Brain, Behavior, and Immunity 37 (2014) 134–141

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Short exposure to a diet rich in both fat and sugar or sugar alone impairs place, but not object recognition memory in rats
Jessica E. Beilharz a, Jayanthi Maniam b, Margaret J. Morris b,⇑ a b

School of Medical Sciences, University of New South Wales, Australia
Department of Pharmacology, School of Medical Sciences, University of New South Wales, Australia

a r t i c l e

i n f o

Article history:
Received 10 October 2013
Received in revised form 11 November 2013
Accepted 25 November 2013
Available online 3 December 2013
Keywords:
Diet
Fat
Sugar
Obesity
Memory
Learning
Hippocampus
Inflammation
Oxidative stress

a b s t r a c t
High energy diets have been shown to impair cognition however, the rapidity of these effects, and the dietary component/s responsible are currently unclear. We conducted two experiments in rats to examine the effects of short-term exposure to a diet rich in sugar and fat or rich in sugar on object (perirhinaldependent) and place (hippocampal-dependent) recognition memory, and the role of inflammatory mediators in these responses. In Experiment 1, rats fed a cafeteria style diet containing chow supplemented with lard, cakes, biscuits, and a 10% sucrose solution performed worse on the place, but not the object recognition task, than chow fed control rats when tested after 5, 11, and 20 days. In Experiment
2, rats fed the cafeteria style diet either with or without sucrose and rats fed chow supplemented with sucrose also performed worse on the place, but not the object recognition task when tested after 5, 11, and 20 days. Rats fed the cafeteria diets consumed five times more energy than control rats and exhibited increased plasma leptin, insulin and triglyceride concentrations; these were not affected in the sucrose only rats. Rats exposed to sucrose exhibited both increased hippocampal inflammation (TNF-a and IL1b mRNA) and oxidative stress, as indicated by an upregulation of NRF1 mRNA compared to control rats.
In contrast, these markers were not significantly elevated in rats that received the cafeteria diet without added sucrose. Hippocampal BDNF and neuritin mRNA were similar across all groups. These results show that relatively short exposures to diets rich in both fat and sugar or rich in sugar, impair hippocampaldependent place recognition memory prior to the emergence of weight differences, and suggest a role for oxidative stress and neuroinflammation in this impairment.
Crown Copyright Ó 2013 Published by Elsevier Inc. All rights reserved.

1. Introduction
Many people in developed countries eat a diet that is rich in saturated fat and refined sugar (Kearney, 2010). Excessive intake of this diet leads to increased body weight, progression into obesity, and the development of metabolic and cardiovascular disorders
(Bruce-Keller et al., 2009; Haslam and James, 2005). Epidemiological data has also associated the consumption of high energy diets with an increased risk of age related deficits and neurological diseases including dementia and Alzheimer’s disease (Eskelinen et al.,
2008; Grant et al., 2002; Solfrizzi et al., 2010). Studies with rodents have confirmed that the long-term intake of such a diet produces cognitive deficits, especially in spatial tasks that require the hippocampus (Heyward et al., 2012; McNay et al., 2010; Ross et al., 2009). Some of these studies have also identified changes in the hippocampus that may mediate the link between obesity and cognitive deficits. Such changes include oxidative stress,

⇑ Corresponding author. Tel.: +61 2 9385 1560; fax: +61 2 9385 1059.
E-mail address: m.morris@unsw.edu.au (M.J. Morris).

neuroinflammation, as well as decreased levels of neurotrophins
(Molteni et al., 2004; Pistell et al., 2010).
Moreover there is very recent evidence that cognitive deficits can not only be produced by long-term excessive intake of a high energy diet but also by relative short exposures to such a diet.
Holloway et al. (2011) reported that healthy adults who ate a high fat diet for 5 days performed worse on tasks measuring attention and speed of retrieval than they had prior to the diet. Kanoski and Davidson (2010) also found that rats exposed to a high fat and glucose diet performed worse on a spatial task than chow fed control rats after only 3 days whereas 30 days exposure was necessary for non-spatial memory impairments.
The present experiments constituted a further study of the effects of relatively short-term exposure to a high energy diet on cognition in rats. We set out to confirm and extend previous reports that the consumption of diets high in both fat and sugar is accompanied by impaired memory retention. The modern diet is replete with a diverse range of foods that are rich in fat and sugar and for this reason in the first experiment we chose a cafeteria style diet (cakes, biscuits, lard) supplemented with a 10% sucrose solution. To compare non-spatial and spatial memory we

0889-1591/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbi.2013.11.016 J.E. Beilharz et al. / Brain, Behavior, and Immunity 37 (2014) 134–141

employed the object and place recognition tasks, which are respectively dependent on the perirhinal cortex and the hippocampus
(Aggleton and Brown, 2005). We assessed performance on both tasks from 5 to 21 days diet exposure to examine changes in the magnitude of specific deficits. There is some evidence that the hippocampus is particularly sensitive to changes in dietary intake and impairments have been reported on hippocampal-dependent tasks prior to substantial weight gain (Kanoski and Davidson, 2010;
Murray et al., 2009). Here we specifically aimed to test short, rather than the more commonly studied long term dietary interventions
(3–12 months).
To assess the relative contributions of fat and sugar to the behavioral effects observed, our second experiment included two additional diet groups, a regular diet supplemented with 10% sucrose and the cafeteria style diet without sucrose. At the end of the study we assessed plasma leptin, insulin and triglycerides as well as hippocampal expression of inflammatory markers; tumor necrosis factor (TNF-a) and interleukin 1 (IL-1b), oxidative stress markers; nuclear respiratory factor 1 (NRF1), and sirtuin 3 (SIRT3), as well as brain-derived neurotrophic factor (BDNF) and neuritin.
These markers were selected as CNS inflammation has been reported after as little as 1–3 days on a high energy diet (Hansen et al., 1998; Thaler et al., 2012) and obesity itself is characterized by a state of low grade, chronic inflammation (Bastard et al.,
2006). In addition, oxidative stress is believed to be one of the first events following the consumption of a high energy diet and it may be responsible for the decreased levels of BDNF reported in long term diet studies (Molteni et al., 2004). NRF1 is involved in the activation of antioxidant response element-dependent genes (Ohtsuji et al., 2008) while SIRT3 has roles in metabolism, oxidative stress and cell survival (Weir et al., 2013). BDNF has been implicated in a number of processes postulated to underlie hippocampal-dependent learning and memory including long term potentiation, neurogenesis and synaptic plasticity (Stranahan et al., 2008; Yamada et al., 2002).
2. Methods
2.1. Subjects
Male Sprague–Dawley rats (Experiment 1: 363–467 g; Experiment 2: 302–366 g; Animal Resource Centre, Perth, WA, Australia) were housed four per polypropylene cage (47 cm  29 cm Â
15 cm) in a temperature controlled (18–20 °C) colony room on a
12 h light/dark cycle (lights on at 07:00 h). Rats were acclimatized for 1 week, during which they were maintained ad libitum on standard rat chow (Gordon’s Premium Rat and Mouse Breeder diet,
NSW, Australia) and tap water. Prior to diet commencement rats were weight matched across groups and two home cages were assigned to each diet condition (n = 8 per group). All experimental procedures were approved by the Animal Care and Ethics Committee at the University of New South Wales.

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(consuming 74% energy as carbohydrate, 17% protein, 9% fat over the course of the experiment) and a modified cafeteria group
(CAF SÀ) that received the same diet as the CAF S+ rats but with access to two water bottles (49% carbohydrate, 4% protein, 47% fat). All food, water and sucrose were available ad libitum and replaced daily. Food and liquid consumption over 24 h was recorded at regular intervals. Items were weighed before presentation to the rats, and the remainder was weighed after 24 h. The amount eaten per cage was converted to energy (kJ) using data provided by the manufacturers and average intake was calculated assuming equal intake for each rat. Rats were weighed at the diet onset and at regular intervals across the experiment.
2.3. Object and place recognition tests
The apparatus consisted of an open-field arena (60 cm Â
60 cm  50 cm) constructed from black PVC plastic which was divided equally into 16 quadrants. A video-camera was positioned directly above the arena to record behavior. The objects used were commercial products (e.g., 1.25 L soft drink bottles, coffee mugs, lunch box containers), made of a variety of materials (aluminium, glass, plastic and porcelain) and varying in both height (7.5–29 cm) and width (4.5–13 cm). Rats were acclimatized to the empty arena for 10 min per day on 2 consecutive days, and testing commenced the next day. Specifically, half of the rats from each diet group were assessed on the object task and the other half on the place recognition task on the first day of testing and vice versa for the second day of testing. For both tasks, each rat was placed into the center of the arena with two identical objects (located in two of the middle four quadrants) and allowed to explore for 5 min (familiarization phase). The rat was then removed for 5 min (retention phase) and the arena and objects were cleaned with 80% ethanol. For the object test phase, two objects were placed into the same positions as the familiarisation trial. One of these objects was identical to the sample object previously presented and the other was a novel object. In the place test phase, two identical objects to those used in familiarisation were presented; one remained in its original location while the other was moved to one of the four corner quadrants of the arena. The test phase lasted for 3 min. Following the completion of baseline measures, the diets were commenced and rats received 3 additional object and place memory tasks after
5–6, 11–12 and 20–21 days.
Each object was only used for one trial per rat. Rats were tested at the same time of day and the object order and location were counterbalanced between rats and across trials. Exploration was defined as the rat’s head within 2 cm of the object with the neck extended and vibrissae moving around the object. Data were scored using Macropod ODlogÒ software by the experimenter and a second observer naïve to group allocation. The correlation between the two observers was high (r > .90). Data are reported as the Exploration Ratio, which was the time spent exploring the novel object divided by the time spent exploring both objects
(tnovel/(tnovel + told).

2.2. Diets

2.4. Sample collection

The regular diet (RD) rats received chow (59% carbohydrate,
26% protein, 15% fat) and access to two water bottles in each cage throughout the experiment. The rats on the cafeteria diet (CAF S+) received chow, and access to one sucrose bottle (10% solution) and one water bottle in each cage. This was supplemented with a container (7.5 cm  7.5 cm) of lard, and a selection of biscuits and cakes that changed every day (50% carbohydrate, 5% protein, 45% fat). For the second experiment, four diets were compared: RD and CAF S+ as above; a sugar group (S+) that received chow and access to one sucrose bottle (10% solution) and one water bottle

In Experiment 2, rats were anesthetized (xylazine/ketamine 15/
100 mg/kg i.p.) between 09:00 and 13:00 h, 6–8 days after the completion of their final object and place recognition task. All rats had access to their respective diets until cull. Body weight, nasoanal length and girth (base of ribcage) were measured. A cardiac blood sample was centrifuged (Microspin 12) and the plasma separated and stored at À20 °C for subsequent determination of plasma leptin, insulin and triglycerides. Rats were then killed by decapitation. White adipose tissue (WAT) (retroperitoneal (RP), visceral, and gonadal deposits), and organs (adrenal, kidney, liver)

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were dissected and weighed. Brains were removed and coronal incisions made at the level of the optic chiasm, and the rostral border of the hypothalamus (Bregma -1.30 to -4.52 mm). The hippocampus was dissected from this section. The hippocampus was snap frozen in liquid nitrogen and stored at À80 °C for determination of mRNA expression of genes of interest.
2.5. Plasma hormone measurements
Plasma leptin and insulin were analyzed using commercial kits according to manufacturer’s instructions (Millipore RL-83K and
Millipore RI-13K, Zug, Switzerland). Plasma triglyceride was measured spectrometrically using RocheÒ triglyceride reagent and SigmaÒ glycerol standard solution.
2.6. Real-time quantitative PCR
RNA was extracted using Tri-reagent (SIGMA ALDRICH) and treated with DNase I (Invitrogen) to remove any contaminating genomic
DNA and stored at À80 °C. Spectrophotometric measurements were done using the Nanodrop Spectrophotometer (Nanodrop Technologies Wilmington, DE, USA) to determine RNA concentration and purity. One microgram of RNA was reverse transcribed to cDNA using Omniscript Reverse Transcription kit (QIAGEN) and stored at À20 °C. Quantitative real time PCR (qPCR) was performed with 50 ng cDNA using custom designed gene expression assays for TNF-a (RN 99999017_m1), IL-1b (Rn00580432_m1),
NRF1 (RN01455958_m1), SIRT3 (Rn01501410_m1), BDNF
(Rn01484924_m1) and Neuritin (Rn00584304_m1) from Applied
Biosystems. Five different housekeepers were assessed (B2M: beta-2-microglobulin, HPRT-1: hypoxanthine phosphoribosyltransferase 1, YWAZ: tyrosine 3-monooxygenase/tryptophan
5-monooxygenase activation protein, zeta polypeptide, TBP: TATA box binding protein, PPIA: peptidyl prolylisomerase A). The stability of each reference gene was analysed using the Normfinder software
(Andersen et al., 2004). As YWAZ was identified as the most stable housekeeper gene, the genes of interest were normalised against
YWAZ. Analysis of the relative expression was performed using the DDCT method (Livak and Schmittgen, 2001) with a chow fed rat used as the calibrator. Similar results were obtained when different control rats were used as the calibrator.
2.7. Statistical analysis
Results are expressed as mean ± SEM. A one-way ANOVA followed by post hoc Tukey tests were used to evaluate baseline measures of body weight, energy intake, total exploration time, exploration ratios and kill data. If data were not normally distributed they were log transformed. ANOVA with repeat measures followed by post hoc Tukey was used to evaluate body weight, energy intake, total exploration time and exploration ratios across the diet period. P < .05 was considered significant. Data were analyzed with
IBM SPSS Statistics 20.
3. Results
3.1. Experiment 1
3.1.1. Effect of diet on body weight and energy intake
A one-way ANOVA of baseline body weight revealed that there was no significant difference between diet conditions (F < 1). All rats gained weight across the diet period (F(5,60) = 243.09, p < .05), and the CAF S+ rats gained weight more rapidly than the RD rats
(F(5,60) = 34, p < .05). The main effect of diet was not significant
(F(1,12) = 3.07, p = .105). A post hoc analysis revealed that the CAF

S+ rats weighed significantly more than control rats after 16 and
21 days on the high energy diet (p < .05). Total energy intake over the experimental period was 8498 and 31,359 kJ in chow and CAF
S+ rats, respectively (data not shown).
3.1.2. Effect of diet on object and place memory
On the object and place tasks, baseline exploration ratios (F < 1) and baseline exploration times (Object task F(1,12) = 1.473, p = .248;
Place task F < 1) were comparable between the RD and CAF S+ rats.
As shown in the top panel of Fig. 1, total exploration time on the object (Time F(1,12) = 1.227, p = .270; Time à diet F < 1; Diet F < 1) and place (Time F(1,12) = 1.781, p = .207; Time à diet F(1,12) = 2.978, p = .110; Diet F < 1) tasks was not affected by diet or time and there were no interactions. However, as displayed in the bottom panel of
Fig. 1, exploration ratios on the place task were lower among the
CAF S+ rats than the RD rats, but on the object recognition task these ratios were similar, or slightly higher, among the CAF S+ rats.
A repeat measures ANOVA revealed a significant effect of diet on the place task (F(1,12) = 10.459, p < .05), and this was of borderline significance for the object task (F(1,12) = 4.493, p = .06). In addition, there was a significant effect of time for the place task
(F(1,12) = 11.606, p < .05), but not for the object task (F < 1), indicating that exploration ratios decreased over time on the place but remained stable on the object task.
3.2. Experiment 2
3.2.1. Effect of diet on body weight and energy intake
A one-way ANOVA of baseline body weight revealed that there was no significant difference between diet conditions (F < 1). Fig. 2
(left) shows that all rats gained weight over the diet period
(F(1,28) = 726.226, p < .05), there was a main effect of diet
(F(3,28) = 3.021, p < .05), and a significant diet  time interaction
(F(3,28) = 20.743, p < .05). A post hoc analysis revealed that the
CAF S+ and CAF SÀ rats weighed significantly more than the RD rats after 15 and 20 days on their respective diets (p < .05). Fig. 2
(right) also shows that energy intake was significantly different between the diet groups (F(3,4) = 221.603, p < .05) with post hoc tests revealing that rats supplemented with cafeteria items consumed more kJ than the S + and RD rats (p < .05), while there were no significant differences in energy intake between the S + and RD rats.
As can be seen, over a 24 h period, the CAF S+ and CAF SÀ rats consumed approximately 5 times more kJ than the RD and S+ rats. On average the RD rats consumed around 30 g of chow over a 24 h period whilst the CAF S+, CAF SÀ and S+ rats decreased their chow intake to 16 g, 14 g and 20 g respectively. Sucrose was avidly consumed over water, with rats in the CAF S+ groups drinking around
37 ml of sucrose and 10 ml of water over 24 h whilst on average a
S + rat consumed 113 ml of sucrose and 2 ml of water. The RD and
CAF S+ rats drank around 38 ml and 31 ml of water respectively over 24 h.
3.2.2. Effect of diet on object and place memory
Fig. 3 shows that baseline exploration ratios and total exploration times were similar across the diet conditions on both the object and place tasks (largest F = 2.683). All diet conditions had similar total exploration times across sessions on both the object and place tasks (largest F = 3.02; Fig. 3 top). When the high energy diets were available, all rats continued to spend more time exploring the novel than familiar objects on the object task (largest
F = 2.041). However, there was a significant effect of diet on the place task (F(3,28) = 32.754, p < .05; Fig. 3 bottom). Specifically, while the RD rats displayed a novelty preference, the CAF S+, S+ and CAF SÀ rats equally distributed their time between the novel and familiar places from 5 to 21 days on each of the diets. No other main effects or interactions were significant (F < 1).

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Object Task

Exploration time (s)

30

Place Task

20

10

0
Day 0

5-6

11-12

20-21

Day 0

5-6

11-12

20-21

Exploration ratio

0.8
0.7
0.6

RD
CAF S+

0.5
Day 0

5-6

11-12

20-21

Day 0

5-6

11-12

20-21

Fig. 1. Total exploration times (top) and exploration ratios (bottom) of the RD (open circle) and CAF S+ (closed circle) groups on the object (left) and place (right) tasks at 0, 5–
6, 11–12 and 20–21 days after the commencement of the diet. Data are expressed as mean ± SEM, with n = 7 rats per group. Data were analyzed by repeated measures
ANOVA.

4000

525

3000

kJ/24 h/rat

Weight (g)

575

475
425

CAF SCAF S+

2000
1000

S+
RD

375
0
0

5

10
15
Days on diet

20

1

5

10
15
Days on diet

20

Fig. 2. Body weight (left) and energy intake (right) of the RD (open circle), CAF S+ (closed circle), S+ (open triangle) and CAF SÀ (solid square) rats. Data are expressed as mean ± SEM, n = 8 rats per group. Energy intake calculations are based on two home cages of four rats for each diet condition. Data were analyzed by repeated measures
ANOVA followed by post hoc Tukey.

3.2.3. Body weight, fat and organ mass
Table 1 shows that at the completion of Experiment 2 there was a significant effect of diet on body weight (F(3,28) = 6.196, p < .05) and adiposity (F(3,26) = 14.716, p < .05). The CAF S+ rats had heavier body weights and more WAT than the RD and S+ rats (p < .05) while the CAF SÀ rats weighed more than the RD rats and had more WAT than the RD and S+ rats (p < .05). Naso-anal length, girth, adrenal weight, liver mass and kidney mass did not differ among the diet conditions (largest F = 2.903) (data not shown).
3.2.4. Leptin, insulin and triglyceride concentrations
As shown in Table 1, there was a statistically significant difference in plasma leptin concentrations among the diet groups
(F(3,27) = 20.651, p < .05). Post hoc tests indicated that plasma leptin was significantly higher in the cafeteria diet groups than in the RD and S+ rats (p < .05). As expected, higher plasma leptin was correlated with higher body weight (r = 0.69, p < .05, n = 31), and more
WAT (r = 0.91, p < .05, n = 31). Leptin concentration was negatively correlated with novelty preferences on the final place task
(r = À0.41, p < .05, n = 31). Plasma insulin concentrations also differed between the diet groups (F(3,26) = 5.866, p < .05). Specifically, insulin concentrations were higher in the CAF S+ rats compared to the RD and S+ rats (p < .05). Plasma insulin levels were also correlated with body weight (r = 0.596, p < .05, n = 30), WAT (r = 0.541, p < .05, n = 30) and lower novelty preferences on the final place

task (r = À0.376, p < .05, n = 30). Finally, plasma triglyceride concentrations differed among the groups (F(3,27) = 5.396, p < .05) with concentrations higher in the cafeteria diet groups compared to controls (p < .05). Triglyceride concentrations were correlated with body weight (r = 0.57, p < .05, n = 31), WAT (r = 0.71, p < .05, n = 30) and with impaired performance on the final place task (r = À0.382, p < .05, n = 31).
3.2.5. Hippocampal mRNA expression
We were interested in the inflammatory and oxidative stress response to the various diet interventions. As shown in Fig. 4, there was a significant effect of diet on hippocampal TNF-a
(F(3,24) = 4.086, p = .018) and IL-1b (F(3,25) = 4.905, p = .008) mRNA expression. Both markers were significantly elevated in the CAF
S+ rats compared to controls (p < .05) and were of borderline significance in the S+ rats for TNF-a (p = .062) and IL-1b (p = .071) compared to the RD rats. The CAF SÀ rats TNF-a (p = .789) and
IL-1b (p = .963) mRNA expression were comparable to controls.
As shown in Fig. 5, higher TNF-a mRNA levels were strongly correlated with lower novelty preferences on the final place task
(r = À0.70, p < .05, n = 27). In addition, we found a significant effect of diet on hippocampal NRF1 (F(3,24) = 4.045, p = .018) but not SIRT3
(F(3,24) = 2.360, p = .097) mRNA expression. Post hoc tests revealed that NRF1 hippocampal mRNA was significantly elevated in the
CAF S+ and S+ rats compared to the RD rats (p < .05). NRF1 mRNA

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Exploration time (s)

40

Object Task

Place Task

30
20
10
0
Day 0

5-6

11-12

20-21

Day 0

5-6

11-12

20-21

Exploration ratio

0.8
0.7

RD
0.6

CAF SS+
CAF S+

0.5
Day 0

5-6

11-12

20-21

Day 0

5-6

11-12

20-21

Fig. 3. Total exploration times (top) and exploration ratios (bottom) of the RD (open circle), CAF S+ (closed circle), S+(open triangle) and CAF SÀ (solid square) groups on the object (left) and place (right) tasks at 0, 5–6, 11–12 and 20–21 days after the commencement of their respective diets. Data are expressed as mean ± SEM, with n = 8 rats per group. Data were analyzed by repeated measures ANOVA.

Table 1
Mean body parameters at endpoint.
RD
Body weight (g)
Total white adipose (g)
Leptin (ng/ml)
Insulin (ng/ml)
Triglycerides (ng/ml)

CAF S+

S+

CAF SÀ

479.6 ± 18.2
8.79 ± 0.59
4.21 ± 0.18
0.07 ± 0.04
1.26 ± 0.16

587.3 ± 18.3a,b
26.54 ± 3.22a,b
10.40 ± 0.84a,b
0.54 ± 0.14a,b
2.69 ± 0.35a

510.0 ± 18.4
13.41 ± 0.47
5.10 ± 0.31
0.08 ± 0.05
1.61 ± 0.25

558.5 ± 22.2a
24.96 ± 2.82a,b
9.32 ± 0.92a,b
0.28 ± 0.08
2.44 ± 0.33a

Data are expressed as mean ± SEM, n = 8 rats per group. RD total white adipose, leptin, insulin and triglycerides, S + insulin, n = 7. Data were analyzed by one-way ANOVA followed by post hoc Tukey. a p < .05 versus RD. b p < .05 versus S+.

levels were also correlated with lower exploration ratios on the final place task (r = À0.57, p < .05, n = 27). There were no significant differences in hippocampal BDNF or neuritin mRNA expression across groups (all Fs < 1).

4. Discussion
Our results confirm and extend previous studies by showing that rats maintained on high energy diets display impaired memory retention. First, the present study showed comparable deficits in the rats place recognition memory following exposure to a cafeteria diet supplemented with 10% sucrose (in two independent cohorts), a regular diet supplemented with 10% sucrose and a cafeteria diet without sucrose. Second, these deficits emerged prior to any differences in body weight. Critically, this persistent deficit was selective to place recognition memory as rats in these three dietary conditions performed just as well as chow fed rats on the object recognition task from 5 to 20 days on the diets. Finally, our results show an association between deficits in the hippocampal-dependent place recognition task and increased hippocampal markers of inflammation (TNF-a and IL-1b mRNA) and oxidative stress (NRF1 mRNA).
There are limited data on the short-term effects of high energy diets on cognitive performance. Our results are consistent with the early and sustained spatial impairments reported by Kanoski and

Davidson (2010) on the radial arm maze following 3–90 days on a high fat and glucose diet. In addition, we found that our relatively short diet duration was not sufficient to impair non-spatial, object recognition memory. This was also reported by Kanoski and Davidson (2010) who only found deficits on the non-spatial maze task after 30 days on the diet. In addition, the fact that we observed no differences in total exploration time as a result of diet, and similar exploration ratios for the object recognition task provides evidence against a selective effect of diet on motivational factors that could have affected exploration behavior. Future studies will include additional behavioral measures to further validate this. Together these results support the claim that the hippocampus is especially sensitive to changes in dietary energy intake.
Many studies reporting diet-induced cognitive impairments have used diets that are high in both fat and sugar and thus do not allow the impairments to be ascribed to either fat or sugar, or to their combined effect. The present study demonstrated comparable impairment of place recognition following relatively brief exposures to the CAF S+, S+ and CAF SÀ diets. To the best of our knowledge, only two other studies have directly compared fat and sucrose (32% solution) supplemented diets. These studies reported impaired spatial performance after 5 weeks on the Morris maze (Jurdak et al., 2008) and impaired object recognition memory after 8 weeks (Jurdak and Kanarek, 2009) in the sucrose but not the fat supplemented rats. These findings are consistent with the present results showing that a 10% sucrose solution is sufficient to

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TNF-α

3

IL-1β a a

Hippocampal mRNA (relative expression)

2

1

0
RD

S+

CAF S- CAF S+

2.0

a

S+

NRF1

1.5

RD

CAF S- CAF S+

SIRT3 a 1.0
0.5
0.0
RD

S+

1.5

CAF S- CAF S+

RD

S+

BDNF

CAF S- CAF S+

Neuritin

1.0

0.5

0.0
RD

S+

CAF S- CAF S+

RD

S+

CAF S- CAF S+

Fig. 4. Hippocampal TNF-a, IL-1b, NRF1, SIRT3, BDNF and neuritin mRNA levels of the RD (open bar), S+ (grey bar), CAF SÀ (striped bar) and CAF S+ (filled bar) groups after
27–29 days on their respective diets. Data are expressed as mean ± SEM, n = 5–8 rats per group. Data were analyzed by one-way ANOVA followed by post hoc Tukey. ap < .05 versus RD.

Object Task

Exploration Ratio

0.9

Place Task

0.8

r = -0.70 p < 0.0001

0.7
0.6
0.5
0.4

0

1

2

3

4

1

2

3

4

5

TNF-α mRNA (relative expression)
Fig. 5. Relationship between hippocampal TNF-a mRNA expression and exploration ratios on the object (left) and place (right) recognition tasks at 20–21 days on the diets. n = 5–8 rats per group. TNF-a mRNA was negatively correlated with exploration ratios for the place task.

impair hippocampal-dependent place recognition memory. It would be of interest to test the effects of a low carbohydrate diet supplemented with palatable fat to directly assess the causal role of fat in the deficits observed. Moreover, it is possible that temporal differences may emerge amongst the diet conditions if place recognition memory is assessed prior to 5 days, or if object recognition memory is assessed after 21 days on the diets.
To explore the mechanisms that may underlie the deficits observed after short-term exposure to such diets, we examined markers of inflammation and oxidative damage in the hippocampus. Obesity is characterized by a state of low grade, chronic

inflammation (Bastard et al., 2006; Gregor and Hotamisligil,
2011) and CNS inflammation has been reported after as little as one to 3 days on high energy diets (Hansen et al., 1998; Thaler et al., 2012). Our sucrose supplemented rats (CAF S+ and S+) exhibited increased hippocampal inflammation, as evidenced by elevated TNF-a and IL-1b mRNA expression. Moreover, strong negative correlations were observed between TNF-a expression and exploration ratios on the final place task. This association was strongest in rats which received the cafeteria items plus the sucrose solution. In addition, we observed significant upregulation of hippocampal NRF1 mRNA in our sucrose supplemented rats

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which is indicative of oxidative stress (Hertel et al., 2002). Across all groups NRF1 expression was negatively correlated with exploration ratios on the final place task, mainly as a result of the strong effects in our CAF S+ rats. It is noteworthy that these inflammatory and oxidative stress markers were not significantly elevated in rats that received the cafeteria diet without sucrose despite the fact that these rats demonstrated comparable place memory deficits.
The rapidity of the inflammatory response (Hansen et al., 1998;
Thaler et al., 2012), in conjunction with evidence from longer term diet studies (Baune et al., 2012; Morrison et al., 2010; Pistell et al.,
2010) suggest that oxidative stress and inflammation may both contribute to the hippocampal-dependent memory impairments seen, at least, in our sucrose supplemented rats. Whilst the CAF
SÀ rats did not receive sucrose to drink, sugar was still present in their diet (e.g., cakes and biscuits) and thus it is difficult to tease apart the relative contribution of fat and sugar in this group. It would be of interest in future studies to collect brains at an earlier time point when the memory impairments are first evident to determine the levels of inflammation and oxidative stress.
We also measured BDNF and neuritin mRNA expression because of their important roles in the survival, maintenance and growth of many types of neurons (Naeve et al., 1997; Yamada et al., 2002). We found no effect of diet on hippocampal BDNF or neuritin mRNA levels over the time frame examined. There is abundant evidence that a dysfunction of the BDNF system affects memory however, the diet periods used in these studies range from 2 months to 2 years (e.g., Kanoski et al., 2007; Molteni et al., 2002; Stranahan et al., 2008). It has been proposed that oxidative stress is one of the earliest insults that ensues following consumption of a high energy diet and that formation of reactive oxygen species may play a role in reducing BDNF and cognitive dysfunction (Molteni et al., 2004). Therefore, whilst our sucrose supplemented rats displayed elevated levels of NRF1 mRNA, indicative of oxidative stress, the diet period was not sufficiently long to capture later decreases in BDNF levels.
Finally, we measured plasma leptin, insulin and triglyceride concentrations. Each of these markers were moderately correlated with impaired performance on the place recognition task but were strongly correlated with higher body weight and adiposity. We would also have expected much more modest increases after
5 days on each of the diets, when the memory impairments were first evident, than when the rats were culled at 27–29 days. Our results therefore suggest that the associations between place recognition memory and plasma leptin, insulin and triglycerides are more likely to be an outcome of the diet length rather than a precipitating cause in the acute place recognition impairments.
Previous studies have reported that damage to the hippocampus can increase food intake, body weight gain and disrupt the relationship between meal size and latency to start the next meal
(Davidson et al., 2009; Henderson et al., 2013). Given that high energy foods can impair hippocampal function, excessive intake of these foods may contribute to body weight gain by interfering with hippocampal-dependent higher-order processes including episodic memory (i.e., remembering what we have eaten) and our sensitivity to internal hunger and satiety cues (Davidson et al., 2007;
Francis and Stevenson, 2011). The rapidity of the memory impairments in the current study warrant further empirical testing of the claim that impaired hippocampal function may be both a cause and consequence of overeating and obesity.
We have shown that high fat and sugar or sugar supplemented diets rapidly and selectively impair hippocampal-dependent place memory after as little as 5 days whilst perirhinal-dependent object memory is spared for periods up to 21 days. Hippocampal inflammation and oxidative stress markers were significantly elevated in rats supplemented with sucrose (CAF S+ and S+) and exhibited strong negative correlations with hippocampal-dependent place

recognition memory. Future studies should focus their attention on post hoc style interventions to combat diet-induced cognitive impairments. That is, once the deficits are evident, what can be done to reduce or eliminate them? A promising starting point is past studies which have presented a pharmacological (e.g., resveratrol) or dietary (e.g., curcumin) intervention in conjunction with a high energy diet and have prevented the subsequent increases in oxidative stress, inflammatory markers and memory impairments
(Jeon et al., 2012; Wu et al., 2006).
Acknowledgments
We would like to thank Fred Westbrook for his invaluable advice and assistance with designing and implementing the behavioral tasks. This work was supported by project grant funding from the National Health and Medical Research Council of Australia to Morris and Westbrook.
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