Chronic high fat diet consumption impairs sensorimotor gating in mice

Psychoneuroendocrinology (2013) 38, 2562—2574

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j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p s y n e u e n

Chronic high fat diet consumption impairs sensorimotor gating in mice Marie A. Labouesse *, Ulrike Stadlbauer, Wolfgang Langhans, Urs Meyer Physiology and Behavior Laboratory, Swiss Federal Institute of Technology (ETH) Zurich, Schorenstrasse 16, 8603 Schwerzenbach, Switzerland Received 15 March 2013; received in revised form 4 June 2013; accepted 5 June 2013

KEYWORDS Antipsychotics; Dopamine; High fat diet; Obesity; Prepulse inhibition; Schizophrenia

Summary Chronic intake of high fat diets (HFD) has been long recognized to induce neuronal adaptations and impair elementary cognitive functions. Yet, the consequences of chronic HFD consumption on central information processing remain elusive. The present study thus explored the impact of chronic HFD consumption on pre-attentive central information processing using the paradigm of prepulse inhibition (PPI) of the acoustic startle reflex in mice. Animals were fed an experimental diet with 60% of its calories derived from fat, and were compared to control low fat diet (LFD, 10% calories from fat) fed animals. A first experimental series demonstrated that adult mice exposed to chronic HFD throughout adolescent development displayed significant deficits in PPI compared to LFD-fed mice. Identical chronic HFD treatment further led to presynaptic dopaminergic abnormalities in the form of increased tyrosine hydroxylase density in the nucleus accumbens core and shell subregions. Moreover, we found that tyrosine hydroxylase density in the nucleus accumbens shell negatively correlated with the mean PPI scores, suggesting a potential contribution of the accumbal dopamine system to HFD-induced PPI deficits. This impression was further supported by an additional series of experiments showing that the HFD-induced attenuation of PPI can be mitigated by systemic administration of the dopamine receptor antagonist haloperidol. Finally, HFD feeding was sufficient to disrupt PPI when its exposure was restricted to the peripubertal period, whilst the same manipulation failed to affect PPI when limited to adulthood. In conclusion, our findings emphasize that pre-attentive information processing as assessed by the PPI paradigm is highly sensitive to nutritional factors in the form of chronic HFD consumption, especially when initiated during peripubertal maturation. It is likely that the disrupting effects of HFD on sensorimotor gating involve, at least in part, dopaminergic mechanisms. # 2013 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: +41 44 655 74 50; fax: +41 44 655 72 06. E-mail address: [email protected] (M.A. Labouesse). 0306-4530/$ — see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.psyneuen.2013.06.003

High fat diet impairs prepulse inhibition

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1. Introduction Chronic intake of high fat diets (HFD) is strongly associated with the development of a number of metabolic disturbances, including obesity, type-2 diabetes, and cardiovascular disease (Hu et al., 2001). Converging evidence from experimental work in animals and clinical investigations in humans indicates that excessive consumption of such ‘‘Western diets’’ can also lead to neuronal adaptations and impair elementary cognitive functions (Francis and Stevenson, 2013). Dietary effects on the hippocampus and prefrontal cortex have received wide appreciation in this context because cognitive performance critically depends on the integrity of these brain areas (Kanoski et al., 2007; Kanoski and Davidson, 2011). Owing to its negative impact on brain functions, chronic HFD intake has also been associated with an elevated risk of neurological disorders that are characterized by (progressive) cognitive impairments, most notably Alzheimer’s Disease (AD) and other forms of dementias in aging (Hildreth et al., 2012). In the present study, we set out to explore the impact of chronic HFD consumption on pre-attentive central information processing using the paradigm of prepulse inhibition (PPI) of the acoustic startle reflex. PPI refers to the reduction of the startle reaction to a startle-eliciting stimulus (pulse) when it is shortly preceded by a weak stimulus (prepulse) (Hoffman and Searle, 1965; Graham, 1975). PPI is an operational measure of sensorimotor gating, in which central gating mechanisms protect the processing of the information contained in the initial prepulse from distraction by the subsequent pulse stimulus (Swerdlow et al., 2000). PPI thus reflects the ability to filter or gate intrusive sensory-motor information, and this phenomenon can be readily demonstrated in a variety of species, including humans and rodents (Swerdlow et al., 1999). A direct association between excessive intake of dietary fat and abnormalities in sensorimotor gating has thus far not been established. However, this possibility seems likely given

some findings linking metabolic disturbances to PPI attenuation. First, significant PPI deficits have been reported in db/ db mice, which harbor an autosomal recessive point mutation in the leptin receptor gene and are characterized by multiple metabolic dysfunctions, including hyperphagia, progressive hyperglycemia, and obesity (Sharma et al., 2010). Second, an indirect link between hyperphagia, excess fat deposition, and emergence of PPI deficits has also been established in an inflammation-mediated developmental pathogenesis model in mice (Pacheco-Lo ´pez et al., 2013). Deficits in PPI are also commonly (but not exclusively) observed in patients with schizophrenia (Braff et al., 2001), a chronic mental illness characterized by widespread psychopathological symptoms. Schizophrenic patients often display metabolic disturbances even prior to the initiation of chronic antipsychotic medication (Thakore et al., 2002; Verma et al., 2009; Kirkpatrick et al., 2012) and are frequently reported to consume saturated fat diets more excessively than healthy controls (reviewed in Dipasquale et al., 2013). Yet, the extent to which excessive HFD intake in this clinical population may actually contribute to the emergence of psychopathological symptoms such as PPI deficiency remains elusive. These considerations prompted us to seek evidence for a possible causal relationship between chronic HFD consumption and sensorimotor gating dysfunctions in mice. To mimic chronic HFD intake, animals were fed an experimental diet with 60% of its calories derived from fat, whereas control low fat diet (LFD) animals were fed a diet with only 10% of its calories from fat. First, we compared the effects of chronic HFD or LFD feeding given throughout adolescent development on PPI in adulthood. In animal models, cognitive effects of chronic HFD exposure have mostly been studied following a dietary intervention restricted to adulthood (Winocur and Greenwood, 2005). Recent findings suggest that chronic HFD exposure during peripubertal development may exert a more extensive negative impact on cognitive functions compared to identical dietary exposure in adulthood (Boitard et al.,

Table 1 Summary of the different experimental series. Animals in cohort 1 were exposed to chronic low fat diet (LFD) or high fat diet (HFD) for 8 weeks throughout adolescent development, that is, from postnatal days (PND) 28 to 84. Behavioral testing was conducted on PND 84 before they were sacrificed for the purpose of post-mortem immunohistochemical analyses 1 week later on PND 91 (not shown in table). Animals in cohort 2 were also exposed to LFD or HFD throughout adolescent development for 8 weeks from PND 28 to 84. These animals were pre-treated with the dopamine receptor antagonist haloperidol (HAL) or vehicle (VEH) before prepulse inhibition testing. Animals in cohort 3 were exposed to HFD or LFD for 4 weeks either between PND 28 and 56 (puberty), or between PND 70 and 98 (adulthood exposure). Prepulse inhibition testing was conducted on the last day of the pubertal or adult dietary intervention (i.e., on PND 56 or 98). Cohort

Experimental series

1

Chronic exposure throughout adolescence

Age when diet was given

Number of weeks on diet

Experimental groups

Group Size

PND 28—84

8

LFD HFD

10 10

PND 28—84

8

LFD/VEH LFD/HAL HFD/VEH HFD/HAL

PND 28—56

4

PND 70—98

4

2 Influence of haloperidol after chronic exposure throughout adolescence 3 Influence of developmental timing of dietary exposure

LFD/Puberty HFD/Puberty LFD/Adulthood HFD/Adulthood

8 9 8 9 12 12 12 12

2564 2012). To target both (and perhaps equally important) stages of maturation, our first series of investigations included HFD exposure throughout adolescent development covering both peripubertal and adult stages. We also correlated PPI scores in HFD and LFD animals with presynaptic dopamine-related changes in dorsal and ventral striatal regions based on the well-established functional role of the striatal dopamine system in sensorimotor gating (Swerdlow et al., 1992, 1994). In addition, we determined whether the dopamine receptor antagonist haloperidol (HAL) might be effective in mitigating the anticipated disrupting effects of HFD on PPI. Finally, we also performed a direct examination of whether the impact of chronic HFD consumption on sensorimotor gating might be influenced by the precise timing of postweaning diet exposures (Boitard et al., 2012). This was achieved by exposing mice to HFD or LFD diets specifically during the peri-pubertal or the adult period before assessment of sensorimotor gating functions.

2. Materials and methods

M.A. Labouesse et al. or LFD either during pubescence (4 weeks from PND 28 to 56) or early adulthood (4 weeks from PND 70 to 98). The pubescent time period (starting from PND 28) was chosen based on previous studies in mice showing that this maturational time window is highly sensitive to the deleterious effects of HFD on brain and behavioral functions (Boitard et al., 2012). Furthermore, this pubescent period is characterized by various maturational changes in the mesolimbic dopamine system (Andersen et al., 1997; Tarazi and Baldessarini, 2000), a neurotransmitter system strongly implicated in the regulation of PPI (Swerdlow et al., 1992, 1994). Longitudinal body weight changes of all cohorts, and longitudinal energy intake of pubescent and adult mice (cohort 3) are reported in Supplementary Figures 1 and 2. Each cohort of mice consisted of animals from multiple independent litters (at least four per diet condition/cohort) in order to minimize confounds arising from litter effects (Zorrilla, 1997). The number of animals included in each experimental condition/cohort is outlined in Table 1, which also summarizes the duration and developmental timing of the dietary manipulations.

2.1. Animals C57BL/6N mice were used throughout the study. C57BL/6N male and female breeding pairs were originally obtained from Charles River (Sulzfeld, Germany) and maintained in our animal facility for the generation of sufficient animals for the different experimental series. All animals were maintained in groups (2—3 per cage) in a temperature- and humidity-controlled (21  1 8C, 55  5%) vivarium under a reversed light—dark cycle (lights off: 07:00 to 19:00 h). Only male mice were included in all experimental series in order to avoid potential confounds arising from sexual dimorphism. All procedures were approved by the Cantonal Veterinary Office of Zurich and are in agreement with the principles of laboratory animal care in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86-23, revised 1985). All efforts were made to minimize the number of animals used and their suffering.

2.2. Chronic HFD and LFD feeding Animals were maintained on standard chow (KLIBA 3433, Provimi Kliba, Kaiseraugst, Switzerland) until the commencement of the HFD or LFD treatments. Experimental diets included a HFD (KLIBA 2127, Provimi Kliba) with 60, 26, and 14% of the energy from fat, carbohydrate and protein, respectively, and a control ingredient and micronutrientmatched LFD (KLIBA 2125, Provimi Kliba), with 10, 65, and 25% of the energy from fat, carbohydrate, and protein, respectively. Diets and water were always accessible ad libitum throughout all experiments. In the first and second experimental series (cohorts 1 and 2), mice had access to HFD or LFD for 8 weeks starting from postnatal day (PND) 28 (Table 1). Hence, these cohorts of animals were exposed to HFD or LFD throughout adolescent development, covering pre-pubertal and post-pubertal stages of maturation (Spear, 2000). These stages are defined based on the gradual attainment of sexual maturity and age-specific behavioral discontinuities from younger to older animals (Spear, 2000). In the third experimental series (cohort 3), the animals had access to HFD

2.3. Prepulse inhibition of the acoustic startle reflex Sensorimotor gating was assessed using the paradigm of prepulse inhibition (PPI) of the acoustic startle reflex. The apparatus consisted of four startle chambers for mice (San Diego Instruments, San Diego, CA) as fully described elsewhere (Meyer et al., 2005). In the demonstration of PPI of the acoustic startle reflex, the animals were presented with a series of discrete trials comprising a mixture of four trial types. These included pulse-alone trials, prepulse-plus-pulse trials, prepulse-alone trials, and no-stimulus trials in which no discrete stimulus other than the constant background noise was presented. The pulse and prepulse stimuli used were in the form of a sudden elevation in broadband white noise level (sustaining for 40 and 20 ms, respectively) from the background (65 dBA), with a rise time of 0.2—1.0 ms. In all trials, three different intensities of pulse (100, 110, and 120 dBA) and three intensities of prepulse (71, 77, and 83 dBA, which corresponded to 6, 12 and 18 dBA above background, respectively) were used. The stimulus-onset asynchrony of the prepulse and pulse stimuli on all prepulse-pluspulse trials was 100 ms (onset-to-onset). The protocol used for the PPI test was extensively validated in our laboratory before (e.g., Vuillermot et al., 2010, 2011; Pacheco-Lo ´pez et al., 2013). The animals were placed into the Plexiglas enclosure and adapted to the apparatus for 2 min before the first trial began. The first six trials consisted of six startle-alone trials; such trials served to habituate and stabilize the animals’ startle response and were not included in the analysis. Subsequently, the animals were presented with 10 blocks of discrete test trials. Each block consisted of (i) three pulse-alone trials (100, 110, or 120 dBA), (ii) three prepulse-alone trials (+6, +12, or +18 dBA above background), (iii) nine possible combinations of prepulse-plus-pulse trials (three levels of pulse  three levels of prepulse), and (iv) one no-stimulus trials. The 16 discrete trials within each block were presented in a pseudorandom order, with a variable interval of 15 s on average (ranging from 10 to 20 s). For each

High fat diet impairs prepulse inhibition of the three pulse intensities (100, 110, or 120 dBA) PPI was indexed by percent inhibition of the startle response obtained in the pulse-alone trials by the following expression: 100%  (1 [mean reactivity on prepulse-plus-pulse trials/mean reactivity on pulse-alone trials]), for each animal, and at each of the three possible prepulse intensities (+6, +12, or +18 dBA above background). The test of PPI was always conducted during the dark phase of the reversed light-dark cycle and was performed at the end of the duration of HFD or LFD exposure as outlined in Table 1. Hence, the animals had access to HFD or LFD on the day of PPI testing. Animals in cohorts 1 and 3 were tested without exposure to any other manipulation, whereas animals in cohort 2 were pre-treated with HAL or vehicle (VEH) 45 min prior to PPI testing (see below).

2.4. Drugs In the second experimental series (cohort 2), we assessed the effects of systemic administration of the dopamine receptor antagonist HAL relative to VEH in HFD and LFD animals. HAL was obtained from Janssen-Cilag (Baar, Switzerland) in the form of ampoules consisting of 5 mg of HAL in 1 ml of saline solvent containing minimal amounts of lactic acid. It was further diluted in 0.9% physiological NaCl (=saline) solution to obtain the desired concentration. Saline solution served as the corresponding VEH treatment. HAL was administered at a dose of 0.2 mg/kg based on previous dose responses (e.g., Yee et al., 2005). All solutions were injected via the intraperitoneal (IP) route using an injection volume of 5 ml/kg. They were administered 45 min before PPI testing according to injection protocols established before (Yee et al., 2005). The HAL solution was freshly prepared on the day of testing.

2.5. Collection of brain samples In the first experimental series (cohort 1), the animals were sacrificed for the purpose of post-mortem immunohistochemical investigations (see below) 1 week following the completion of PPI testing. A 1-week resting period between PPI testing and sacrifice was allowed to minimize potential confounds arising from possible stress induced by prior behavioral testing. During this resting period, the animals had access to HFD or LFD until immediately before sacrifice. The animals were deeply anesthetized with an overdose of sodium pentobarbital (Cantonal Pharmacy, Zurich, Switzerland) and perfused transcardially with 0.9% NaCl, followed by 4% phosphate-buffered paraformaldehyde solution containing 15% picric acid. The dissected brains were postfixed in the same fixative for 6 h and processed for antigen retrieval involving overnight incubation in citric acid buffer (pH 4.5) followed by a 90 s microwave treatment at 480 W according to previously established protocols (Vuillermot et al., 2010, 2011). The brains were then cryoprotected using 30% sucrose in PBS, frozen with powdered dry ice, and stored at 80 8C until further processing.

2.6. Immunohistochemistry Perfused brain samples were cut coronally at 30 mm thickness from frozen blocks with a sliding microtome. Eight series of

2565 sections were collected, rinsed in PBS, and stored at 20 8C in antifreeze solution (30% glycerol and 30% ethylene glycol in PBS at 25 mM and pH 7.4) until further processing. For immunohistochemical staining, the slices were rinsed three times for 10 min in PBS, and blocking was done in PBS, 0.3% Triton X-100, 5% normal serum for 1 h at room temperature. Rabbit anti-tyrosine hydroxylase (TH; Santa Cruz Biotechnology, Heidelberg, Germany; diluted: 1:500) diluted in PBS containing 0.3% Triton X-100 and 2% normal serum was used as the primary antibody. The sections were incubated free floating overnight at room temperature, and after three washes with PBS (10 min each) incubated for 1 h with the biotinylated secondary antibody diluted 1:500 in PBS containing 2% NGS and 0.3% Triton X-100. Sections were washed again three times for 10 min in PBS and incubated with Vectastain Kit (Vector Laboratories) diluted in PBS for 1 h. After three rinses in Tris—HCl 0.1 M, pH 7.4, the sections were stained with 1.25% 3,3-diaminobenzidine and 0.08% H2O2 for 10— 15 min, rinsed again four times in PBS, dehydrated, and coverslipped with Eukitt (Kindler, Freiburg, Germany). The chosen immunohistochemical method has been validated before and has been proven to be an efficient and reliable procedure to stain and quantify TH protein in striatal tissue of adult C57BL/6 mice (e.g., Vuillermot et al., 2010, 2011).

2.7. Optical densitometry Quantification of TH immunoreactivity in dorsal and ventral striatal regions was achieved by means of optical densitometry using ImageJ software (NIH). Optical densitometry was chosen because TH protein is highly enriched at synaptic sites in the areas of interest. Digital images were acquired at a magnification of 2.5  (NA 0.075) using a digital camera (Axiocam MRc5, Zeiss) mounted on a Zeiss Axioplan microscope. Exposure times were set so that pixel brightness was never saturated. Pixel brightness was measured in the respective areas of one randomly selected brain hemisphere. In addition, pixel brightness was measured in the corpus callosum as background area. The background-corrected optical densities were averaged per brain region and animal. Five to six coronal brain sections per animal were analyzed. All immunohistochemical preparations were quantified in the dorsal striatum (=caudate putamen, CPu), nucleus accumbens core (NAc core), and nucleus accumbens shell (NAc shell) with the experimenter being blind to the experimental conditions.

2.8. Delineation of brain areas All brain areas of interest were delineated according to ‘‘The Mouse Brain in Stereotaxic Coordinates’’ by Franklin and Paxinos (2008). The analyses conducted in the NAc core included sections ranging from bregma +1.94 to +0.74 mm. The dorsal border of the NAc core lined the ventral side of the CPu, while its ventral border followed the dorsal side of the NAc shell. The lateral border of the NAc core lined the medial border of the intermediate endopiriform claustrum, and its medial border was adjacent to the lateral side of the NAc shell. The analyses conducted in the NAc shell included sections ranging from bregma +1.94 to +0.86 mm. The dorsal side of the NAc shell followed the ventral border of the NAc

2566 core, except for the most rostral section (bregma +1.94 mm), in which it lined the ventral border of the CPu. The ventral side of the NAc shell lined the dorsal border of the ventral pallidum. The lateral border of the NAc shell was adjacent to the medial side of the NAc core, and the medial NAc shell border followed the lateral border of the lateral septal nucleus. The analyses conducted in the CPu included sections ranging from bregma +1.94 to +0.14 mm. The dorsal border of the CPu lined the ventral side of the forceps minor of the corpus callosum for rostral slices (i.e., from bregma +1.94 to +1.34 mm), and the ventral side of the external capsule for more caudal sections (i.e., from bregma +1.18 to +0.14 mm). The ventral border of the CPu lined the dorsal border of the NAc core for rostral slices (i.e., from bregma +1.94 to +1.70 mm). For more caudal regions of the CPu (i.e., from bregma +1.54 to +0.14 mm), a horizontal line was drawn at the tip of the lateral ventricle to delineate its ventral border. The lateral and medial borders of the CPu were always adjacent to the medial border of the lateral part of the

M.A. Labouesse et al. forceps minor of the corpus callosum, and to the lateral border of the medial part of the forceps minor of the corpus callosum, respectively.

2.9. Statistical analyses In the first experimental series (cohort 1), % PPI was analyzed using a 2  3  3 (diet  prepulse level  pulse level) parametric ANOVA, and reactivities to pulse-alone trials and prepulse-alone trials were analyzed using 2  3 (diet  pulse pulse level) and 2  2 (diet  prepulse level) parametric ANOVAs, respectively. For cohort 2, % PPI was analyzed using a 2  2  3  3 (diet  drug  prepulse level  pulse level) parametric ANOVA, and reactivities to pulse-alone trials and prepulse-alone trials were analyzed using 2  2  3 (diet  drug  pulse level) and 2  2  2 (diet  drug  prepulse level) parametric ANOVAs, respectively. For cohort 3, % PPI was analyzed using a 2  2  3  3 (diet  period  prepulse level  pulse level) parametric ANOVA, and reactivities to

Figure 1 Prepulse inhibition deficits in adult mice chronically exposed to high fat diet (HFD) compared to low fat diet (LFD) throughout adolescent development. (A) The line plot shows percent prepulse inhibition (% PPI) as a function of the three distinct pulse levels (P-100, P-110, and P-120, which correspond to 100, 110 and 120 dBA) and prepulse levels (+6, +12 and +18 dBA above background of 65 dBA); and the bar plot depicts the mean % PPI across all prepulse and pulse stimuli used. *p < 0.001, reflecting the main effect of diet on mean % PPI. (B) The line plot illustrates prepulse-elicited reactivity (in arbitrary units, AU) as a function of prepulse intensity. *p < 0.05, reflecting the significant difference between HFD and LFD mice at the +18 dBA prepulse condition. (C) The line plot shows startle reactivity (in arbitrary units, AU) to pulse-alone trials for the three different pulse levels. N = 10 per group; all values are means  SEM.

High fat diet impairs prepulse inhibition pulse-alone trials and prepulse-alone trials were analyzed using 2  2  3 (diet  period  pulse level) and 2  2  2 (diet  drug  prepulse level) parametric ANOVAs, respectively. These analyses were followed by Fisher’s LSD post hoc comparisons or restricted ANOVAs whenever appropriate. Based on the findings obtained in cohort 1, a priori comparisons between vehicle-treated LFD and HFD and between HAL- and VEH-exposed HFD animals were also conducted using independent Student’s t tests (two-tailed) with Bonferroni—Holm corrections. TH-immunoreactivity in striatal areas was analyzed using independent Student’s t tests (twotailed). Correlative analyses between mean % PPI measures and TH-immunoreactivity in striatal areas were first performed using Pearson’s product moment correlations, followed by first-order partial correlations partialling for the two diet conditions. The partial correlations were used to control for the effects of the independent variable ‘‘diet’’. p < 0.05 was considered significant, except for the a priori comparisons using the Bonferroni—Holm test ( pcorrected < 0.0167). All statistical analyses were conducted using the statistical software SPSS version 13.0 (SPSS Inc., USA).

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3. Results 3.1. Chronic HFD consumption throughout adolescent development impairs PPI in adulthood First, we explored the consequences of chronic HFD given throughout adolescent development (PND 28—84) on PPI in adulthood. The analysis of % PPI revealed that HFD mice displayed a significant overall reduction in % PPI compared to LFD mice (main effect of treatment: F (1,18) = 6.63, p < 0.05). As summarized in Fig. 1A, the mean PPI scores were 25% lower in HFD than in LFD mice. The PPI-disrupting effects of HFD largely emerged independent of the precise pulse and prepulse stimuli used, i.e., the interactions between diet and pulse or prepulse levels failed to reach statistical significance. As expected, the reactivity to prepulse-alone trials increased with increasing prepulse levels (main effect of prepulse levels: F (2,36) = 12.46, p < 0.001). This effect was influenced by diet as indicated by the significant interaction between diet and prepulse levels (F (2,36) = 6.02, p < 0.01). Indeed, HFD mice displayed a lower ( p < 0.05) prepulseinduced reactivity at the highest prepulse level (+18 dB from

Figure 2 Striatal tyrosine hydroxylase (TH) immunoreactivity in adult mice chronically exposed to high fat diet (HFD) or low fat diet (LFD) throughout adolescent development. (A) The bar plots show the relative optical density of TH (in arbitrary units, AU) measured in dorsal striatal regions (caudate putamen, CPu) and ventral striatal regions (nucleus accumbens core and shell, NAc core and shell) in LFD and HFD mice. *p < 0.05 and ***p < 0.001. N = 9—10 per group; all values are means  SEM. (B) Coronal brain sections of representative LFD and HFD mice stained with anti-TH antibody. The sections are taken at the level of the striatum: 1 = CPu, 2a = NAc core, 2b = NAc shell. Note the increase of TH immunoreactivity emerging especially in the NAc core and shell subregions of HFD mice (indicated by the arrow heads) relative to LFD mice.

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Figure 3 Correlations between mean percent prepulse inhibition (mean % PPI) and the relative optical density of tyrosine hydroxylase (TH OD) in adult mice chronically exposed to high fat diet (HFD) or low fat diet (LFD) throughout adolescent development. Scatter plots are depicted for correlations between mean % PPI and TH OD in dorsal striatal regions (caudate putamen, CPu) and ventral striatal regions (nucleus accumbens core and shell, NAc core and shell). The correlations were performed using first-order partial correlations, controlling for diet [LFD, white symbols; HFD, gray symbols). The regression lines represent the partial correlative analyses adjusted for dietary treatment. N = 19 (9 LFD, 10 HFD) and df = 16 for each correlation.

background) than LFD mice (Fig. 1B). On the other hand, the diet did not affect the startle reaction to the distinct pulse used (Fig. 1C). Startle reactivity generally increased as a function of increasing pulse levels (main effect of pulse: F (2,36) = 14.19, p < 0.001), and this effect was similar in HFD and LFD mice (Fig. 1C).

3.2. Chronic HFD consumption throughout adolescent development increases THimmunoreactivity in the adult NAc: correlation with PPI-disrupting effects of HFD Deficits in PPI have often been causally linked to increased dopaminergic activity in the ventral striatum (Swerdlow et al., 1992, 1994). Here, we compared the relative density of TH-positive fibers in the dorsal (CPu) and ventral (NAc core and shell) striatum of adult mice chronically exposed to HFD or LFD throughout adolescent development to assess possible influences of the diet on presynaptic dopaminergic abnormalities. TH is the rate-limiting enzyme of dopamine synthesis in vivo and can therefore be used as a presynaptic dopaminergic marker in striatal regions (Bacopoulos and Bhatnagar, 1977). HFD mice had a significantly higher level of TH-immunoreactivity than LFD mice especially in the NAc core (t17 = 2.59, p < 0.05) and NAc shell (t17 = 4.85, p < 0.001) (Fig. 2). No group differences were detected with respect to TH-immunoreactivity in the CPu (Fig. 2). To further substantiate a possible role of striatal dopaminergic abnormalities in the emergence of HFD-induced PPI deficits, we correlated the mean % PPI scores of HFD and LFD mice (Fig. 1) with TH immunoreactivity in CPu, NAc core, and NAc shell (Fig. 2). Initial unprotected Pearson’s product moment correlations revealed a significant negative correlation between mean % PPI and TH immunoreactivity in the NAc shell (r = 0.67, df = 17, n = 19, p < 0.01). Importantly, this correlation remained significant using first-order partial correlations controlling for the between-subjects factor of diet (r = 0.53, df = 16, n = 19, p < 0.05; Fig. 3). Neither the

correlation between mean % PPI and TH immunoreactivity in the NAc core, nor between mean % PPI and TH immunoreactivity in the CPu, reached statistical significance using Pearson’s product moment or first-order partial correlations (Fig. 3).

3.3. HFD-induced PPI deficits are attenuated by systemic treatment with the dopamine receptor blocker HAL The presence of increased TH immunoreactivity in the NAc of HFD-exposed animals (Fig. 2) and the negative correlation between mean % PPI and NAc shell TH density indicates that the HFD-induced PPI deficiency may result from increased (accumbal) dopaminergic activity. To seek pharmacological evidence for this possibility, we compared the effects of acute systemic administration of the dopamine receptor antagonist HAL (0.2, mg/kg, i.p) or vehicle (VEH = saline) on PPI in HFD and LFD mice. Consistent with our initial findings (Fig. 1A) we found that HFD exposure led to a significant reduction in % PPI, as indicated by the significant main effect of diet (F (1,30) = 9.59, p < 0.01) and by the a priori comparison between VEH-treated HFD and VEH-treated LFD mice ( p < 0.01; Fig. 4A). As expected (Flood et al., 2011), HAL treatment generally led to a significant increase in the mean % PPI scores as supported by the presence of a significant main effect of drug treatment (F (1,30) = 8.60, p < 0.05). Notably, HAL treatment was effective in significantly increasing % PPI scores in HFD mice, as supported by the a priori comparison between VEH- and HAL-treated HFD mice ( p < 0.01; Fig. 4A). The reactivity to prepulse-alone trials increased with increasing prepulse levels (main effect of prepulse levels: F (2,60) = 23.85, p < 0.001). In line with our initial findings (Fig. 1B), VEH-treated HFD animals displayed a significant decrease in prepulse-induced reactivity specifically at the highest prepulse condition (+18 dB from background) compared to VEH-treated LFD mice (Fig. 4B). HAL administration did not mitigate this effect, but instead, it significantly

High fat diet impairs prepulse inhibition decreased prepulse-induced reactivity at the highest prepulse condition in LFD mice (Fig. 4B). This pattern of results led to a significant interaction between diet and drug (F (1,30) = 6.04, p < 0.05) and between diet, drug and prepulse intensity (F (2,60) = 4.58, p < 0.05). Subsequent post hoc group comparisons at the highest prepulse condition (+18 dB from background) confirmed the significant difference between VEH-treated LFD mice and all other groups (all p < 0.01). The analysis of the responses to pulse-alone trials revealed increased startle reactivity as a function of increasing pulse levels (main effect of pulse levels: F (2,60) = 55.46, p < 0.001). HFD mice generally displayed increased startle reactivity especially at the highest (120 dB) pulse conditions (Fig. 4C). This effect of diet emerged independently of the drug conditions, leading to a significant main effect of diet (F (1,30) = 7.64, p < 0.01) and its interaction with pulse levels (F (2,60) = 12.74, p < 0.001). Post hoc comparisons of startle reactivity confirmed the significant difference between HFD and LFD animals ( p < 0.01) at the highest pulse condition (Fig. 4C).

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3.4. Peripubertal but not adult HFD exposure impairs PPI Recent investigations in mice suggest that chronic HFD exposure during peripubertal development may exert a more extensive negative impact on cognitive functions compared to identical dietary exposure in adulthood (Boitard et al., 2012). To test whether the precise timing of post-weaning diet exposures may similarly influence the impact of HFD consumption on sensorimotor gating, we exposed mice for 4 weeks to HFD or LFD either between PND 28 and 56 (pubertal exposure) or between PND 70 and 98 (adult exposure) and explored the influence of these dietary manipulations on PPI as described before. As shown in Fig. 5A, we found that pubertal but not adult HFD exposure markedly reduced % PPI scores. This impression was supported by the presence of a significant interaction between diet and period (F (1,44) = 6.05, p < 0.05) and by subsequent post hoc group comparisons verifying a significant difference between pubertal LFD and pubertal HFD exposure

Figure 4 Effects of systemic haloperidol treatment on prepulse inhibition in adult mice chronically exposed to high fat diet (HFD) compared to low fat diet (LFD) throughout adolescent development. The animals were pre-treated with haloperidol (HAL; 0.2 mg/kg, IP) or vehicle (VEH) solution 45 min prior to testing. (A) The line plot shows percent prepulse inhibition (% PPI) as a function of the three distinct pulse levels (P-100, P-110, and P-120, which correspond to 100, 110 and 120 dBA) and prepulse levels (+6, +12 and +18 dBA above background of 65 dBA); and the bar plot depicts the mean % PPI across all prepulse and pulse stimuli used. **p < 0.01, reflecting significant differences in mean % PPI based on Holm—Bonferroni comparisons. (B) The line plot illustrates prepulse-elicited reactivity (in arbitrary units, AU) as a function of prepulse intensity. **p < 0.01, reflecting the significant difference between LFD/VEH mice and all other groups at the +18 dBA prepulse condition. (C) The line plot shows startle reactivity (in arbitrary units, AU) to pulse-alone trials for the three different pulse levels. §p < 0.01, reflecting the significant increase in startle reactivity at the highest (120 dBA) pulse displayed by HFD compared to LFD mice. N = 8—9 per group; all values are means  SEM.

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Figure 5 Influence of the developmental timing of high fat diet (HFD) compared to low fat diet (LFD) exposure on prepulse inhibition. Mice were exposed for 4 weeks to HFD or LFD either between postnatal days (PND) 28 and 56 (puberty), or between PND 70 and 98 (adulthood exposure) before testing. (A) The line plot shows percent prepulse inhibition (% PPI) as a function of the three distinct pulse levels (P-100, P-110, and P-120, which correspond to 100, 110 and 120 dBA) and prepulse levels (+6, +12 and +18 dBA above background of 65 dBA); and the bar plot depicts the mean % PPI across all prepulse and pulse stimuli used. *p < 0.05 and **p < 0.01, based on post hoc group comparisons. (B) The line plot illustrates prepulse-elicited reactivity (in arbitrary units, AU) as a function of prepulse intensity. §p < 0.01, reflecting the significant increase in prepulse-induced reactivity at the highest prepulse level (+18 dBA above background) displayed by animals exposed to the dietary interventions in adulthood compared to puberty. (C) The line plot shows startle reactivity (in arbitrary units, AU) to pulse-alone trials for the three different pulse levels. #p < 0.01, reflecting the significant increase in startle reactivity at the middle (110 dBA) and highest (120 dBA) pulse displayed by HFD compared to LFD mice. N = 12 per group; all values are means  SEM.

( p < 0.01) and between pubertal and adult HFD mice ( p < 0.01). Neither pubertal nor adult HFD exposure significantly affected prepulse-induced reactivity. Mice with the adult dietary (HFD or LFD) exposure generally showed increased prepulse-induced reactivity, especially at the highest (+18 dB above background) prepulse condition compared to animals with peripubertal diet exposure (Fig. 5B). This led to a significant main effect of period (F (1,44) = 11.21, p < 0.01) and its interaction with prepulse levels (F (2,88) = 3.90, p < 0.05). Subsequent post hoc comparisons confirmed the significant main effect of period on prepulse-induced reactivity at the highest prepulse condition ( p < 0.01). The analysis of pulse-alone trials confirmed that the overall startle reactivity increased with increasing pulse levels, leading to a significant main effect of pulse (F (2,88) = 49.36, p < 0.001). Mice with the peripubertal dietary (HFD or LFD) exposure generally showed increased startle reactivity at the 110-dB and 120-dB pulse conditions compared to animals

with adult diet exposure (Fig. 5C). These impressions were supported by the presence of a significant main effect of period (F (1,44) = 12.43, p < 0.01) and its interaction with pulse levels (F (2,88) = 15.17, p < 0.001). Post hoc group comparisons verified the significant main effect of period on pulse-induced startle reactivity at 110-dB and 120-dB pulse conditions (both p < 0.01).

4. Discussion The present study is the first demonstration that chronic HFD consumption in mice impairs sensorimotor gating in the form of PPI of the acoustic startle reflex. Our findings thus add to a growing body of literature documenting negative influences of chronic HFD exposure on behavioral and cognitive functions in various rodent models and human clinical conditions (reviewed in Winocur and Greenwood, 2005; Francis and Stevenson, 2013). Perhaps even more importantly, our study

High fat diet impairs prepulse inhibition extends those previous reports in as much as we identify a novel impact of chronic HFD on a specific and elementary form of pre-attentive information processing that is conserved amongst a variety of species, including rodents and humans (Swerdlow et al., 1999). In neuropsychological terms, sensorimotor gating reflects the ability to filter intrusive sensory-motor information (Hoffman and Searle, 1965; Graham, 1975), and the underlying inability to gate intrusive sensory-motor information is often reflected by the presence of overt PPI impairments as seen here in HFD-exposed mice (Braff et al., 2001). The neural circuitry of PPI appears to be highly complex and likely involves multiple neurotransmitter systems (Swerdlow et al., 2001). However, converging evidence supports a key role of the central dopamine system in the regulation and modulation of PPI (e.g., Mansbach et al., 1988; Swerdlow et al., 1992, 1994; Ralph-Williams et al., 2003; Yee et al., 2005). The current consensus is that manipulations leading to enhanced presynaptic dopamine release and/or dopamine receptor stimulation especially in ventral striatal structures promote the disruption of PPI, whereas blockade of dopamine receptor signaling potentiates PPI and/or restores PPI deficits associated with dopaminergic imbalances. Here, we show that the same animals that display significant PPI deficits following HFD exposure throughout adolescent development also exhibit increased TH immunoreactivity in the NAc core and shell subregions. TH is the rate-limiting enzyme of presynaptic dopamine synthesis (Bacopoulos and Bhatnagar, 1977), so that changes in the expression of this enzyme is often interpreted as and associated with increased presynaptic dopaminergic activity in dopaminergic systems such as the mesoaccumbal dopamine pathway (e.g., Vuillermot et al., 2010). Consistent with previous findings in mice (Vuillermot et al., 2011), we further found that the magnitude of PPI negatively correlated with the level of TH immunoreactivity in the NAc shell. Together, these findings suggest that dopaminergic imbalances in ventral striatal structures may assume an important role in precipitating the deficits in PPI following chronic HFD exposure. Additional support for this hypothesis is obtained by our findings showing that the HFD-induced PPI deficits are rectified by acute systemic treatment with the dopamine receptor antagonist HAL. It would be interesting to further explore the efficacy of intraaccumbal dopamine receptor antagonist infusions relative to infusions into other selected brain areas in order to further substantiate a role for dopaminergic imbalances in the ventral striatum in the present model of HFD-induced PPI deficiency. HAL is known to exert sedative and motor-depressing effects especially when given at high doses (Duncan et al., 2006). In the present study, HFD-VEH animals displayed reduced levels of % PPI as compared to HFD-HAL treated animals, but these two groups showed similar startle reactivities to pulse-alone trials, suggesting that HAL treatment at the chosen dose did not induce severe motor impairments. Furthermore, these results indicate that the beneficial effects of HAL on % PPI in HFD mice emerge independently of possible effects on startle reactivity. However, we found that baseline startle reactivities were not readily consistent between the different HFD cohorts. HFD mice did not display altered startle reactivity compared to LFD mice when they were tested at basal conditions, i.e. in the absence of

2571 additional interventions before PPI testing. In contrast, HFD exposure increased startle reactivity compared to LFD when the animals received additional substance (VEH or HAL) administration before the PPI test. It thus appears that the dietary manipulation does not induce robust effects on startle reactivity. Rather, differences in pulse-induced startle reactivity following chronic HFD may only be unmasked by additional exposure to (mild) stress such as injections, a phenomenon that has been documented previously (Vuillermot et al., 2010). It is important to further point out that both VEH- and HAL-treated HFD mice showed increased startle reactivity, but only VEH-treated (but not HAL-treated) HFD mice displayed a significant reduction in % PPI. This dissociation of effects suggests that HAL rescued the HFDinduced PPI deficits independently of the differences in pulse-induced startle reactivity. Our study further emphasizes that the precise developmental timing critically determines the extent to which HFD can impair sensorimotor gating. Indeed, we found that a 4week HFD regimen during peripubertal development (PND 28—56) was sufficient to induce significant PPI deficits. On the other hand, identical HFD exposure restricted to early adulthood (PND 70—98) spared PPI. These findings readily suggest that the peripubertal period is more sensitive than early adulthood with respect to the negative influences of chronic HFD consumption on sensorimotor gating. It is intriguing to note that a similar age-dependent association has recently been documented in a mouse model comparing peripubertal versus adult HFD exposure on hippocampus-dependent cognitive functions: Boitard et al. (2012) found that chronic HFD exposure across peripubertal (starting PND 21) but not adult (starting PND 84) age led to impaired relational memory flexibility as assessed in the radial arm maze and to impaired hippocampal neurogenesis. The present findings thus further support the hypothesis that the peripubertal period is a critical maturational window that is highly sensitive to the negative influences of chronic HFD consumption on brain and behavioral functions (Boitard et al., 2012). This concept of a ‘‘sensitive peripubertal window’’ is consistent with findings from a number of studies suggesting that peripuberty is particularly vulnerable to the disrupting effects of other environmental influences such as stress (Spear, 2009; Giovanoli et al., 2013) or drugs of abuse (Richetto et al., 2013). Differences in the precise timing of chronic HFD exposure may also explain some of the seemingly discrepant effects of the diet on dopaminergic parameters. As discussed above, the present findings are generally indicative of increased dopaminergic activity following chronic HFD consumption. These outcomes are congruent with the findings by Naef et al. (2008) who demonstrated that HFD exposure during the perinatal period, including the pre-weaning lactation period, leads to enhanced TH levels in the NAc and ventral tegmental area. However, such ‘‘hyperdopaminergic’’ effects contrast several reports of blunted dopaminergic activity following chronic HFD exposure in rats or mice when the dietary manipulations were initiated once the animals had reached adulthood (e.g., Davis et al., 2008; Li et al., 2009; Vucetic et al., 2012; Sharma et al., 2011). For example, Sharma et al. (2011) found that chronic HFD exposure in adult mice reduced accumbal TH levels, together with compensatory changes of dopamine receptors. An additional investigation of striatal TH levels in mice exposed to HFD in pubescence

2572 alone, and in mice fed HFD in adulthood alone, could be used to strengthen the presumed links between HFD exposure, dopaminergic changes, and disruption of PPI discussed above. One expectation would be that animals with HFD exposure in adulthood may not show increased (accumbal) TH levels as they do not show impairments in sensorimotor gating. Additional work seems thus highly warranted in order to advance our understanding of how different maturational stages can influence the sensitivity to and/or nature of diet-induced changes in brain and behavior, including dopamine-associated functions. Related to this, the molecular mechanisms underlying the emergence of HFD-induced dopaminergic changes and associated behavioral abnormalities such as PPI deficiency also largely remain elusive. In the mouse, dopaminergic innervation of the dorsal and ventral striatal target regions is mostly completed by the end of the first postnatal week (Van den Heuvel and Pasterkamp, 2008). Therefore, it seems unlikely that the dopaminergic changes in HFD-exposed animals described herein stem from a disruption of the initial formation of the mesolimbic dopamine system. Yet, the central dopamine system undergoes considerable structural and functional modifications across peripubertal maturation, including striatal dopamine receptor pruning (Teicher et al., 1995; Andersen et al., 1997) and increased dopaminergic innervation of the medial prefrontal cortex (Naneix et al., 2012). One possibility would therefore be that excessive HFD consumption, especially when initiated during peripubertal stages of life, could interfere with the normal maturation of the mesolimbic dopamine system. An alternative (but not mutually exclusive) possibility is that the molecular mechanisms by which HFD exposure alters dopamine-associated functions involve epigenetic modifications of relevant dopaminergic targets. A proof of concept of such epigenetic modifications has recently been obtained in a mouse model of diet-induced obesity, in which mice continuously exposed to HFD from weaning displayed altered DNA methylation patterns in the promoter regions of TH and of the dopamine transporter (Vucetic et al., 2012). Finally, a number of metabolic circulating factors such as leptin, insulin and glucose, functionally interact with dopamine signaling (Bello and Hajnal, 2006; Fulton et al., 2006; Laboue `be et al., 2013; Perry et al., 2010) and could therefore contribute to the changes seen in accumbal dopaminergic activity and PPI performance after chronic HFD. Indeed, these endocrine factors are strongly affected by chronic HFD consumption (Liu et al., 2012), regardless of whether the dietary manipulation is initiated in puberty or adulthood (Boitard et al., 2012). We acknowledge that the lack of endocrine measurements is another limitation of our study. Therefore, additional studies are thus highly warranted in order to directly explore the potential involvement of such endocrine factors in dopamine-related imbalances after peripubertal or adult HFD exposure, and their (causal) relation to sensorimotor gating deficits. Chronic exposure to protein restriction may yet provide another potential mechanism that contributes to the emergence of HFD-induced PPI impairments. In our experimental settings, there is a 9% difference in protein contents between HFD and LFD treatments resulting from substitution of protein by increased amounts of fat. It has been shown before that early protein restriction (starting from prenatal periods) can alter dopamine circuitry (Vucetic et al., 2010),

M.A. Labouesse et al. highlighting the possibility that such processes may also be operational in our model. One possibility to directly explore this hypothesis would be to use HFD in which fat is mainly substituted by carbohydrates (instead of protein), thus allowing maintenance of protein contents at normal levels. We would also like to emphasize that our findings may be relevant to certain aspects of schizophrenia and related disorders. Indeed, deficits in PPI are frequently (but not exclusively) observed in patients with schizophrenia (Braff et al., 2001) and in a plethora of preclinical animal models of the disease (Swerdlow et al., 2000; Peleg-Raibstein et al., 2012). The fact that the HFD-induced PPI deficits can be mitigated by the clinically effective antipsychotic drug HAL further adds to this notion. It is also of note that schizophrenic patients often display an excessive consumption of saturated fat diets compared to healthy controls (reviewed in Dipasquale et al., 2013). By identifying a causal relationship between chronic HFD and emergence of PPI deficiency, our findings thus support the hypothesis that adverse nutritional factors may contribute to the clinical manifestation of behavioral abnormalities relevant to schizophrenia. This interpretation still needs to be met with caution, however, because the extent to which chronic HFD can induce other schizophrenia-relevant behavioral and cognitive disturbances still awaits direct exploration. In conclusion, our findings emphasize that pre-attentive information processing as assessed by the PPI paradigm is highly sensitive to chronic HFD consumption, especially when initiated during peripubertal maturation. It is likely that the disrupting effects of HFD on sensorimotor gating involve, at least in part, dopaminergic mechanisms. Our study adds further weight to the emerging role of dietary influences on brain and behavioral functions and draws particular attention to the negative impact of excessive HFD consumption across peripubertal brain maturation.

Conflict of interest All authors declare that they have no conflicts of interest to disclose.

Acknowledgements We remain indebted to Elisabeth Weber for her technical assistance in the immunohistochemical analyses. This work was supported by ETH Zurich and partially by the European Union Seventh Framework Programme (FP7/2007—2011) under Grant Agreement No. 259679 awarded to UM.

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.psyneuen.2013.06.003.

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