Behavioral features of mice fed with a cholesterol-enriched diet： Deficient novelty exploration and unaltered aggressive behavior

Ekaterina Veniaminova， Raymond Cespuglio， Nataliia Markova，5， Niall Mortimer， Chi Wai Cheung， Harry W. Steinbusch， Klaus-Peter Lesch，， Tatyana Strekalova（?）

1Department of Neuroscience， School for Mental Health and Neuroscience， Maastricht University， Universiteitssingel 40， Maastricht 6200 MD， the Netherlands

2Institute of General Pathology and Pathophysiology， Russian Academy of Medical Sciences， Baltiyskaya 8， Moscow 125315， Russia

3Faculty of Medicine， Neuroscience research Center of Lyon， C. Bernard University， 8 Av. Rockefeller， Lyon 69373， France

4Department of Anatomy， Guangzhou Medical University， Guangzhou 511436， China

5Laboratory of Biomolecular Screening， Institute of Physiologically Active Compounds， Russian Academy of Sciences， Moscow Region， Russia

6Division of Molecular Psychiatry， Laboratory of Translational Neuroscience， Department of Psychiatry， Psychosomatics and Psychotherapy， University of Wuerzburg， Fuechsleinstrasse 15， Wuerzburg 97080， Germany

7Department of Anaesthesiology， the University of Hong Kong， Queen Mary Hospital， Hong Kong， China

Behavioral features of mice fed with a cholesterol-enriched diet： Deficient novelty exploration and unaltered aggressive behavior

Ekaterina Veniaminova1，2， Raymond Cespuglio3，4， Nataliia Markova1，2，5， Niall Mortimer6， Chi Wai Cheung7， Harry W. Steinbusch1， Klaus-Peter Lesch1，6， Tatyana Strekalova1（?）

1Department of Neuroscience， School for Mental Health and Neuroscience， Maastricht University， Universiteitssingel 40， Maastricht 6200 MD， the Netherlands

2Institute of General Pathology and Pathophysiology， Russian Academy of Medical Sciences， Baltiyskaya 8， Moscow 125315， Russia

3Faculty of Medicine， Neuroscience research Center of Lyon， C. Bernard University， 8 Av. Rockefeller， Lyon 69373， France

4Department of Anatomy， Guangzhou Medical University， Guangzhou 511436， China

5Laboratory of Biomolecular Screening， Institute of Physiologically Active Compounds， Russian Academy of Sciences， Moscow Region， Russia

6Division of Molecular Psychiatry， Laboratory of Translational Neuroscience， Department of Psychiatry， Psychosomatics and Psychotherapy， University of Wuerzburg， Fuechsleinstrasse 15， Wuerzburg 97080， Germany

7Department of Anaesthesiology， the University of Hong Kong， Queen Mary Hospital， Hong Kong， China

ARTICLE INFO

Received： 20 May 2016

Revised： 27 May 2016

Accepted： 31 May 2016

? The authors 2016. This article

is published with open access

at www.TNCjournal.com

Western diet；

cholesterol；

novelty exploration

aggression；

mouse

Objectives： Previous studies involving mice have demonstrated that a cholesterolenriched diet evokes liver steatosis， dystrophy， inflammation， and aspects of nonalcoholic fatty liver disease （NAFLD）. These changes are accompanied by the activation of pro-inflammatory brain and liver molecular pathways， as well as anxiety and depressive-like behaviors. Given previously reported evidence for the neurobiological relationship between the above-mentioned molecular changes and abnormalities in coping with environmental stimuli， such as interactions with other individuals and new environmental contexts， we hypothesized that novelty exploration and aggressive behavior are affected in a mouse NAFLD model.

Methods： To test this hypothesis， young female C57BL/6J mice were fed with a regular chow or a diet containing 0.2% cholesterol for 3 weeks. The mice were then assessed for new object and novel cage exploration， and social interaction in a food competition test.

Results： We found reduced object exploration in mice on the cholesterol-enriched diet. This reduction was not related to whether the new object was placed in an anxiogenic or non-anxiogenic environment. These changes were accompanied by diminished exploration of the new environment in a novel cage， and delayed approach to food after a period of food deprivation. Mice on the regular chow or cholesterolenriched diet showed no differences in aggressive behavior towards a counter-partner in a food competition test. Food intake and body weight did not differ between the groups， thus， excluding their potential as confounders in the measured behaviors.

Conclusions： We conclude that a diet enriched with cholesterol reduces novelty exploration irrespective of the anxiogenic level of the environment and does not induce aggressive behavior in female mice.

1 Introduction

The archetypal “Western diet”， a diet enriched with saturated fat and cholesterol， can lead to obesity and obesity-related outcomes such as hypercholesterolemia， insulin resistance， metabolic syndrome， and numerous other medical conditions. It is also associated with a high prevalence of affective disorders［1-3］. In humans， high trans-fat consumption has been associated with behavioral irritability and aggression［4］. Clinical studies suggest that a preference for a high-fat diet is connected to increased aggression and inflammation［4-6］. Rodent-based studies have also reported increased aggression levels after the administration of “Western diet” model food. In male mice and rats， a three-week exposure to a high-fat diet （43% kcal from fat vs. 16% in control diet） was found to potentiate aggression［7］. A potential developmental and cross-generational effect of diet was highlighted by the increased aggression， depressive-like behavior， and locomotor activity of the offspring of mice with a high intake of polyunsaturated fatty acids during pregnancy［8］. However， in monkeys， high fat and cholesterol intake was shown to decrease aggressive behavior［9］. Aggressive behavior can also be accompanied by reduced novelty exploration. Novelty exploration is closely associated with aggressive trait features， such as impulsivity and attention deficit［10-15］.

In addition to having a modulatory effect on aggression， the high intake of fat and sugars is known to induce metabolic syndrome， which has been linked to depressive symptoms. Some forms of metabolic syndrome have also been associated with increased rates of aggression and violent behavior［16-21］. Mice that have been experimentally induced to display depressive symptoms also show decreased novelty exploration， anxiety， and aggression［22， 23］. Multiple studies have shown that low-level novelty-seekers exhibit increased anxiety［24-28］， depression-like behaviors， and vulnerability to chronic stress［29， 30］. In summary， clinical and animal studies suggest a strong interrelationship between the symptoms of depression， aggression， and diminished interest in novelty exploration.

The neurobiological factors underlying such behavioral changes during exposure to the “Western diet” are believed to be associated with inflammatory factors in the central and peripheral nervous system［31-36］. For example， inflammation was shown to impair motivational responses to novelty in humans［37］and reduce novelty exploration in rodents［38， 39］. In humans， a relationship between inflammatory markers in both the plasma and cerebrospinal fluid， and aggression was recently reported［5， 6］. Interestingly， it has been found that dietary intake of anti-inflammatory n-3 fatty acids leads to lower hostility in young adulthood［40］.

In the present study， we used a recently established non-alcoholic fatty liver disease （NAFLD） model， in which young female C57BL/6J mice are exposed to a diet containing 0.2% cholesterol for 3 weeks. This NAFLD model has been shown to evoke behavioral despair， anxiety-like and impulsive behaviors， and upregulation of Toll-like receptor 4 in the brain and liver［36， 41， 42］. Given the substantial pro-inflammatory changes demonstrated in this paradigm， we hypothesized that aggressive behavior and novelty exploration could be altered in this model. To test this hypothesis， young female C57BL/6J mice were fed with regular chow or a cholesterol-enriched diet for 3 weeks and tested in new object and novel cage exploration tests， and social interaction in a food competition test.

2 Methods

2.1 Animals

Studies were performed using 3-month-old female C57BL/6J mice. Mice were obtained from RAS， Moscow， a provider licensed by Charles River （http：//www.spf-animals.ru/about/providers/animal）. C57BL/6J mice were housed five per cage during the study， under a 12-h light-dark cycle （lights on： 19：00） with food and water ad libitum， under controllable laboratory conditions （22 ± 1°C， 55% humidity）. Experiments were carried out in accordance with the European Communities Council Directive for the care and use of laboratory animals and were approved by the relevant local governmental bodies.

2.2 Study flow and dietary challenge

For three weeks prior to and throughout behavioral testing， twenty-six mice were exposed to either aregular laboratory diet （n = 12） with an energy content of 3.0 kcal/g （“Chara”， Agro， Moscow region， Russia） or to a diet with 0.2% （w/w） cholesterol （n = 14） and an energy content of 4.6 kcal/g （Research Diet Inc.， New Brunswick， NJ， USA， Figure 1a）. The content of other nutrients was similar between two diets （Figure 1b）. Behavioral testing included previously established protocols of novel cage and new object exploration testing on Day 21 of the experimental procedure and a food competition test on Day 26 that was prefaced with a 24 h period of food deprivation. Body weight was evaluated on Day 1， 9， and 16 of the study. Dietary intake was measured on Day 20 and 25 for each cage and recalculated per mouse per day.

Figure 1 Schematic of the study （a） and composition of the diets， % of total calories （b）.

2.3 Behavioral testing

2.3.1 Novel cage

The novel cage test was performed to assess exploration of a new environment. Mice were introduced into a standard plastic cage （21 cm × 27 cm × 14 cm）， under 5 Lux lighting. The number of exploratory rears was counted per minute during the 5-min test by visual observation， as described elsewhere［36， 43］.

2.3.2 New object exploration test

The detailed protocol for the new object exploration test has previously been described［44］. The apparatus for the new object exploration test consisted of a plastic cage （21 cm × 27 cm × 14 cm） which was placed near a wall corner at a height of 80 cm. Two identical objects made of plastic （? 2.5 cm， height 10 cm） were placed symmetrically 2 cm away from the cage wall and 30 cm from each other. Object close to the wall （in 2 cm away from the wall corner） was counted as being in the “non-anxiogenic” area while object far from the wall （in 32 cm away from the wall corner） was noted as being in the “anxiogenic” area， a suitable lighting level was used. During the test， a mouse was placed in the box at an equal distance from both objects and allowed to explore the area freely for 15 min. The duration of object exploration， which was defined as the mouse's nose being directed towards the object while it is situated at a distance ＜2 cm from the object， was scored off-line for each object and for both objects combined. The latency period until first exploratory act was also recorded.

2.3.3 Food competition test

This test was used to investigate aggressive behavior， which is induced in the context of a competition between two individual female mice， after 24 h of food deprivation， for a highly palatable food （a small piece of meat）. Two food-deprived mice from different cages and the same experimental group， were placed in a plastic cage （21 cm × 27 cm × 14 cm） which contained a piece of meat （2 g） for 10 min under suitable lighting （5 Lux）. The latency period until the first act of eating a piece of meat was recorded. The presence and number of attacks was used as a measure of aggressive behavior and the latency period until the first attack was noted.

2.4 Statistics

Data were analyzed using GraphPad Prism version 5.0 （San Diego， CA， USA） using the t-test， Mann- Whitney test， and Shapiro-Wilk normality test. The Mann-Whitney test was used in cases of non-normally distributed data. The level of significance was set at P ＜ 0.05.

3 Results

3.1 Body weight and food intake

Body weight and average food intake did not differbetween the groups （Day 1： t = 0.18， P = 0.856， Day 9： t = 0.00， P = 0.999， Day 16： t = 1.01， P = 0.319， t-test， and Days 16-20： t = 0.37， P = 0.746， Days 21-25： t = 0.66， P=0.576， t-test， Figures 2a and 2b）.

Figure 2 Mouse body weight （a） and average food intake （b） during the study. Control—standard diet， HCD—high-cholesterol diet. Data are shown as mean±SEM.

3.2 Effects of exposure to diet enriched with cholesterol on behavior in novel cage test

In the novel cage test， the number of rears per minute did not differ significantly between the groups （1st min： t = 0.45， P = 0.653， 2nd min： t = 0.53， P = 0.605， 3rd min： t = 1.48， P = 0.148， 4th min： t = 0.80， P = 0.428， 5th min： t = 1.61， P = 0.119， t-test， Figure 3a）. There was a trend of decreased total number of rears in the cholesterol-enriched diet group， but the difference was not statistically significant （t = 1.51， P = 0.073， t-test， Figure 3b）. Total number of rears during 3 to 5 min was significantly decreased in the diet-challenged group compared with the control group （t = 2.07， P = 0.024， t-test， Figure 3c）.

3.3 Effects of exposure to diet enriched with cholesterol on behavior in new object exploration test

In the new object exploration test， exploration time of the object in the “non-anxiogenic” area did not differ significantly between groups （t = 1.67， P =0.110， t-test， Figure 4a）. There was a strong trend of decreased exploration time for mice on the cholesterol-enriched diet when the object was placed in the “anxiogenic” area， but the difference was not statistically significant （t = 1.97， P = 0.061， t-test， Figure 4b）. A significant difference between the groups was found in the summarized time of exploration for both objects （t = 2.16， P = 0.042， t-test， Figure 4c）， with decreased exploration time in mice fed with the cholesterol- enriched diet compared with control mice. Latency of object exploration in the anxiogenic and non-anxiogenic areas did not differ between control and cholesterol- enriched diet groups （t = 0.08， P = 0.941 and t = 0.22， P = 0.828， respectively， t-test， Figures 4d and 4e）.

3.4 Effects of exposure to diet enriched with cholesterol on behavior in food competition test

In the food competition test， the latency of attack did not differ significantly between groups （U = 60.0， P ＞ 0.999， Mann-Whitney test）， as no attacks were observed during the test in either group. There was a significant difference in the latency of eating behavior， where the mice fed with the cholesterol-enriched diet showed an increased time before the first act of eating a piece of meat compared with control mice （U = 10.0， P = 0.0002， Mann-Whitney test， Figure 5a）. Averaged food intake after deprivation did not differ significantly between groups （t = 0.18， P = 0.880， t-test， Figure 5b）.

Figure 3 Effects of high-cholesterol diet on mouse behavior in the novel cage test： number ofrears per minute （a）， total number of rears （b），and summarized number of rears during 3 to 5 min （c）. Control—standard diet， HCD—high-cholesterol diet. *P≤0.05 vs. control group，t-test. Data are shown as mean±SEM.

Figure 4 Effects of high-cholesterol diet on mouse behavior in object exploration test： duration of object exploration in non-anxiogenic area （a） and in anxiogenic area （b）， total duration of object exploration （c）， latency of object exploration in non-anxiogenic area （d） and in anxiogenic area （e）. Control—standard diet， HCD—high-cholesterol diet. *P≤0.05 vs. control group， t-test. Data are shown as mean±SEM.

Figure 5 Effects of high-cholesterol diet on mouse behavior in food competition test： latency of eating behavior （a） and average food intake after food deprivation （b）. Control—standard diet， HCD—high-cholesterol diet. ***P≤0.001 vs. control group， Mann- Whitney test. Data are shown as median with interquartile range and mean±SEM.

4 Discussion

In this study， we showed that 3-week exposure of young female mice to a high cholesterol diet reduces novelty exploration in novel cage and new object exploration tests and delays approach to food after food deprivation. Reduced object exploration was not related to whether the new object was placed in an anxiogenic or non-anxiogenic environment， despite our previous experiments showing increased anxiety in O-maze and dark/light box tests in mice fed on the high cholesterol diet［36， 42］. Given previously demonstrated findings that the cholesterol-enriched diet used in this study evokes upregulation of Toll- like receptor 4 in the brain and liver［36， 42］， it could be suggested that mechanisms of neuroinflammation may underlie the changes in novelty exploration. This suggestion is supported by other work in mice that shows inflammation can impair new object exploration［39］and augment appetitive neophobia［38］. Clinical studies revealed that experimentally induced inflammation impairs motivational responses to novelty［37］. A similar mechanism of action in mice could potentially explain our finding of impaired novelty exploration.

In the current study， we found no induction of aggressive behavior in a food competition test in mice challenged with a cholesterol-enriched diet. Previously， it has been shown that both male and female mice of certain strains display competitive aggression when a pellet of standard food is placed in the cage after food deprivation［45］. Our unpublished observations on genetically modified mice with deficient serotonin synthesis revealed these effects as well （Costa-Nunes and Strekalova， unpublished data）. Since the food used in the induced aggression food test was unfamiliar， it is possible that the lack of aggressive behavior could be related to the neophobic response of animals to the unfamiliar food. Despite the expectation that palatable food can provoke greater competition between mice， no aggressive behavior was observed in the current experiment in either experimental group. Further studies are required to test the question of potentially altered aggression in mice fed on the cholesterol-enriched diet， using familiar food and animals of male gender. The latter factor can be of high relevance since female mice are known to be highly neophobic in comparison to males.

The importance of testing in male mice is supported by the literature， which reports that in rodent models， the effect of a high-fat diet on aggressive behavior can be found only in males［7］. Furthermore， female mice do not typically display aggressive behavior toward strangers except during the peripartum period［46-48］. In contrast， a diet enriched with cholesterol has limited effects on the measures of inflammation inthe liver and CNS of male mice， as compared with female mice［41］.

In summary， our work demonstrates that 3-week exposure to a cholesterol-enriched diet reduces novelty exploration， irrespective of the anxiogenic levels of the environment， and does not induce aggressive behavior in female mice. Further studies are required to address the neurobiological mechanisms of these changes and such studies are currently under way.

Acknowledgements

We acknowledge the important contribution of Dr. Elena Shevtsova （Institute of Physiologically Active Compounds）.

Conflict of interests

All contributing authors have no conflict of interests.

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Veniaminova E， Cespuglio R， Markova N， Mortimer N， Cheung CW， Steinbusch HW， Lesch KP， Strekalova T. Behavioral features of mice fed with a cholesterol-enriched diet： Deficient novelty exploration and unaltered aggressive behavior. Transl. Neurosci. Clin. 2016， 2（2）： 87-95.

? Corresponding author： Tatyana Strekalova， E-mail： t.strekalova@maastrichtuniversity.nl.

Supported by the European Community （EC： AGGRESSOTYPE FP7/No. 602805） and Russian Foundation for Basic Research （15-04-03602）.