Behavioral changes in male mice fed a high-fat diet are associated with IL-1β expression in specific brain regions

    Behavioral changes in male mice fed a high-fat diet are associated with IL-1β expression in specific brain regions Camila P. Almeida-Suhett, Alice Graham, Yifan Chen, Patricia Deuster PII: DOI: Reference:

S0031-9384(16)31063-0 doi: 10.1016/j.physbeh.2016.11.016 PHB 11550

To appear in:

Physiology & Behavior

Received date: Revised date: Accepted date:

3 May 2016 18 October 2016 18 November 2016

Please cite this article as: Almeida-Suhett Camila P., Graham Alice, Chen Yifan, Deuster Patricia, Behavioral changes in male mice fed a high-fat diet are associated with IL-1β expression in specific brain regions, Physiology & Behavior (2016), doi: 10.1016/j.physbeh.2016.11.016

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Behavioral Changes in Male Mice Fed a High-Fat Diet are Associated with IL-1 Expression in Specific Brain Regions

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Military and Emergency Medicine

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Consortium for Health and Military Performance

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Camila P. Almeida-Suhett1*, Alice Graham1, Yifan Chen1, Patricia Deuster1

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F. Edward Hébert School of Medicine Uniformed Services University of the Health Sciences 4301 Jones Bridge Road

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Bethesda, MD 20814, USA

*Corresponding Author:

Camila P. Almeida-Suhett, PhD

Military and Emergency Medicine

Consortium for Health and Military Performance F. Edward Hébert School of Medicine Uniformed Services University of the Health Sciences 4301 Jones Bridge Road Bethesda, MD 20814 Phone: (301)295-1244 Fax: (301)295-5914 Email: [email protected] 1

ACCEPTED MANUSCRIPT ABSTRACT High-fat diet (HFD)-induced obesity is associated with not only increased risk of

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metabolic and cardiovascular diseases, but cognitive deficit, depression and anxiety disorders.

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Obesity also leads to low-grade peripheral inflammation, which plays a major role in the development of metabolic alterations. Previous studies suggest that obesity-associated central

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inflammation may underlie the development of neuropsychiatric deficits, but further research is

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needed to clarify this relationship. We used 36 male C57BL/6J mice to investigate whether chronic consumption of a high-fat diet leads to increased expression of interleukin-1 (IL-1) in

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the hippocampus, amygdala and frontal cortex. We also determined whether IL-1 expression in those brain regions correlates with changes in the Y-maze, open field, elevated zero maze and

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forced swim tests.

After 16 weeks on dietary treatments, HFD mice showed cognitive impairment on the Y-

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maze test, greater anxiety-like behavior during the open field and elevated zero maze tests, and increased depressive-like behavior in the forced swim test. Hippocampal and amygdalar expression of IL-1 were significantly higher in HFD mice than in control mice fed a standard diet (SD). Additionally, hippocampal GFAP and Iba1 immunoreactivity were increased in HFD mice when compared to SD controls. Cognitive performance negatively correlated with level of IL-1 in the hippocampus and amygdala whereas an observed increase in anxiety-like behavior was positively correlated with higher expression of IL-1 in the amygdala. However, we observed no association between depressive-like behavior and IL-1 expression in any of the brain regions investigated. Together our data provide evidence that mice fed a HFD exhibit cognitive deficits, anxiety and depressive-like behaviors. Our results also suggest that increased expression of IL2

ACCEPTED MANUSCRIPT 1 in the hippocampus and amygdala may be associated with the development of cognitive

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deficits and anxiety-like behavior, respectively.

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ACCEPTED MANUSCRIPT KEYWORDS

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 High-fat diet  Hippocampus  Amygdala  Cognitive deficit  Anxiety-like behavior  Interleukin-1

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ACCEPTED MANUSCRIPT 1.

INTRODUCTION Dietary fat intake has significantly increased from 1991 through 2008 [1] and obesity has

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almost doubled since 1980 [2]. In addition to increasing the risk of metabolic syndrome (Type II

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diabetes, hyperlipidemia, hypercholesterolemia and hypertension), obesity has been associated with deficits in executive function and memory [3-8] and development of psychiatric conditions,

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such as anxiety disorders [9-12] and depression [9, 13-16]. In rodents, diet-induced obesity leads

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to deficits similar to those observed in humans. Mice fed a high-fat diet develop metabolic syndrome [17-20] and display signs of learning and memory impairment [21], as well as

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depressive- [22-25] and anxiety-like behavior [26-29].

Evidence from rodent studies show that intake of a high-fat diet leads to disruption of

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cerebral vascular function [30, 31], activation of microglia and astrocytes [32, 33], and increased expression of cytokines [34, 35] in different brain regions. The hippocampus, which serves a

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major role in learning and memory, seems particularly susceptible to high-fat diet-induced functional and morphological changes [34-36]. Furthermore, consuming a high-fat diet chronically impairs expression of long-term potentiation [37, 38] and disrupts hippocampal neurogenesis [38-40]. Existing data suggest that hippocampal inflammation is the most probable mechanism responsible for cognitive changes in animals consuming high-fat diets [36]. Nonetheless, more evidence is needed to elucidate the relationship between neuroinflammation and high-fat diet-induced behavioral alterations. The amygdala is a collection of several nuclei located deep within the temporal lobe, involved in the processing and modulation of emotional behavior [41-43], and serves a particularly important role in the development of anxiety disorders. Neuroimaging studies show that amygdalar hyperactivity is frequently observed in patients with such psychiatric conditions

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ACCEPTED MANUSCRIPT [44]. Increased anxiety-like behavior in rodents has also been associated with inflammatory responses in the amygdala [45-49]. Despite increasing evidence in the literature showing an

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association between anxiety-like behavior in rodents and overconsumption of fat, few studies

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have investigated the impact of a high-fat diet on the amygdala [50-53]. To the best of our knowledge, no study to date has investigated whether changes in anxiety behavior induced by a

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high-fat diet are associated with expression of inflammatory markers in the amygdala.

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The purpose of the current study is to investigate the impact of chronic consumption of a high-fat diet on behavioral changes associated with emotional and cognitive dysfunction and

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expression of the pro-inflammatory cytokine - interleukin-1 (IL-1) - in different brain regions. Cognitive deficits in leptin receptor-deficient db/db mice have been accounted for by elevated

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levels of IL-1 in the hippocampus [54]. Here, we sought to determine whether levels of IL-1 in the hippocampus and amygdala correlate with working memory deficit and anxiety-like

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behavior, respectively in male mice fed a high-fat diet. We found that after consuming a high-fat diet for 16 weeks, mice developed signs of cognitive deficit and depressive and anxiety-like behaviors. Levels of IL-1 in the hippocampus and amygdala correlated with cognitive deficit. In addition, development of anxiety-like behavior was associated with increased expression of IL-1 in the amygdala. Moreover, hippocampal expression of GFAP and Iba1were significantly increased in HFD mice. Our results suggest that neuroinflammation following chronic consumption of fat might contribute to the development of behavioral alterations in mice.

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ACCEPTED MANUSCRIPT 2.

METHODS

2.1. Animals and dietary treatments

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Male C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) were purchased at 5 weeks

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of age. Upon delivery they were housed in pairs in an environmentally controlled room (2023ºC, ~44% humidity, 12-h light/12-h dark cycle [350-400 lux], lights on at 6:00 am) with food

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and water available ad libitum at the Uniformed Services University of Health Sciences. They

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were acclimated to the environment for 4 days with standard laboratory diet (Harlan Teklad Global Diet 2018, 18% protein rodent diet; Harlan Laboratories; Indianapolis, IN) prior to

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beginning the specialized dietary treatments. Mice were then randomly assigned to receive one of two dietary treatments (n = 24/group): standard diet (STD – Harlan Teklad Global Diet 2018,

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18% protein rodent diet) or high-fat diet (HFD – TD.06414, Harlan Laboratories; Indianapolis, IN). Nutritional information for each diet is summarized in Table 1. The animals were weighed

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at the start of their specialized dietary treatments and then weekly throughout the remaining 18 weeks. Behavioral testing was conducted on the 16th week; in week 17, a glucose tolerance test was performed. At the end of the 18th week, all 36 mice were euthanized for tissue collection. All procedures and experiments were conducted with approval and oversight of the Institutional Animal Care and Usage Committee of the Uniformed Services University of the Health Sciences. 2.2. Behavioral Tests On completion of the 16th week of dietary treatment 36 mice (18 per group) underwent behavioral testing. The tests were performed in the order presented bellow and all mice performed all 4 behavioral tests.

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ACCEPTED MANUSCRIPT 2.2.1. Open Field On the first day of behavioral testing, anxiety-like behavior was assessed in each mouse

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by using an open field apparatus (40 cm x 40 cm) with black opaque walls (Stoelting, Wood

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Dale, IL) [55, 56]. Each mouse was placed individually in the center of the open field and locomotor activity was recorded for 20 minutes by an overhead camera linked to a computer

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with ANY-maze behavioral tracking software (Stoelting, Wood Dale, IL). The first 10 min of

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each session were for acclimation; data collected during the remaining 10 min were used for assessment of anxiety-like behavior. Total movement time, total distance traveled, average

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speed, percent mobile time spent in the center, and number of center entries in the open field were analyzed. The center is defined as a 20 cm x 20 cm area located 10 cm away from each

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wall. An entry was counted when the animal head and 80% of its body were within the area delineated as center. Anxiety-like behavior was assessed by quantification of the percentage of

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time spent in the center and the number of entries in the center. 2.2.2. Elevated Zero Maze

One hour after completion of open field experiments, anxiety-like behavior was also assessed using an elevated zero maze (50 cm inner diameter, 5 cm wide track, 50 cm from the floor; Stoelting, Wood Dale, IL) test [57]. The maze is a circular apparatus equally divided in four quadrants, two of which have high, opaque walls. Each mouse was individually placed at the edge of one of the high wall sections of the maze, facing the inside of the quadrant. The mouse was then allowed to explore the maze freely for 5 min. Animal activity was automatically tracked by an overhead camera linked to a computer with ANY-maze software. The number of entries and time spent in the non-high wall quadrant were used for assessment of anxiety-like behavior. An entry was counted when the head and all four legs of the animal were in the non-

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ACCEPTED MANUSCRIPT high wall quadrant. On completion of elevated zero maze test, mice were returned to their cages and rested for 2 days before next behavioral experiments.

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2.2.3. Y-maze

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Spontaneous alternation in the Y-maze was used as a hippocampal-dependent test of working memory [54, 58, 59]. The Y-maze apparatus (Stoelting, Wood Dale, IL) consists of

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three closed arms, 50 cm long, 11 cm wide and 10 cm high, made of opaque Plexiglas, connected

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to each other in the shape of a Y. Visual cues placed in the maze walls of each arm were kept constant throughout the testing sessions. Testing sessions were 5 min long and initiated by

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placing the subject into one of the three maze arms. The animal was allowed to freely explore the maze for 5 min while an overhead camera linked to a computer with ANY-Maze software

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automatically recorded its activity. An arm entry was counted when the head and all four legs of the animal were inside an arm. An opportunity was tallied every three entries, and a perfect

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alternation was considered as entry into all three arms given one opportunity. Results were calculated as the number of perfect alternations divided by total opportunities. 2.2.4. Forced Swim Test

One hour after completion of Y-maze experiments, mice were submitted to the Porsolt forced-swim test (FST) to assess depressive-like behavior [60-62]. Each mouse was placed in a cylinder filled with warm water (10 cm deep, 251 C) and allowed to move freely within the cylinder. Any period longer than 1,500 milliseconds without movement at 65% sensitivity was considered immobile time. A camera recorded animal activity for 6 min. The initial 2 min was for acclimation. During the remaining 4 min, immobility time was automatically scored using ANY-maze software. Increased immobility time has been proposed as a behavioral reflection of despair and used as measure for depressive-like behavior in mice [60-62].

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ACCEPTED MANUSCRIPT 2.3. Glucose Tolerance Test Upon completion of behavioral tests animals rested for at least 3 days. In the 17th week

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after initiation of dietary treatments they underwent glucose tolerance testing after overnight

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fasting. Animals were transferred to a clean cage containing no food or feces but with access to water at all times for 16 hours prior to test. Blood sample were collected from a single wound

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made by cutting the tip of the tail. The first 4 drops of blood were discarded and the 5th one was

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used to measure glucose level by using a commercial glucometer kit (AlphaTRAK, Abbott Animal Health) at baseline and five time points after glucose administration (15, 30, 60, 90, 120

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min). Mice were injected intraperitoneally with glucose 20% in sterile saline for a final dose of 2 mg of glucose/g of body weight. Upon completing glucose tolerance testing, each mouse was

2.4. Blood Collection

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placed in a new, clean cage with free access to water and the diet they were originally fed.

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Blood collection was performed as described by Islam et al. [63], one week after the glucose tolerance test. Once mice were fully anesthetized by using isoflurane (2.5%), their carotid artery was exposed and blood was collected through a small incision in the artery by using a heparin-primed cannula leading to a heparin-primed tube. Blood samples were centrifuged immediately and then plasma was transferred to clean tubes, snap-frozen on dry ice and stored at -80 ºC until the day of analysis.

2.5. Brain Dissection Upon completion of blood collection, mice were decapitated. Bilateral dissection of frontal cortex, hypothalamus, amygdala and hippocampus was modified from [64]. Brain was placed on a cold metal plate and after removal of olfactory bulb, 2 mm of the frontal cortex was

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ACCEPTED MANUSCRIPT collected using a blade. Frontal cortex was then dissected from the middle dark area with the use of a tissue corer, transferred to a microcentrifuge tube and snap-frozen on dry ice. Next,

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hypothalamus was dissected from the ventral surface of the brain by sliding a thin curved

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tweezers around it. Hypothalamus was then stored and frozen as described for frontal cortex. Brain was mounted to the tissue slicers (Vibratome, 1000 Plus) and 500 µm-thick slices were cut

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using a tissue slicer in ice-cold cutting solution consisting of (in mM): 125 NaCl, 2.5 NaCl, 1

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CaCl2, 2 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, 25 D-Glucose. Amygdala was dissected by cutting a triangle-shaped piece of tissue using a scalpel blade. One side of the triangle was cut from the

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end of the external capsule to the optical tract and the other side was cut from the end of the external capsule on a straight line excluding the piriform cortex. The hippocampus was then

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dissected by detaching it from the surrounding tissue. Amygdala and hippocampus were transferred separately to microcentrifuge tubes and then snap-frozen on dry ice. Samples were

2.6. ELISA

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stored at -80 ºC until the day of analysis.

Brain-tissue cytokine levels and plasma levels of cytokines and adipocytokines were measured using enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems (Minneapolis, MN). Frozen brain tissue were homogenized in CelLytic™ MT (Sigma-Aldrich St. Louis, MO) supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO) at a 1:10 ratio of cold lysis buffer to tissue weight. After centrifugation for 10 min at approximately 14,000 x g and 4 ºC, the supernatants were transferred to new tubes and stored at -80 ºC for further analysis. Levels of IL-1 in the hippocampus, amygdala, frontal cortex and hypothalamus were quantified following manufacture’s instructions and normalized to total protein in each

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ACCEPTED MANUSCRIPT sample, which was determined using the Lowry assay (Bio-Rad, Hercules, CA). The intra-assay coefficient of variation (CV) was 9.12% and the inter-assay CV was 10.3%.

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2.7. Immunohistochemistry

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2.7.1. Fixation and Tissue Processing

One week after the glucose tolerance test, 6 mice per group were deeply anesthetized

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using isofurane (2.5%) and transcardially perfused with phosphate buffered saline (PBS, 20 mL)

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followed by 4% paraformaldehyde (50 mL). Brains were removed, post-fixed in 4% paraformaldehyde overnight, and routinely processed in paraffin for immunohistochemistry.

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Coronal sections containing the hippocampus and amygdala were cut at 5 m thickness in a 50 m interval.

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2.7.2. GFAP and Iba1 Immunohistochemistry Immunohistochemical staining was performed as previously described [65]. Briefly,

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paraffin sections mounted on charged glass slides were deparaffinized and rehydrated using xylene and decreasing series of ethanol. Following, antigen epitopes were retrieved at 90 – 97 ºC for 30 min in an acidic solution (R&D Systems, Mineapolis, MN). Brain slices were incubated in a blocking solution containing 10% normal goat serum (Millipore Bioscience Research Reagents, Temecula, CA), 1% BSA and 0.03% Triton X-100 in PBS for 1 hour at room temperature. Sections were then incubated with anti-GFAP (glial fibrilary acid protein, 1:500, AbCam, Ab4674, Cambridge, MA) and anti-Iba1 (ionized calcium binding adaptor molecule 1, 1:500, Wako, Richmond, VA) antibodies diluted in PBS overnight. After rinsing three times for 10 min each in PBS, the sections were incubated with Alexa Fluor 594-conjugated goat antirabbit (1:1000, Jackson ImmunoResearch, West Grove, PA) and Alexa Fluor 488-conjugated donkey anti-chicken (1:1000, Jackson ImmunoResearch, West Grove, PA) antibodies in PBS for

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ACCEPTED MANUSCRIPT 1 hour at room temperature. After rinsing three times for 10 min each in PBS, slides were coverslipped with ProLong Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA).

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2.7.3. Image Quantification

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Image quantification of GFAP and IBA1 stained cells was performed using ImageJ software as previously described [66]. Fluorescent images were acquired using a Leica AF6000

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microscope under a 20X objective. A total of 10 pictures of the hippocampus and 5 pictures of

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the amygdala were taken for each animal. All pictures were taken using the same camera setting and exposure time for each channel and 8-bit images were used for quantification. After a fixed

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background subtraction (Rolling Ball Radius: 50.0 pixel), we applied a fixed threshold to all images. Following, the labeled area was quantified and averaged for each animal.

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2.7. Statistical Analysis

Statistical values are presented as mean ± standard error (SE). Results from standard diet

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and high-fat diet animals were compared using independent t-test. Multiple comparisons between weight gain tertiles were performed using ANOVA followed by Bonferroni post-hoc test. The relationships between different variables were determined using Pearson’s correlation, Statistical significance for all statistical analysis was considered when p < 0.05 (two-tailed). Sample sizes (n) refer to the number of mice.

3.

RESULTS

3.1. High-fat diet increases body weight gain We have previously compared the effects of a standard chow diet and a low-fat purified ingredient diet on physiological, metabolic and behavioral alterations (unpublished data). Because we found that chow diet and a low-fat diet have similar impact on weight gain, glucose

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ACCEPTED MANUSCRIPT intolerance, cytokine expression and behavioral alterations we decided to use regular chow as the control diet in the present study. The average body weight of animals in each diet group did not

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differ at arrival (19.28 ± 0.29 g for SD animals, 19.8 ± 0.28 g for HFD animals). Animals were

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then weighed weekly. Differences in body weight between the two groups increased over time [F(1,46)=64.86, p<0.001; Figure 1A]. Average body weight did not differ significantly between

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SD and HFD mice until the end of week 7 after starting dietary treatments, but HFD mice

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became significantly heavier in weeks 8 – 18. When the behavioral experiments were performed at week 16, average body weight was 31.3  0.45 g for SD animals and 41.5  1.0 g for HFD

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animals (p<0.0001). Over the course of the study, HFD animals gained significantly more weight

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(24.6  1.0 g) than SD mice (12.3  0.4 g; p<0.0001; Figure 1B).

3.2. High-fat diet induces glucose intolerance in mice.

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Consistent with previously published data [17-20], mice fed a high-fat diet had higher baseline glucose (14.89  0.3 mmol/L) compared to mice fed a standard diet (11.12  0.4 mmol/L; p < 0.0001). Additionally, HFD mice had a larger area under the glucose tolerance curve (3,787  126 mmol/L120 min) than SD mice (2,444  68 mmol/L120 min, p < 0.001; Figure 2).

3.3. Cognitive performance (Y-maze) We next examined whether long-term high-fat feeding affects cognitive performance. Specifically, we used the Y-maze sequence test to assess working memory [58]. Perfect alternation, counted every time an animal entered all three arms of the maze sequentially, was expressed as a percentage of the total opportunities taken by each animal in a 5-min test period. 14

ACCEPTED MANUSCRIPT HFD animals performed fewer perfect alternations (54.5  3.3) when compared to SD (68.3  2.3; p<0.01; Figure 3). However, the total distance traveled did not differ between SD (5.19 

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0.38) and HFD (4.98  0.35) groups.

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3.4. Anxiety-like behaviors (open field, elevated zero maze)

Anxiety-like behavior was assessed in mice at week 16 by using the open field apparatus.

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Animals fed a high-fat diet spent less time moving in the center of the field (6.6  0.6 % mobile

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time in the center; Figure 4A) and entered the center area fewer times (23.8  1.4; Figure 4B) compared to those fed with a standard diet (9.2  0.9 % mobile time in the center; 29.1  1.9

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center entries; p<0.05). SD mice also moved at a higher average speed (0.052  0.002 m/s;

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Figure 3C) when compared to HFD mice (0.043  0.001 m/s; p<0.05). Although SD animals

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consistently traveled a longer distance (30.7  1.2 m; Figure 4D) compared to HFD mice (26.66  1.15 m; p<0.05), animals in the two groups had similar movement times (447.7 ± 8.6 sec for SD mice, 424.2 ± 10.4 sec for HFD mice; Figure 4E). Anxiety-like behavior in animals fed a high-fat diet was further assessed using the elevated zero maze (EZM) test. During the 5-min EZM testing session, SD mice traveled similar distance (8.56 ± 0.4 m) compared to HFD mice (7.59 ± 0.5 m). Consistent with anxiety-related behavioral alterations observed in the open field test, HFD mice entered fewer times (6.0  0.6; Figure 5A) and spent less time in the open zone (61.1  6.5 sec; Figure 5B) compared to SD mice (9.2  0.6 entries; p<0.001; 84.6  6.2 sec; p<0.05).

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ACCEPTED MANUSCRIPT 3.5. Depressive-like behavior (forced swim test) The forced swim test (FST) was used to assess depressive-like behavior in SD and HFD

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mice. Immobility time was recorded for each animal as a measure of behavioral despair. Animals

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fed with a high-fat diet showed a significantly higher immobility time (165.6  4.4 sec; Figure 6)

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in the FST test when compared to those fed with a standard diet (138.7  6.6 sec; p<0.01).

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3.6. Expression of IL-1 in the hippocampus and amygdala

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We next used ELISA to determine whether long-term high-fat diet feeding contributes to increased expression of pro-inflammatory cytokines in the hippocampus, amygdala, frontal

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cortex and hypothalamus. Functional and morphological changes in these specific regions

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function relate either directly or indirectly to cognitive impairments and development of anxiety and depressive-like behaviors [21, 58, 67-69]. We could not quantify levels of interleukin-6 (IL-

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6), tumor necrosis factor- (TNF-) and C-C motif ligand-2 (CCL2) in all brain regions examined, in both SD and HFD mice. It is likely that these cytokines/chemokine were expressed at levels below the detection limit of the assays. However, we found that expression of IL-1 in the hippocampus of HFD animals was significantly increased (18.2  2.4 pg/mg; Figure 7A) when compared to SD animals (7.7  1.2 pg/mg; p<0.05). Similarly, amygdalar expression of IL1 was higher in HFD (19.7  3.9 pg/mg; Figure 7B) than in SD mice (9.3  1.8 pg/mg; p<0.05). Expression of IL-1 was higher in the frontal cortex (20.8  9.1 pg/mg; Figure 7C) and hypothalamus (35.6  15.3 pg/mg; Figure 7D) of animals fed a high-fat diet when compared to those fed a standard diet (9.3  2.1 pg/mg for frontal cortex; 10.6  2.8 pg/mg for hypothalamus); however, this increase was not statistically significant.

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ACCEPTED MANUSCRIPT We used correlation analyses to look for associations between the level of IL-1 in different brain regions and behavioral outcomes (Table 2). Cognitive performance in the Y-maze

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test measured as % perfect alternation negatively correlated with levels of IL-1 in the

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hippocampus (r = -0.56; r2 = 0.32, p < 0.001) and amygdala (r = -0.59; r2 = 0.35, p < 0.01).

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Anxiety-like behavior, evaluated by the number of entries in the center of the open field and in the open zone of the elevated zero maze, was associated with levels of IL-1 in the amygdala (r

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= -0.43; r2 = 0.18, p < 0.05 for entries in the center of the open field; r = -0.48; r2 = 0.23, p < 0.05

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for entries in the open zone of EZM). Immobility time in the forced swim test did not correlate with levels of IL-1 in any brain region examined in this study (data not shown).

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3.7. Quantification of GFAP+ and Iba-1+ stained area in the hippocampus and amygdala Immunofluorescence was used to assess reactive astrocytes and microglia in the

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hippocampus and amygdala (Figure 8). Quantification of the stained area in the hippocampus revealed that GFAP expression is significantly increased in mice fed the HFD (HFD: 119.9  16.9 vs. SD 69.9  6.3 m2; p < 0.05; m2) compared to mice fed the SD (Figure 8A, B, C). Consistently the hippocampal Iba1 stained area in HFD mice (HFD: 275.5  24.2 vs SD: 205.8  21.1 m2; p < 0.05) was increased relative to SD mice (Figure 8D, E, F). Quantification of stained areas in the amygdala of mice fed a HFD did not reveal any significant difference in the expression of GFAP (43.0  3.4) or Iba1 (314.6  54.2) compared to mice fed the SD (GFAP: 32.6  7.2 m2; Iba1: 254.9  47.8 m2; Figure 8G, H, I, J, L, M).

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ACCEPTED MANUSCRIPT 3.8. Effect of differential weight gain on metabolic and behavioral outcomes in the HFD group

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Lastly, we sought to distinguish between the effects of chronic consumption of HFD and

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increased weight gain and associated metabolic alterations. We observed that on average 30% of all mice fed the HFD did not gain excessive weight over the course of the study. A tertile

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analysis of mice within the HFD group according to their weight gain yielded three groups of

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equal sample size (n = 8): 1 = low weight gain (19.7 ± 0.73 g); 2 = mid weight gain (23.5 ± 0.5 g); and 3 = high weight gain (30.3 ± 1.4 g). Multiple comparison analyses of all variables across

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the three groups showed group 3 gained significantly more weight than group 1 (p < 0.0001) and 2 (p < 0.001; Table 3). Similarly, group 2 gained significantly more weight than group 1 (p <

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0.05). A correlation analysis across weight gain and all the other variables in the HFD group indicated weight gain correlated with area under the glucose tolerance curve (AUCglucose) (r = -

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0.53; r2 = 0.28, p < 0.01; Table 4) and number of entries in the open zone of the EZM (r = -0.59; r2 = 0.35, p < 0.01; Table 3). We did not find any other association between weight gain and the additional variables (data not shown).

4.

DISCUSSION

The contribution of dietary fat to alterations in behavior and inflammation is important given the high prevalence of obesity and metabolic disorders. Moreover, relationships between obesity, high fat diets, and deficits in executive function and memory are of great interest. In the present study we investigated the effects of chronic high-fat feeding on behavioral alterations and development of neuroinflammation in mice. Our data indicate that chronic high-fat feeding results in a decline in cognitive performance and an increase in anxiety and depressive-like

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ACCEPTED MANUSCRIPT behaviors in male mice. This was accompanied by, and may be the result of, increased expression of pro-inflammatory cytokines in the hippocampus and amygdala. Additionally,

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hippocampal expression of IL-1 negatively correlates with cognitive performance. Importantly,

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tests are associated with levels of IL-1 in the amygdala.

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we report, for the first time, that anxiety-like behaviors in the open field and elevated zero maze

In humans, body mass index and waist circumference, indicators of obesity, are inversely

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associated with learning, memory and executive functioning [3-8]. Although we cannot easily define obesity in rodents, mice fed a HFD, where 60% of the total energy is from fat, gain

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excessive weight over time and develop glucose tolerance, hyperinsulinemia, hyperleptinemia, hyperlipidemia and hypercholesteromia, which mimic human metabolic alterations associated

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with obesity [17-20]. Consistently, in the present study mice maintained on HFD for 16 weeks consistently gained excessive weight and developed glucose intolerance. Substantial evidence

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indicates that diet-induced obesity leads to deficits in learning and memory processes in rodents [34, 35, 70-73]. Here, we found that mice fed a HFD display signs of impaired working memory performance. Although we cannot directly compare the design and outcome of tests used to assess cognition in humans and rodents, our study is consistent with data showing that obesity is associated with poorer working memory in humans [3-6]. In the present work we focused on the effects of chronic high-fat feeding on working memory performance assessed by spontaneous alternation behavior (SAB) [58], rather than on learning and memory acquisition, consolidation and retrieval. Studies investigating the effects of HFD on SAB in rodents have had mixed results. Short-term exposure (one week) of C57BL/6 mice to 60% HFD resulted in decreased spontaneous alternation [74], whereas Long-Evans rats fed either a “Western Diet” or 60% HF diet did not develop any impairment in the Y-maze test

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ACCEPTED MANUSCRIPT [75]. Another study has shown that long-term (10 – 12 weeks) feeding with high fat/high fructose diet did not alter SAB in C57BL/6J male mice [76]. Our data indicate that long-term (16

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that chronic consumption of HFD impairs working memory.

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weeks) exposure to a HFD significantly reduced SAB in male C57BL/6J mice, which suggests

Working-memory performance in the Y-maze test requires activation of networks in

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different brain regions including hippocampus, septum, basal forebrain, prefrontal cortex and

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cerebellum [21, 58]. In particular, hippocampal inflammation has been previously associated with changes in the Y-maze performance [54, 77, 78]. Moreover, among all the overexpressed

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pro-inflammatory cytokines, IL-1 has been suggested as a correlate for adiposity and cognitive deficits in leptin receptor-deficient mice. Intra-hippocampal injection of the IL-1 receptor

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antagonist (IL1ra) completely prevented the development of cognitive impairments in db/db mice [54]. Here, we aimed to investigate whether HFD-induced working memory deficits in the

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Y-maze test would be associated to increased expression of IL-1 in the hippocampus. We observed that hippocampal expression of this cytokine was significantly increased in mice fed a high-fat diet. Consistent with this finding, immunoreactivity for GFAP and Iba1, markers for astrocytes and microglia, respectively [66], were significantly increased in the hippocampus. Astrocytes and microglia induce cytokines synthesis and become hypertrophic in response to chemical and/or physical to the brain [79]-[80]. Together, increased GFAP and Iba1 immunoreactivity and elevated expression of IL-1 in the hippocampus indicate that chronic HFD intake leads to neuroinflammation. Moreover, hippocampal levels of IL-1 levels negatively correlated with SAB in the Y-maze. Our data therefore is in line with previous studies suggesting that increased expression of IL-1 in the hippocampus may be one underlying mechanism for the cognitive deficits associated with obesity [54]. 20

ACCEPTED MANUSCRIPT The relationship between obesity and anxiety in humans is still unclear. Although some studies report a strong association between these two conditions [9, 10], some researchers report

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no special linking [81-83]. Rodent studies, however, consistently show that anxiety-like

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behaviors are increased in two major models of obesity: genetic [84, 85] and HFD-induced [2629] models. The present study likewise shows that mice fed a HFD for 16 weeks display clear

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signs of anxiety-like behavior in the open field and elevated zero maze tests.

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The amygdala plays a major role in the pathogenesis of anxiety [41-43]. Functional and pathological alterations in the amygdala have often been identified as the etiology of anxiety

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[67]. One study in particular showed that LPS-induced alterations in amygdalar neuronal activity (assessed by intracerebral electroencephalogram recordings and c-Fos expression) and

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expression of IL-1 were associated with increased anxiety-like behavior of freely moving rats in the open field [46]. Although the anxiogenic effect of IL-1 has been extensively reported in

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the literature [45-49], whether anxiety-like behavior in animals fed a HFD is associated with increased expression of IL-1 in the amygdala has not been determined yet. Here, we show for the first time that IL-1 levels are increased in the amygdala of mice fed a HFD and correlate with anxiety-like behaviors in the open field and elevated zero maze tests. Additionally, increased amygdalar expression of IL-1 also correlated with reduced alternation in the Y-maze test. A direct involvement of the amygdala in the modulation of SAB has not been previously reported. However, it is possible that amygdalar pathophysiological changes affect hippocampaldependent behavioral tasks due to functional and physical connections of both brain regions. Unlike what we observed in the hippocampus, GFAP- and Iba1-stained areas were not significantly increased in the amygdala of mice fed a HFD compared to a SD. Nonetheless, increased expression of IL-1 was observed in the amygdala of HFD mice. It has been 21

ACCEPTED MANUSCRIPT previously shown that increased brain expression of cytokines following systemic injection of LPS precedes morphological activation of microglia and astrocytes [80]. Therefore, it is possible

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that amygdalar expression of GFAP and Iba1 could significantly increase over time. Moreover,

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surgical lesions to the hippocampus induces IL-1 and TNF- expression in the striatum, cortex

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and basal ganglia, which is not accompanied by the presence of reactive astrocytes and microglia in those brain regions [86]. Other studies not investigating whether or not activation of astrocytes

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and microglia takes place, have reported that cytokine signals propagate to brain regions far from the site where kainate was injected [87] or controlled cortical impact was delivered [88].

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Therefore, cytokine signals may propagate through the brain by volume transmission and wiring [89]. Volume transmission indicates that cytokines diffuse through cerebral spinal fluid, nerve

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bundles and perivascular space. Wiring, on the other hand, suggests that diffusion occurs through neuronal projections and gap junctions. The hippocampus and the amygdala make reciprocal

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connections with each other, which would enable diffusion of IL-1 produced in one region to the other through wiring. Additionally, this cytokine may also propagate through CSF in the lateral ventricle, which borders both the hippocampus and amygdala. This possibility remains to be investigated.

The association between obesity and depression is well documented in humans [9, 1316]. In the present study we used the forced swim test (FST) to investigate whether mice fed a HFD develop depressive-like behavior and in agreement with previously published data [22, 23, 25], our study showed that chronically feeding mice a HFD lead to increased immobility time during the FST, which may reflect depressive-like behavior. There are limitations in using the FST to assess depressive-like behavior in mice with increased adiposity. We cannot exclude the possibility that increases in immobile behavior reflect reduced physical mobility caused by

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ACCEPTED MANUSCRIPT oversized bodies. However, total movement time was not affected by dietary treatment and increased body weight in the open field test, which challenges the possibility that mobility is

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directly affected by weight gain. Immobile time in the HFD group may also reflect better

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buoyancy due to reduced body density. Immobility in the FST has been suggested as a measure of learned helplessness in response to inescapability [60-62, 90, 91], but we should also consider

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that increased adiposity should affect the ability of a mouse to float. Nonetheless, increased

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immobile time in the FST may reflect the development of depressive-like behavior in mice fed a HFD [22, 23, 25] and in leptin-deficient ob/ob mice [95]. This idea is supported by the lack of

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correlation between weight gain and immobility time [present study, 22] and by the observation that leptin administration ameliorates depressive-like symptoms in ob/ob mice without affecting

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body weight [95].

Despite the number of studies investigating the impact of chronic HFD intake on

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pathophysiological alterations in the brain, it is still unclear how excessive dietary fat, weight gain and associated metabolic alterations individually contribute to the development of behavioral alterations. In the present study, 30% of all mice fed a HFD gained significantly less weight compared to the average weight gain of the upper tertile and the whole HFD group. Although comparison of metabolic and behavioral outcomes across all three tertiles in the HFD group did not reveal any significant results, weight gain and AUCglucose negatively correlated with each other. This suggests that a lower weight gain in mice fed a HFD did not translate into a better metabolic profile. Additionally, anxiety-like behavior measured by entries in the open zone of the EZM was the only behavioral outcome negatively correlated with weight gain the HFD group. Overall, statistical analyses of the present data suggest that excessive weight gain is not necessary for the deleterious effect of HFD on metabolic and behavioral changes to take

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ACCEPTED MANUSCRIPT place. Consistently, it has been previously reported that a 72-hour intake of high fat feeding reduces central insulin sensitivity without any significant change in body weight or body

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adiposity [98]. Moreover, one-day high-fat feeding is sufficient to induce brain inflammation in

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mice [99]. In accordance with the literature our data suggest that excessive weight gain is not necessary for the development of metabolic and behavioral alterations in mice fed a HFD. More

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research is needed, however, to distinguish between the direct effect of fat consumption and

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concomitant insulin resistance on the development of neuroinflammation and behavioral alterations.

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In future studies it would be interesting to investigate whether the use of knockout mouse models for IL-1 receptor (IL-1R) or intracerebral administration of IL-1R antagonist prevents

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or at least ameliorates behavioral alterations following chronic HFD intake. The present study does not establish causation between expression of IL-1 and behavioral alterations, but rather

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shows that these two variables are associated. Manipulation of IL-1 signaling pathway would help to determine whether increased expression of IL-1 contributes to the underlying mechanisms leading to changes in behavior. Finally, future studies need to include female mice to investigate differences in gender susceptibility to chronic HFD intake. Gender inclusion in animal research is imperative as results showing both significant or no sex difference provide important knowledge about the etiology and treatment of various health conditions. In conclusion, the results from this study suggest that chronic consumption of a HFD leads to cognitive deficits and increased anxiety and depressive-like behaviors in mice. Our data confirm the association between high-fat feeding and hippocampal inflammation and also show that levels of IL-1 in the hippocampus and amygdala correlate with working memory deficits in mice. Moreover, we showed for the first time that the development of anxiety-like behavior in 24

ACCEPTED MANUSCRIPT mice is associated with increased amygdalar, but not hippocampal or hypothalamic, expression of IL-1. With the high incidence of cognitive and psychiatric disorders being reported among

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the obese population, it is essential to understand the mechanisms underlying their development.

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Although we did not investigate mechanisms triggering the inflammatory response, the results

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described above suggest that brain function is susceptible to the effects of chronic low-grade inflammation subsequent to high-fat feeding. Finally, our data suggest that excessive weight gain

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is not necessary for neuroinflammation and behavioral alterations to take place in mice fed a HFD. In summary, our study supports the hypothesis that neuroinflammation as a consequence

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of chronic high-fat consumption may serve a role in the development of cognitive and

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psychiatric deficits, which has major public health implications.

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ACCEPTED MANUSCRIPT DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). The

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the Department of Defense or the United States Government.

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opinions expressed herein are those of the authors and should not be construed as representing

ACKNOWLEDGEMENTS

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We gratefully acknowledge Marissa Rescott and Dr. Tianzheng Yu (USUHS) for

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technical assistance throughout the duration of these experiments.

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GRANTS

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The authors acknowledge the Department of Defense in the Center Alliance for Dietary

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Supplement Research for financially supporting the present work. Grant# NC91FD15. URL of funder’s website: https://www.usuhs.edu/mpcrn/center-alliance-for-dietary-supplement. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

AUTHOR CONTRIBUTIONS Y.C., P.D. Acquired funding for the research; C.P.A.-S., conception and design of research; C.P.A.-S., A.G. performed experiments; C.P.A.-S., A.G. analyzed data; C.P.A.-S., A.G. interpreted results of experiments; C.P.A.-S. prepared figures; C.P.A.-S. drafted manuscript; P.D. edited and revised manuscript; C.P.A.-S., A.G., Y.C., P.D. approved final version of manuscript. 26

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ACCEPTED MANUSCRIPT Table 1. Nutrient profile of standard (SD) and high-fat (HFD) diets HFD (TD.06414)

Protein (g/Kg)

182.4

234.6

Protein (% kcal)

24.0

Carbohydrate (g/Kg)

452.5

Carbohydrate (% kcal)

18.0

Fat (g/Kg)

59.9

Fat (% kcal)

18.0

Fiber (NDF), g/Kg

147.2 3.2

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18.4

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ME (kcal/g)

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SD (T.2018)

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Nutrients

SFA (g/Kg)

273.1 21.3 342.7 60.3 65.5 5.1

8.9

124.8

12.4

160.5

32.5

54.0

3.0

5.5

29.5

48.2

Trace

4.7

6.8

80.2

0.1

9.6

1.6

39.3

12.0

146.8

18:2 (g/Kg)

29.5

47.0

18:3 (g/Kg)

3.0

5.5

-

347.5

MUFA (g/Kg)

14:0 (g/Kg) 16:0 (g/Kg) 16:1 (g/Kg) 18:0 (g/Kg) 18:1 (g/Kg)

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PUFA (g/Kg)

Cholesterol (mg/Kg)

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ACCEPTED MANUSCRIPT Table 2. Correlations between IL-1 levels in different brain regions and behavioral outcomes. Working Memory

Elevated Zero Maze

Y-maze

r2

r2

r2

Hippocampus

0.01

Hypothalamus *p < 0.05; **p < 0.01

0.01

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0.01

0.32**

0.01

0.001

0.02

0.043

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Frontal Cortex

0.36**

0.19*

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0.1869*

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Open Field

Amygdala IL-1

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Anxiety

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ACCEPTED MANUSCRIPT Table 3. Multiple comparisons of metabolic and behavioral outcomes between weight gain in the HFD group tertiles. Weight Gain Weight Gain Tertile Tertile 1 2

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Dependent Variable

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1

2

3

0.000

1

0.026

3

0.000

1

0.000

2

0.000

2

0.026

3

0.000

1

0.026

3

0.000

1

0.000

2

0.000

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Final Weight

0.026

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Weight Gain

Significance

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Weight Gain HFD Group (r2) 0.281**

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AUCglucose % Alternation – Y-maze

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0.193

Center Entries – Open Field

0.003

% Time Mobile in the Center – Open Field

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Open Zone Entries – Elevated Zero Maze

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% Distance in the Center – Open Field

0.002 0.012

0.348** 0.115

Immobility Time – Forced Swim Test **p < 0.01

0.0004

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Open Zone Time – Elevated Zero Maze

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Figure 1 – Weight gain. (A) Body weight of SD or HFD mice for 18 weeks. HFD mice became significantly heavier over time [F(1,46)=64.86, p<0.0001], starting at week 8. (B) Average body

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weight gain over 18 weeks of dietary treatment. **p<0.01; ***p<0.001; ****p<0.0001; n = 24 for

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each group.

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Figure 2 – Glucose Tolerance Test. (A) Changes in plasma glucose during intraperitoneal glucose tolerance test (2 mg of glucose/g body weight) following an overnight (18 h) fast. (B)

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Area under the glucose tolerance curve (AUCglucose) was calculated for glucose by using the

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trapezoidal rule. ****p < 0.0001; n = 24 for each group.

Figure 3 – Cognitive performance in the Y-maze test. Graph shows % perfect alternation during a 5 min trial. A decrease in percent perfect alternation was observed in HFD mice. ** p<0.01; n = 18 for each group.

Figure 4 – Anxiety-like behavior and locomotor activity in the open field test. Mice acclimated to the apparatus for 10 min and data were recorded for additional 10 min. HFD mice showed reduced preference for the center area as measured by reduced % time spent in the center (A) and reduced number of entries in the center (B). HFD mice also moved at a lower average speed (C) and traveled a shorter distance (D). No significant differences were found between SD and HFD mice in total mobile time (E). *p<0.05; n = 18 for each group.

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ACCEPTED MANUSCRIPT Figure 5 – Anxiety-like behavior in the elevated zero maze test. HFD mice showed reduced preference for open zone of the elevated zero maze as measured by reduced number of entries

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(A) and reduced time spent in the open zone (B) in a 5 min trial. *p<0.05; ***p<0.001; n = 18 for

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each group.

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Figure 6 – Depressive-like behavior in the forced swim test. Mice acclimated to the water for

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2 min and immobility time was recorded for additional 4 min. HFD mice showed increased

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immobile behavior in the forced swim test. **p<0.01; n = 18 for each group.

Figure 7 – Neuroinflammation in the hippocampus and amygdala. Expression of IL-1β was

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significantly increased in the hippocampus (n = 16 for each group) (A) and amygdala (n = 12 for

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each group) (B) of HFD mice. *p<0.05

Figure 8 – Quantification of GFAP+ and Iba1+ stained areas in the hippocampus and amygdala. GFAP and Iba1 stained areas significantly increase in the hippocampus but not in the amygdala of HFD mice. Sample micrographs of GFAP-stained hippocampus from (A) SD and (B) HFD mice. Quantification of GFAP-stained area in the hippocampus of SD and HFD mice (C). Sample micrographs of Iba1-stained hippocampus from (D) SD and (E) HFD mice. Quantification of Iba1-stained area in the hippocampus of SD and HFD mice (F). Sample micrographs of GFAP-stained amygdala from (G) SD and (H) HFD mice. Quantification of GFAP-stained area in the hippocampus of SD and HFD mice (I). Sample micrographs of Iba1stained amygdala from (J) SD and (L) HFD mice. Quantification of Iba1-stained area in the hippocampus of SD and HFD (M) mice. *p<0.05; n = 6 mice for each group.

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Figure 1

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Figure 5

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Figure 7

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Mice fed a high-fat diet (HFD) develop cognitive deficit and anxiety-like behavior Expression of IL-1 is increased in the hippocampus and amygdala of HFD mice Cognitive deficit correlates with levels of IL-1 in the hippocampus and amygdala Levels of IL-1 in the amygdala correlates with anxiety-like behavior Excessive weight gain is not necessary metabolic and behavioral changes to occur.

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HIGHLIGHTS

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