growth and acute FR caused weight loss Combined ANOVA-R showed that chronic FR led to a significant main effect of time (F(2, 90) =
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102.434, P < 0.001) and group (F(3, 45) = 32.670, P < 0.001), and a significant interaction of time × group (F(6, 90) = 46.340, P < 0.001) on the body weights of mice (Fig. 3). All chronic FR groups had significantly lower weights than the control group from P32 to P74 after FR (P < 0.01 for all). The weight growth index showed significant group difference before each onset of the three Y-maze tasks (2-min ITI: F(3, 49)=23.86, P < 0.001; 1-h ITI: F(3, 49)=38.09, P < 0.001; 4-h ITI: F(3, 48)=36.69, P < 0.001) (Table 1).
Figure 3 Table 1
Similarly, acute FR resulted in significant main effect of time (F(2, 90) = 13.445, P < 0.001) and group (F(4, 45) = 238.759, P < 0.001), and a significant interaction of time × group (F(8, 90) = 5.216, P < 0.001) on the body weight of mice. The weight of acute FR mice was significantly decreased before each Y-maze 6
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onset, compared with that of the control mice (2-min ITI: F(4, 49)=56.29, P < 0.001; 1-h ITI: F(4, 49)=99.34, P < 0.001; 4-h ITI: F(4, 49)=149.68, P < 0.001) (Table 2).
Table 2
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3.2. Severe chronic FR impaired memory retention The percentages of time, visits and ambulation spent in the novel arm during the retention test were
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evaluated as an index of novelty exploration and compared with chance level (33.3%). After 2-min ITI, the control group and all three chronic FR groups had scores significantly higher than chance level for the three
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indices (P < 0.01 for all, Fig. 4A-C left). In contrast, after 1-h ITI, only the control and FR30 groups showed significantly higher scores than chance level (P < 0.05 for all), and no significant difference was observed
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for both FR50 and FR70 groups (P > 0.05 for all) (Fig. 4A-C middle). In addition, after 4-h ITI, the control group and both FR30 and FR50 groups had either one or two indices with significantly higher scores than chance level (P < 0.05 for all), but FR70 group did not have (P > 0.05 for all) (Fig. 4A-C right).
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Combined ANOVA-R displayed significant differences in percentage time, number of arm visits and ambulation in the three arms after 2-min, 1-h, and 4-h ITI (P < 0.001 for all, data not shown), except for the
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percentage time after 4-h ITI, which showed a trend towards significant (P = 0.060). After 1-h ITI, a
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significant group × arm interaction was also found in the ambulation distance (P < 0.042). By ANOVA-1, there was a significant main effect of ‘group’ in the percentage of ambulation spent in the novel arm (F(3,
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49)=2.837, P < 0.05). Post-hoc LSD analysis revealed that the FR50 group had a significantly lower score than the control group (P < 0.05; Fig. 4C middle). These results have demonstrated that chronic FR could impair Y-maze spatial recognition memory. Figure 4
We next analyzed the relationship between weight changes of mice and their testing score in the Y maze. Fig. 5 showed the combined results of correlation analysis for the FR groups. The mean scores in novel arm ( i.e., the average percentage of time, visits and ambulation spent in the novel arm during the retention test) increased with the increasing index of the weight of mice, which could be found after 1-h ITI (Fig. 5A) and 4-h ITI (Fig. 5B). There were positive correlations between mean score in novel arm and weight growth index for both 1-h ITI (r = 0.346, P < 0.05) and 4-h ITI (r = 0.353, P < 0.05). The linear regression function of 1-h-ITI for FR groups was: y=13.24x+23.03; and that of 4-h ITI was: y=10.10x+25.21. For these FR mice, 7
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no correlation could be found for the 2-min ITI (r = 0.030, P = 0.859). In addition, there was no correlation for the control group for either 2-min (r = 0.124, P = 0.700), 1-h (r = -0.021, P = 0.948) or 4-h ITIs (r = -0.016, P = 0.961).
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Figure 5
3.3. Acute FR diversely affected memory retention
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Acute FR led to significant differences in percentage time, number of arm visits and ambulation in the three arms with 2-min and 1-h ITIs (P < 0.001 for all). After 4-h ITI, the percentages of distance and number
and the percentage of time was close to significance (P = 0.059).
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of arm visits also showed significant difference among the three arms (distance, P < 0.05; number, P < 0.01),
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As shown in Fig. 6, the percentages of the three indices spent in the novel arm during testing were first compared with 33.3%. The control group and all FR groups had scores significantly higher than chance level
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for 1-3 indices after 2-min ITI (P < 0.05 for all, Fig. 6A-C left). After 1-h ITI, all except FR24 groups still showed higher scores than chance level for 1-3 indices (P < 0.05 for all, Fig. 6A-C middle). After 4-h ITI,
Figure 6
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novel arm (P < 0.05, Fig. 6A-C right).
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only the FR12 group showed significantly higher score than chance level on the percentage of number in the
In addition, correlation analysis was performed between mean score in novel arm of Y-maze and weight loss index for acute FR groups. No significant correlation could be found either after 2-min (r = -0.008, P = 0.963), 1-h (r = 0.171, P = 0.290) or 4-h ITI (r = 0.281, P = 0.079). For the control group, there were also no correlations between mean scores in novel arm and their weight changes (2-min ITI: r = 0.092, P = 0.800; 1-h ITI: r = -0.296, P = 0.406; 4-h ITI: r = 0.328, P = 0.355).
3.4. Acute but not chronic FR decreased locomotor activity Both chronic and acute FR had no effects on the total number of arm visits in the Y-maze (chronic FR: F(3, 49)=0.692, P = 0.561; acute FR: F(4, 49)=0.999, P = 0.418) (Fig. 7A-B). On the other side, the total ambulation distance was a direct index of the locomotor activity. Chronic FR has no significant effect on this index (F(3, 49)=0.838, P = 0.480). In contrast, acute FR decreased the ambulation of the mice (F(4, 8
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49)=6.293, P <) (Fig. 7C-D). Post-hoc LSD analysis revealed that all acute FR groups had significantly lower ambulation distances than the control group (P < 0.05; Fig. 7D). Figure 7
Discussion
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4.
In this study, we mainly found that: (1) both chronic and acute FR had no effect on the preference of
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mice for novelty in Y-maze; (2) severe chronic FR impaired the Y-maze performance, depending on FR intensity and individual tolerability of the FR, and acute FR affected the performance to some extent, either
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improving or impairing the retention scores; (3) acute FR reduced the locomotor activity in mice. The two-trial Y-maze task is a specific and sensitive test of spatial recognition memory in rodents (Dellu
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et al., 2000). We now explore the ambulation as an index for measuring memory of mice in the Y-maze. Compared to the other two indices, it displayed scores greater than 45% during test after 2-min and 1-h ITI, such as for the control mice (chronic FR: 59% for 2-min ITI and 48% for 1-h ITI; acute FR: 50% for 2-min
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ITI and 46% for 1-h ITI). In addition, in acute FR experiment, after 2-min ITI the FR36 group and after 1-h ITI the FR48 group showed higher percentages of ambulation than chance level, which were not observed in
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Y-maze task.
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the other two indices. Thus, the ambulation is a good supplement to the other two indices for this kind of
All chronic and acute FR groups exhibited a preferable level of novelty exploration. The result was
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consistent with the previous open field test revealing no difference in exploratory activity between the CR (e.g., 20%-40% CR) and the control (Cardoso et al., 2016; Wu et al., 2003) . These data showed that FR did not affect the preference of mice for novelty in Y-maze. Severe chronic FR impaired the retention of mice in the two-trial Y-maze tests. Significant correlation was found between the impairment and the slow weight growth induced by FR. The data suggest that chronic FR impairs spatial recognition memory, which is not only related to the FR dosage but also to the individual tolerability of FR. There are limited studies assessing the effects of FR in spatial tasks unrelated to rewards and punishments (Benau et al., 2014; Conrad, 2010). In one study, FR or shock as an unpredictable stress lasting for 4 weeks caused an impairment of spatial memory in rats (Orsetti et al., 2007). In addition, previous studies indicated impairment of chronic stress (e.g., chronic restraint up to 21 d) on spatial memory in rats (Conrad, 2010). Thus, severe chronic FR may serve as a significant stressor, which over time can affect memory of individuals. 9
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Acute FR led to diverse effects on the retention of the Y-maze test in mice. After 4-h ITI, the retention was kept only in FR for 12 h group, but not in the control and other FR groups. Studies with subtle FR, DR or CR have exhibited the enhancement effects (Ko et al., 2015). For example, 2 h of 40% DR for 5 consecutive days increased learning and memory in water maze in adult mice (Rajab et al., 2014). After 1-h ITI, however, acute FR for 24 h impaired the retention. This result is paradoxical because the negative effect
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was not observed on acute FR for longer durations (36-48 h). An important consideration was that the effect of acute FR is dynamic and reversible, which has been raised on changes in the hippocampus caused by
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many chronic stress paradigms (Conrad, 2010). In addition, 12-h FR difference among acute FR groups was involved with natural circadian rhythm. It has reported that the rodents have the ability to integrate spatial
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and temporal information (Lukoyanov et al., 2002), and the effects of FR on learning and memory could be photoperiod-dependent (Steinman et al., 2011). Taken together, acute FR has positive or negative effect on
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spatial recognition memory in mice, which may be dynamic and reversible, or circadian-rhythm-dependent. All mice used in this study were with ages less than 3 months old, which is at an early stage in life. Up to now, few studies were performed in developing rodents to investigate the effects of FR on cognition.
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These limited data, however, exhibit inconsistent results. For example, the male but not female mice at an age of 1 month were fed by 20%-35% DR for 3 months (Wu et al., 2002), or after weaning experienced DR
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for 6 months (Wu et al., 2003), displaying increased learning but not retention in a Y-maze. Herein, the
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Y-maze task is very different from ours, which has foot shocks in start and error arms. However, in female mice after weaning, 40% CR for 4 weeks improved cognitive performance in water maze (Kuhla et al.,
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2013). In the same paradigm, a recent study has reported that the male rats aged 4 weeks were 40% FR for 2 months, and their performance was impaired for spatial learning but not for memory retention (Cardoso et al., 2016) . The divergence may be due to different paradigms and some experimental variables, such as FR intensity and duration. Moreover, the effects of FR may be sex-dependent in developing mice. It will be very interesting in the future to investigate those effects of FR in both male and female animals. On the other side, for severe FR, the rats at an age of 30 days have received 75%-80% less chow than the control rats, and when tested in novel object recognition test after 4 and 14 weeks, their short- and/or long-term memory were improved (Molz et al., 2016). In our chronic study, the mice after weaning experienced 50%-70% FR for 6-7 weeks, displaying impaired recognition memory in the Y-maze test. Both novel object recognition task in that report and Y-maze in our study can test recognition memory basing on natural tendency of rodents to explore novelty, but the underlying neural mechanism may be different. Because it has been reported that object novelty recognition could be performed without an intact 10
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hippocampus, while in this case the Y-maze performance was impaired (Conrad, 2010). This may also explain that FR had no effects on preference of mice for novelty but had influenced their spatial recognition memory in our study. In rodents, the hippocampus is involved in spatial cognition processes (Morris et al., 1982). Previous studies have indicated that hippocampus is one of the brain regions that are more sensitive to the nutritional
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deficits (Cardoso et al., 2013; Hipolito-Reis et al., 2013). A number of previous studies suggest that FR (DR/CR) may lead to alterations of hippocampus (Benau et al., 2014; Conrad, 2010), which further
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contribute to changes of memory (Partadiredja and Bedi, 2011). For example, FR altered hippocampal CB1R expression and function in rats (Talani et al., 2016), and CR enhanced hippocampal neurogenesis and
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remote contextual fear memory in adult mice (Hornsby et al., 2016). Regarding the developing phase, it was published recently that young-onset CR could result in alterations of neurogenesis in the dentate gyrus,
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which may explain some impairment of spatial cognition (Cardoso et al., 2016). These findings also provide an explanation for the current results.
In the present study, the age of the mice approximates to humans less than 20 years old (Harrison, 2011).
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Previous studies have found that the volume of hippocampus has an inverted-U shape relationship with increasing age, which peaks before 20 years old (Krogsrud et al., 2014; Wierenga et al., 2014). At the
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corresponding age phase, we found that both chronic and acute FR treatment affected spatial recognition
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memory in mice, suggesting that FR may affect the key developmental stage of hippocampus since the brain region is essential to spatial cognition. Thus, our data indicate that adolescent animals are sensitive or
issue.
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vulnerable to FR treatment (Cardoso et al., 2016) and the FR routine in this age group should consider this
The ambulation was affected by the acute FR in this study, indicating the decreased locomotor activity. The decreased activity could not explain changes of the retention in the Y-maze, because the total number of arm visits showed no significant difference from the control. On the other hand, previous studies have found that food deprivation for 0-48 h had no effect on the distance traveled in an open field test (Dietze et al., 2016; Pierre et al., 2001). It was assumed that the duration of fasting positively correlates the response rate of animals (Moscarello et al., 2009). Therefore, the decrease of locomotion seems to be an inadequate response at the time when animals are suffering from hunger, which may be detrimental to their survival. However, previous data suggest that exploratory behavior is separable from seeking food and acquiring information associated with environmental aspects (Pierre et al., 2001). In addition, food deprivation for up to 48 h seemed not to alter anxiety or depression behavior (Dietze et al., 2016; Hendriksen et al., 2015). 11
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Thus, the underlying mechanism and function of the influence of acute FR on locomotor activity require further assessment. In conclusion, chronic FR impaired Y-maze spatial recognition memory in developing mice depending on FR intensity and individual tolerability of the FR, and acute FR positively or negatively affected the retention of the memory. Our data give new insights on the effects of FR on spatial cognition in developing
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mice.
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Funding This work was supported by the National Science Foundation of China (81560221, 81560234, 31571109, 31560317, 61502214, and 81460007), the Research Foundation of Kunming University of Science and Technology (KKZ3201560006), and the Natural Science Foundation of Yunnan Province of
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China (2016FB107).
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Acknowledgements We thank Prof. Yuanye Ma (Kunming Institute of Zoology, Chinese Academy of Sciences) for supervision at this project and thank Dr. Junhong Su (Medical Faculty, Kunming University of Science & Technology) for his help with this work. We are especially grateful to Dr. Qiuwei Pan (Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center) for his constructive revising of
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the manuscript.
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Wu, A., Wan, F., Sun, X., Liu, Y. (2002) Effects of dietary restriction on growth, neurobehavior, and reproduction in developing Kunmin mice. Toxicol Sci 70, 238-244.
Yanai, S., Okaichi, Y., Okaichi, H. (2004) Long-term dietary restriction causes negative effects on cognitive functions in rats. Neurobiol Aging 25, 325-332.
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Figure captions Fig. 1. Experimental workflow. (A) In chronic experiment, mice experienced food restriction (FR) from P25 (postnatal day 25) to P74, and three ITIs Y-maze tests were performed on P60, P67, P74, individually. (B) In acute experiment, mice experienced acute FR separately before the three ITIs Y-maze tests, which were performed on P49, P63 and P77. Please refer to the methods for details.
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Fig. 2. Schematic illustration of the 2-trial Y-maze tests. During the first trial (Training), the animal is allowed to explore only two arms of the maze, with the third arm being blocked by a door. During the second trial (Testing), the door is opened and all three arms are accessible.
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Fig. 3. Changes of body weights with time during chronic FR. All FR groups showed signicantly difference from the control group on P32 - P74 (P < 0.01 for all).
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Fig. 4. Differences from chronic FR groups in exploration of novelty after a 2-min (left), 1-h (middle) and 4-h (right) ITIs. Percentages of time (A), visits (B) and ambulation (C) made to the novel arm during the 5 min of the retrieval trial. Comparisons with chance (dotted line, 33.33%): ***P < 0.001; **P < 0.01; *P < 0.05. Comparisons between groups: #P < 0.05.
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Fig. 5. Relationships between changes on weights of mice after chronic FR and their retention performance in the novel arm after 1-h (A) and 4-h (B) ITIs. Weight growth index was calculated as the ratio of weights before and after FR. Mean score in the novel arm (%) was expressed as the average percentage of time, visits and ambulation spent in the novel arm during the retention test. The horizontal dotted lines represent the criteria of 40%, which is a common index of evaluating if mice exhibit a significant exploration of novelty in the two-trial Y-maze, and the vertical dotted lines represent the corresponding weight index from the linear regression functions. Pearson correlation analysis: *P < 0.05.
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Fig. 6. Differences from acute FR groups in exploration of novelty after a 2-min (left), 1-h (middle) and 4-h (right) ITIs. Percentages of time (A), visits (B) and ambulation (C) made to the novel arm during the 5 min of the retrieval trial. Comparisons with chance (dotted line, 33.33%): ***P < 0.001; **P < 0.01; *P < 0.05.
Fig. 7. Effects of chronic (A, C) and acute (B, dD) FRs on locomotor activity. (A) and (B) Total number of arm visits in the Y-maze during the retrieval trial; (C) and (D) Total ambulation in the Y-maze during the retrieval trial. Comparison with the control group: ###P < 0.001; ##P < 0.01; #P < 0.05.
Tables Table 1 Descriptive statistics of weight growth index after chronic FR in mice. Weight Ctrl FR30 FR50 growth index Mean SD N Mean SD N Mean SD
FR70 N
Mean
SD
N
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2-min ITI 1.64 0.19 12 1.53 0.20 13 1.37** 0.18 12 1-h ITI 1.96 0.23 12 1.60*** 0.22 13 1.37*** 0.18 12 4-h ITI 1.92 0.23 12 1.68* 0.24 13 1.37*** 0.18 11 * ** *** P ≤ 0.05, P ≤ 0.01 and P ≤ 0.001; comparisons with the control. P-values were calculated by Post hoc least significant difference (LSD) using ANOVA-1.
1.02*** 0.22 1.07*** 0.22 1.02*** 0.25
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Table 2
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0.93*** 0.03 0.86*** 0.03 0.87*** 0.02
N 10 10 10
Mean
SD
N
0.83*** 0.07 0.86*** 0.02 0.84*** 0.02
10 10 10
FR48 Mean
SD
N
0.83*** 0.04 0.82*** 0.03 0.83*** 0.01
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2-min ITI 1.05 0.03 10 0.98*** 0.02 10 1-h ITI 1.03 0.02 10 0.91*** 0.03 10 4-h ITI 1.02 0.02 10 0.92*** 0.02 10 *** P ≤ 0.001; comparisons with the control. P-values were calculated by post-hoc LSD using ANOVA-1.
FR36
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Descriptive statistics of weight loss index after acute FR in mice. Weight Ctrl FR12 FR24 loss index Mean SD N Mean SD N Mean SD
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Highlights ：
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We used a Y-maze task avoiding reward or punishment to test in developing mice. The levels of chronic and acute food restrictions were systematically varied.
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Severe chronic food restriction caused impairment on spatial recognition memory.
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The impairment significantly correlated with slow weight growth.
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Acute restriction diversely affected memory retention, positively or negatively.
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