the ketogenic diet modifies social and metabolic

E-Mail [email protected]

Original Paper

Dev Neurosci DOI: 10.1159/000362645

The Ketogenic Diet Modifies Social and Metabolic Alterations Identified in the PrenatalValproic Acid Model of Autism Spectrum Disorder

Younghee Ahn a Mariam Narous a Rose Tobias a Jong M. Rho a, b

Richelle Mychasiuk a

Departments of a Pediatrics and b Clinical Neurosciences, Alberta Children’s Hospital Research Institute for Child and Maternal Health, Faculty of Medicine, University of Calgary, Calgary, Alta. , Canada

was tested with the play-fighting paradigm and rats were then sacrificed for mitochondrial bioenergetic analysis. The offspring exposed to VPA prenatally demonstrated a signifi-cant decrease in the number of play initiations/attacks and this was reversed with the KD. Prenatal VPA exposure also disrupted the pattern of play responses; VPA/SD animals used complete rotations more often than saline control ani-mals. Treatment with the KD did not affect the number of complete rotations. In addition, while prenatal exposure to VPA altered mitochondrial respiration, the KD was able to restore aspects of bioenergetic dysfunction. As the KD was able to modify complex social behaviors and mitochondrial respiration, it may be a useful treatment option for ASD. Fu-ture studies will need to examine the effectiveness of the KD to reverse the two additional core deficits of ASD and to ex-plore various treatment regimens to determine optimal treatment duration and formulation.

© 2014 S. Karger AG, Basel

Introduction

Autism spectrum disorder (ASD) is a highly prevalent, pervasive neurodevelopmental disorder characterized by abnormal social interactions, communication deficits and

Key Words

Play behavior · Mitochondria · Autism · Valproic acid · Metabolism · Respiration

Abstract

Autism spectrum disorder (ASD) is a highly prevalent neuro-developmental disorder characterized by abnormal social interactions, communication deficits and stereotyped or re-petitive behaviors. Although the etiology of ASD remains elusive, converging lines of research indicate that mitochon-drial dysfunction may play a substantive role in disease pathophysiology. Without an established causal link, the generation of therapeutic targets for ASD has been relative-ly unsuccessful and has focused solely on individual symp-toms. The ketogenic diet (KD) is a high-fat low-carbohydrate diet that has previously been used for the treatment of in-tractable epilepsy and is known to enhance mitochondrial function. The purpose of this study was to determine if the KD could reverse the social deficits and mitochondrial dys-function identified in the prenatal valproic acid (VPA) rodent model of ASD. Sprague-Dawley dams were administered VPA or saline on gestational day 12.5. The pups were treated with the KD or their standard diet (SD) for 10 days beginning on postnatal day 21 (PD21). On PD35 juvenile play behavior

Received: October 28, 2013 Accepted after revision: April 2, 2014 Published online: July 8, 2014

Richelle Mychasiuk, PhD Department of Paediatrics, University of Calgary Heritage Medical Research Building Room 273, 3330 Hospital Drive, NW Calgary, AB T2N 1N4 (Canada) E-Mail rmmychas @ ucalgary.ca

© 2014 S. Karger AG, Basel0378–5866/14/0000–0000$39.50/0

www.karger.com/dne

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Ahn /Narous /Tobias /Rho /Mychasiuk

Dev NeurosciDOI: 10.1159/000362645

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ritualistic or repetitive behaviors [1–3] . The etiology of au-tism is not known, but it is likely that genetic and environ-mental factors combine to produce the hallmark neurode-velopmental features [4] . Recent studies have reported the development of more clinically relevant animal models of ASD, enabling researchers to rapidly explore the molecu-lar, synaptic and cellular components of the disorder [5] . Valproic acid (VPA) has traditionally been used as a phar-macological anticonvulsant for the treatment of epilepsy and migraine and has recently been implicated in human cases of ASD [6–8] . Analysis of the human epidemiologi-cal literature suggested that VPA exposure was most high-ly correlated with ASD when administered to the mother during pregnancy at the time of neural tube closure in the embryonic period (embryonic day 20–24) [9, 10] . Based on these findings, many rodent studies have been conduct-ed to examine the developmental outcomes and validity of prenatal exposure to VPA as a reliable model of ASD [9, 11–13] . Owing to a robust phenotype that includes struc-tural and behavioral features typically identified in ASD patients, the VPA rodent model of autism has been estab-lished as a reliable tool with construct, face and predictive validity [for a review, see 14 ].

Although there are currently many hypotheses regard-ing the pathophysiology of ASD, several lines of research are beginning to converge on a role of mitochondrial dys-function [for reviews, see 2, 15 ]. Mitochondrial dysfunc-tion not only impairs energy production but also keycellular processes involved in synaptic functioning and neuronal integrity. However, because mitochondrial-based energy production is critically important for neu-rodevelopment, impairment of this critical organelle could contribute to the pathogenesis of ASD [2] . Al-though the VPA model of ASD has been used to examine structural and functional deficits in the developing brain, there appears to be a void in the literature regarding VPA-associated deficits in mitochondrial function and bioen-ergetics. As the strength of an animal model resides in its ability to mimic the human disorder, altered mitochon-drial function in VPA-exposed offspring that resembled abnormalities in human patients with ASD would strengthen the validity of this model.

Interestingly, the antiseizure treatment known as the ketogenic diet (KD) is hypothesized to afford neuropro-tection and alleviate mitochondrial dysfunction by main-taining metabolic homeostasis, thus improving neuronal resistance to metabolic challenge [16, 17] . The KD was first introduced as a therapeutic treatment for epileptic seizures in 1921 [18] and is currently used as an alterna-tive treatment for approximately one-third of all patients

with epilepsy who have failed to respond to other antisei-zure medications [19] . Multiple studies have since been carried out to ascertain a mechanistic understanding of the means by which ketone bodies affect the central ner-vous system [20–22] and, although not completely un-derstood, the KD has been shown to enhance mitochon-drial function [23, 24] .

Recently, reports have also surfaced regarding the ef-ficacy of the KD for patients with Rett syndrome who manifest ASD symptoms [25, 26] . The KD was originally used to control the seizure activity of these patients but beneficial effects were also noted for repetitive behaviors and hyperactivity [25, 27] . A majority of all Rett syn-drome cases are caused by a loss of function mutation in the X-linked methyl-CpG-binding protein 2 (MECP2). Mutations in this gene are also found in patients with oth-er neurological disorders such as Angelman-like syn-drome, motor and learning disabilities, seizures, bipolar disorder, and autism [28–30] . Furthermore, one clinical group in Greece found that high levels of blood ketone bodies following glucose loading were correlated with better outcomes in some ASD patients [31] . Collectively, these findings suggest that the KD may enhance mito-chondrial function and therefore may provide a benefit for children with autism who have underlying mitochon-drial abnormalities.

Abnormal social interactions, including but not lim-ited to peer play, are one of the hallmark traits of ASD. Children with ASD demonstrate both qualitative and quantitative differences in their functional play with peers and adults [32] . Because juvenile play encompasses a complex set of behavioral patterns necessary for healthy socioemotional development and brain maturation [33, 34] , the altered play behavior identified in children with ASD is probably associated with a risk for long-term ad-versity. Furthermore, typical therapeutic agents for ASD have generally been designed to target individual symp-toms, not a complex range of behaviors.

The current study was designed with two primary re-search questions. The first goal of the study was to deter-mine if the prenatal VPA model of autism was associated with mitochondrial dysfunction and abnormalities in play behavior. Following demonstration of these deficits, the second aspect of the study was designed to examine the efficacy of the KD to restore the abnormal social be-haviors and bioenergetic function. To test these hypoth-eses, we examined the play behavior and bioenergetic functioning of offspring exposed to VPA or saline prena-tally that were maintained on their standard diets (SD) or treated with the KD.

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KD Modifies Social and Metabolic Alterations in Prenatal VPA Model of ASD


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Methods

Animals and Dietary Protocols All experimental protocols were in compliance with the Univer-

sity of Calgary Conjoint Faculties Research Ethics Approval Board. Pregnant Wistar Han rats (Jackson Laboratory, Bar Harbor, Maine, USA, or Charles River Laboratories, Wilmington, Mass., USA) were ordered to arrive at our facility on embryonic day 3. The so-dium salt of VPA (Sigma, St. Louis, Mo., USA) was dissolved in 0.9% saline to achieve a concentration of 250 mg/ml. The dosing volume was 500 mg/kg and the dosage was adjusted according to the body weight of the dam on the day of injection. Treated dams (n = 6) received a single intraperitoneal injection of 500 mg/kg VPA sodium salt and control dams (n = 6) received a single intraperito-neal injection of saline on embryonic day 12.5 [35, 36] . The rats were born and reared in a quiet, temperature-controlled room and entrained to a 12-hour light-dark cycle. After weaning at postnatal day 21 (PD21), the rats were placed on either SD or KD (6: 1 fat to carbohydrates plus proteins; Bio-Serv F3666, Frenchtown, N.J., USA) for 10–14 days [37] .

Social Behavioral Testing The animals (male/male pairs) were tested in the play behavior

paradigm between 35 and 38 days of life. The animals were social-ly isolated in standard shoe-box cages (43 × 15 × 28 cm) for 24 h prior to testing as previously described [10, 36] . The play behavior test consisted of placing 2 rats that were previously cage mates (both of which were matched for treatment and diet) into a Plexi-glas ® box (43.2 × 43.2 × 30.5 cm) for a period of 10 min. Play be-haviors were videotaped in the dark and scored by a research ana-lyst (R.M.) who was blinded to the experimental treatment groups. Play behaviors were scored for the play pairing frame by frame and included direct ‘attacks’ to the nape and responses (‘complete rota-tions’, ‘partial rotations’, ‘horizontal rotations’ and ‘evasions’), as described in previous research [38] . The probability of a complete rotation, partial rotation or evasion was calculated as the number of complete rotations, partial rotations or evasions divided by the total number of attacks carried out by the playmate [39] .

Bioenergetic Analysis Mitochondrial Isolation Following play behavior testing, the rats were asphyxiated with

CO 2 and rapidly decapitated; the brain tissue was removed and placed in a beaker of ice-cold isolation buffer (215 n M mannitol, 75 m M sucrose, 0.1% BSA, 1 m M EGTA, 20 m M HEPES at pH 7.2). The neocortex was collected from 7 animals for each experimental group and mitochondria from each animal was used separately for the analysis (n = 7/experimental condition). The neocortex was dissected and the tissue placed in ice-cold isolation buffer until homogenization. Total mitochondria were isolated from the neo-cortex using differential centrifugation, nitrogen disruption and a Ficoll gradient, as reported previously [40–42] . Briefly, the sample was homogenized and then centrifuged at 1,300 g for 3 min at 4 ° C. The supernatant was collected in a fresh tube and centrifuged at 13,000 g for 10 min at 4 ° C. The pellet was resuspended in 600 μl of isolation buffer and placed in a nitrogen bomb at 1,200 p.s.i. for 10 min. The pressure from the nitrogen bomb was released and the sample was placed as the top layer on a Ficoll separation column which consisted of a 10% Ficoll layer and a 7.5% Ficoll layer. The Ficoll column with the sample was centrifuged at 32,000 rpm for

30 min at 4 ° C. Following Ficoll purification, the mitochondrial pellet was resuspended in isolation buffer without EGTA and cen-trifuged at 10,000 g for 10 min at 4 ° C to remove residual Ficoll from the purified mitochondrial sample. The final mitochondrial pellet was resuspended in isolation buffer without EGTA to yield a final concentration of approximately 5 mg/ml and stored imme-diately on ice. Protein concentration for the samples was deter-mined using 96-well BCA protein assay kits measuring absorbance at 562 nm with a Synergy HT Multi-Mode Microplate Reader (BioTek, Winooski, Vt., USA).

Bioenergetic Profiling The Seahorse Bioscience XF24 extracellular flux analyzer was

used to measure mitochondrial function in intact isolated mito-chondria. See figure 1 for a schematic representation of mitochon-drial function and bioenergetic profiling. One day prior to the planned experiment, 1 ml XF calibrant solution (Seahorse Biosci-ence) was added to each of the 24 wells on the calibration plate and stored in a 37 ° C incubator without CO 2 overnight. On the day of the experiment, the injection ports on the sensor cartridge were loaded with the appropriate mitochondrial substrates or inhibitors in respiration buffer (215 m M mannitol, 75 m M sucrose, 0.1% BSA, 20 m M HEPES, 2 m M MgCl 2 , 2.5 m M KH 2 PO 4 at pH 7.2) with5 m M succinate and 2 μ M rotenone as substrates at 10× concentra-tions. The compounds were loaded into the injection ports at the following volumes: port A 50 μl; port B 55 μl; port C 60 μl; port D 65 μl, which yields approximately a 10× dilution for each injection. Once the sensor cartridge was loaded with all of the experimental reagents it was placed into the Seahorse XF24 instrument and cal-ibrated. Following centrifugation of the plates at 2,000 rpm for 20 min at 4 ° C for attachment of the mitochondria (2.5 μg/50 μl) to the XF24 V7 cell culture microplate, 450 μl respiration buffer (37 ° C) was gently added to each well, creating a final volume of 500 μl per well. Each plate was immediately placed into the cali-brated Seahorse XF24 flux analyzer for mitochondrial bioenerget-ic analysis. ADP, oligomycin, FCCP, and antimycin A were in-jected sequentially through ports A–D in the Seahorse Flux Pak cartridges to yield final concentrations of 4 m M ADP, 2 μg/ml oli-gomycin, 4 μ M FCCP, and 2 μ M antimycin A.

Statistical Analysis Means, standard deviations and standard errors of the mean

were determined in a saline-injected control group with SD treat-ment (SAL/SD), a saline-injected control group with KD treatment (SAL/KD), a VPA-injected group with SD treatment (VPA/SD), and a VPA-injected group with KD treatment (VPA/KD). Two-way ANOVAs with treatment (VPA/SAL) and diet (KD/SD) were used to analyze the play behavior and to compare each of the bio-energetic parameters measured. Results were examined for main effects of treatment, diet and interactions between treatment and diet. Alpha levels <0.05 were considered statistically significant.

Results

Demonstration of Ketosis Baseline weights were similar in all 4 groups at PD21

(66.03 ± 1.02 g in SAL/SD, 66.58 ± 1.06 g in SAL/KD, 57.50 ± 1.48 g in VPA/SD, and 62.74 ± 1.86 g in VPA/KD;

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data not shown), but after 10–14 days on their respective diets, KD-fed rats had a significantly reduced body weight gain (117.0 ± 3.036 g in SAL/SD, 18.25 ± 2.763 g in SAL/KD, 129.0 ± 4.358 g in VPA/SD, and 22.79 ± 2.743 g in VPA/KD, p < 0.01; fig. 2 a). However, the KD induced a significant increase in β-hydroxybutyrate levels com-pared to SD between PD35 and 38 (Precision Xtra ketone strips, Mississauga, Ont., Canada; 0.56 ± 0.05 m M in SAL/

SD, n = 5; 1.48 ± 0.16 m M in SAL/KD, n = 6; 0.70 ± 0.04 m M in VPA/SD, n = 6, and 1.58 ± 0.11 m M in VPA/KD, n = 6, p < 0.01; fig. 2 b).

Play Behavior Impairment in social behavior is defined as the most

prominent feature of ASD, with many children demon-strating severe deficits in peer-to-peer interactions such

a

b

Fig. 1. Assessment of bioenergetics using extracellular flux technology. a Schematic representation of a mitochondrial function assay. After baseline OCR is established, se-quential injection of oligomycin, FCCP and antimycin A allows for the determina-tion of the mitochondrial function param-eters; basal respiration, coupled respiration (ATP production), uncoupled respiration (proton leak), maximal respiration, and oxygen consumption which occurs inde-pendent of complex IV (nonmitochondrial respiration). b Schematic representation of the mitochondrial inner membrane with ATP synthase, the complexes of the respi-ratory chain and the mitochondrial carri-ers. The primary purpose of the mitochon-dria is to carry out a set of metabolic pro-cesses designed to convert biochemical energy into ATP.

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as play. The KD recovered some of the social interactions that were altered in the prenatal VPA model of ASD. A total of 30 play pairings were videotaped and scored (SAL/SD = 5, SAL/KD = 4, VPA/SD = 10, VPA/KD = 11) for this experiment. We found that prenatal VPA treat-ment decreased the frequency of play fighting attacks, but the KD was able to increase the amount of rough-and-tumble play in both groups. The two-way ANOVA with treatment (VPA/SAL) and diet (KD/SD) as factors dem-onstrated a main effect of treatment (F 1, 32 = 4.43, p = 0.04) and of diet (F 1, 32 = 11.29, p < 0.01), but the interac-tion was not significant (F 1, 32 = 0.19, p = 0.67; fig. 3 ). In-terestingly, prenatal VPA treatment increased the prob-ability that the rat would defensively respond with a com-plete rotation when compared to saline offspring and the KD did not reverse this. The two-way ANOVA revealed a main effect of treatment (F 1, 32 = 5.56, p = 0.02) but not of diet (F 1, 32 = 1.52, p = 0.23). The interaction also failed to reach significance (p > 0.05; fig. 4 ). The KD did, how-ever, increase the probability that the play partner would respond with a partial rotation, regardless of prenatal treatment ( fig. 4 ). The two-way ANOVA demonstrated a main effect of diet (F 1, 32 = 6.85, p = 0.01) but not of treat-ment (F 1, 32 = 2.02, p = 0.16) or a significant interaction (F 1, 32 = 0.17, p = 0.74). Finally, although not significant in this study, prenatal VPA treatment may decrease the probability that a play partner would evade an attack to the nape, with the KD possibly restoring the evasion re-sponse to normal levels. The two-way ANOVA failed to

show an effect of treatment (F 1, 32 = 3.10, p = 0.08), diet (F 1, 32 = 0.61, p = 0.44) or the interaction (F 1, 32 = 2.87,p = 0.10). Had there been a larger sample size, we may have been able to ascertain whether or not treatment had an effect on levels of evasion with greater confidence ( fig. 4 ).

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Fig. 2. a Average body weight increase expressed as a percentage from baseline to sacrifice for animals in the 4 different treatment groups; comparisons between diet groups are illustrated. * * * p < 0.001. b Average level of β-hydroxybutyrate in blood for animals in the 4 different groups at the time of sacrifice as measured with Preci-sion Xtra ketone strips; comparisons between diet groups are illustrated. * * * p < 0.001.

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Fig. 3. Average number of nape attacks for each treatment condi-tion in a 10-min play session. There was a significant main effect of the treatment (VPA vs. SAL) and a significant main effect ofthe diet (KD vs. SD). Data are expressed as mean ± SEM (n = 5 SAL/SD, n = 4 SAL/KD, n = 10 VPA/SD, n = 11 VPA/KD). * p < 0.05, # p < 0.05.

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Bioenergetics In prior studies, brain growth in children with ASD

was documented through serial head circumference, postmortem and MRI assessments, revealing in particu-lar abnormal temporal and frontal association cortices [43, 44] . It has been suggested that abnormalities in the neocortex may produce core features of ASD [14] . We investigated the effects of the KD on bioenergetics by measuring mitochondrial respiration in isolated mito-chondria from the neocortex. The KD was able to reverse some of the metabolic alterations present in offspring ex-posed to VPA prenatally (p < 0.01; fig. 5 ). The results pre-sented below are all based on the ANOVAs for each of the bioenergetic parameters with treatment (VPA/SAL) and diet (KD/SD) as factors. Basal oxygen consumption levels differed significantly between the SAL and VPA group(p < 0.01; fig. 5 a) and although the KD was able to sig-nificantly reduce basal oxygen consumption rates (OCRs) in the VPA group, the OCR in the VPA/KD group was still significantly higher than that in the SAL/SD and SAL/KD animals (p < 0.01). State III (ADP-stimulated) respi-

ration was also increased in VPA-treated animals (p < 0.01; fig. 5 b), but the KD was able to normalize these OCRs, and VPA/KD respiration did not significantly dif-fer from that in SAL animals during state III. The addition of the ATP synthase inhibitor oligomycin decreased OCR by about 50% in all groups, but due to higher basal rates, VPA animals also had higher OCRs in this state. Higher OCRs following administration of oligomycin generally indicate increased proton leakiness in the mitochondrial membrane. Similar to basal respiration rates, the KD was able to significantly reduce OCR during this state, but the rates in VPA/KD remained significantly higher than those found in SAL animals (p < 0.01; fig. 5 c). Consistent with basal levels, VPA animals exhibited significantly higher maximal mitochondrial respiratory capacity rela-tive to SAL animals (p < 0.01; fig. 5 d), as evidenced by the increase in OCR following the addition of the mitochon-drial uncoupler FFCP. This increased respiratory state, which reflects the maximal capacity of mitochondria, was not affected by the KD. The addition of the complex III inhibitor antimycin A further reduced OCR values in all groups, yielding insignificant differences (p > 0.05; data not shown), thus indicating that the differences in mito-chondrial respiration are not related to complex III activ-ity. Finally, the respiratory control ratio (RCR) was calcu-lated as the ratio of complex II-driven respiration in the presence of 4 m M ADP with and without 1 μ M oligomycin A to determine the overall health and integrity of the mi-tochondria. VPA-exposed animals exhibited significantly lower RCR relative to SAL/SD and no significant differ-ence was observed between VPA/SD and VPA/KD (p < 0.01; fig. 6 ).

Discussion

In the present study, we confirmed that prenatal expo-sure to VPA on day 12.5 of gestation produces identifiable effects on the postnatal behaviors of rats. Compared with the control group, VPA rats demonstrated a diminished number of overall social play behaviors and an alteration in their response to playful attacks. We demonstrated that the KD had a beneficial effect on the playful behaviors ob-served in VPA rats. However, the KD was also found to have an effect on the play behavior of control rats. In ad-dition, we found that prenatal VPA exposure altered the bioenergetic profile of mitochondria from the neocortex, and although the KD was able to ameliorate some of the VPA-induced dysfunction, animals in the VPA/KD group often failed to reach normal OCRs.

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Fig. 4. Average frequency of the play behavior responses to nape attacks/play initiations in the 10-min play session for each of the treatment groups. The two-way ANOVAs demonstrated that there was a strong but nonsignificant main effect identified toward the KD normalizing the percentage of times VPA animals evade (α = 0.08) and a significant effect on the number of partial rotations employed, but there was not a main effect associated with the KD and their elevated use of complete rotations. * p < 0.05: main effect of diet; # p < 0.05: main effect of treatment.

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Prior studies have established the importance of so-cial interactions and juvenile play for the healthy devel-opment of many different species [for a review, see 45 ]. Although neurodevelopment is not contingent upon play, it is believed to be multifunctional, accruing ben-efits in the short term and in adulthood. Studies in rats and humans have demonstrated that depriving individ-uals of play in the juvenile period leads to long-term be-havioral, cognitive and socioemotional deficits [45, 46] . This is particularly significant for the healthy develop-ment of children with ASD who display very limited so-

cialization and peer play activity. Studies have indicated that children with ASD spend less time engaged in play-ful activities and, when they are involved, the play is of-ten simple and lacks complexity [32] . Consistent with the published literature, we found that prenatal expo-sure to VPA decreased the amount of juvenile play in addition to decreasing the complexity of the defensive responses used following a playful attack. VPA-exposed offspring were most likely to respond to a nape attack with a complete rotation (50% of the time), whereas SAL animals only used a complete rotation 30% of the time,

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Fig. 5. Representative traces demonstrating the effects of the KD on mitochondrial respiratory function in the neocortex. The KD revealed some of specific variations in mitochondrial function.n.s. = Nonsignificant. a Basal level: comparing all 4 groups, VPA/SD exhibited a 21% higher rate of complex II-driven basal mito-chondrial oxygen consumption than SAL/SD, and the KD showed a significantly slower rate of oxygen consumption in VPA animals. b ADP: similar to the basal respiration rate, analysis of ADP-de-pendent oxygen consumption revealed significantly higher OCR

in VPA-treated animals. c Oligomycin: VPA/SD exhibited a high-er rate of oxygen consumption in the presence of oligomycin A as an inhibitor of ATP synthase compared to SAL/SD, and the KD reduced OCR in VPA animals. d FCCP: consistent with the above-mentioned data ( a–c ), VPA animals revealed a significantly higher maximal respiratory capacity in the presence of FCCP as a chemi-cal uncoupler of electron transport and oxidative phosphorylation. Data are expressed as mean ± SEM (n = 7 per group). * p < 0.05, * * * p < 0.001.

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employing all of the defensive strategies on a more con-sistent basis.

Play behaviors in the rat generally begin with a simple repertoire and then gradually become more complex with age [39] . From this study, it appears that rats prenatally exposed to VPA have not mastered the behavioral flexi-bility that is present in normal age-matched control rats. Although therapeutic administration of the KD was effec-tive at increasing the amount of juvenile play that the VPA group engaged in, the defensive responding was still predominated by complete rotations. Interestingly, al-though failing to reach statistical significance, the animals that received the KD following VPA exposure exhibited a slight increase in their probability of evasive responses, generating levels similar to those of SAL controls. How-ever, this altered behavior resulted from a decrease in the number of partial rotations used rather than decreasing the probability of a complete rotation – a behavior that appears to be abnormally high in VPA-treated animals. Earlier studies have demonstrated that the primary re-sponse of subordinate rats to playful attacks is complete rotation [45] , possibly indicating that the autistic pheno-type produced by prenatal VPA exposure lowers a rat’s position in the social hierarchy.

In addition to examining the effects of KD treatment on social behavior, our study also investigated the bioen-ergetic profile of rats exposed to VPA prenatally, with or

without KD treatment. As the evidence linking mito-chondrial dysfunction and ASD pathophysiology has grown [2] , investigators have become increasingly inter-ested in metabolic therapies like the KD. Efficient brain bioenergetics would presumably be required to maintain the functionality of highly specialized neural networks that regulate the motivation for play initiation and re-sponse flexibility. Furthermore, because mitochondrial functioning is critical to neuronal development, synaptic transmission and neuroplasticity, deficiencies at this lev-el would modulate an individual’s ability to carry out the complex aspects of juvenile play behavior. In this study, we found abnormal respiration and bioenergetics in the prenatal VPA model of autism, providing further support for this model of ASD and for the hypothesis that mito-chondrial dysfunction may underlie ASD symptomology. The higher OCR in the presence of oligomycin A of VPA animals suggests increased damage to the inner mito-chondrial membrane – specifically, an enhanced proton leak and dissipation of the proton motive force necessary for ATP production. Enhanced proton leak is believed to be compensatory for situations where the proton gradient has become abnormally high, but it has also been associ-ated with mitochondrial dysfunction during aging [47] . These two situations are known to be associated with strain to the cellular system and abnormal function. The reduced OCR by the KD in VPA animals suggests that the KD can help restore normal function in otherwise im-paired mitochondria. The lower RCR and higher basal levels of oxygen consumption identified in VPA-exposed animals are indicative of mitochondrial dysfunction and imply that mitochondrial hyperactivity is required to maintain bioenergetic homeostasis. As the mitochondrial RCR is a measure of the primary function of mitochon-dria, to idle at low rates and then respond when needed with high throughput [48] , the lowered RCR in VPA/SD animals demonstrates an inability to reach maximal out-put when the brain requires energy for complex func-tions. As identified in other neurodegenerative diseases such as Alzheimer’s disease, the abnormal metabolic state combined with a chronic increase in oxidative stress as-sociated with the reduced RCR impairs neuronal func-tioning and the ability of the cells to respond to stress, leading to neuropsychiatric complications and dysfunc-tion.

Recognizing that mitochondrial oxidative phosphory-lation is the primary source of cellular ATP in the brain, and that perturbations to the system ultimately impair brain metabolism, the KD is an ideal treatment strategy because it provides an alternative energy source for mito-

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SAL/SD SAL/KD VPA/SD VPA/KD

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Fig. 6. Representative traces of the RCR in isolated mitochondria. The RCR was significantly decreased in the VPA/SD group com-pared to the control SAL/SD group and, although there was a trend toward normalization in the VPA/KD group, the KD failed to completely return the RCR to control levels. Data are expressed as mean ± SEM (n = 7 per group). * p < 0.05.

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chondria [49] . Typical brain cells (neurons, glia, etc.) will readily adapt to using ketone bodies as their energy source in the absence of glucose [50] , improving mitochondrial function in many circumstances. As demonstrated in studies of epilepsy, the KD increases the brain’s capacity to maintain ATP production in the face of increased physiological demand [17] . As VPA animals demonstrat-ed elevated basal levels of oxygen consumption, the pri-mary mechanism of the KD in these animals may be to lower the cellular stress load required to maintain this elevated level of activity. Consistent with this notion, our results demonstrate that the KD does favorably modulate OCRs, moving them toward normal levels. Although OCR values were still significantly different than those from control (SAL) animals, they were improved in VPA/KD offspring. This finding is particularly interesting be-cause the KD was able to significantly modulate social behavior without returning OCRs to actual baseline lev-els, suggesting nonmitochondrial actions.

There are two possible reasons why the KD was able to enhance mitochondrial respiration and social behavior without completely normalizing either outcome. First and foremost, both social behavior and bioenergetics are complex systems with many intervening factors. It is pos-sible that although the KD has a recognized ability to move systems toward homeostasis, the complexity of the two outcomes examined exceeded the scope of the KD. It is probable that because the VPA-induced deficits are ex-

tensive and involve many developmental pathways, an ef-fective treatment regimen would require the KD in com-bination with behavioral therapies. The second possible reason why we may have identified significant improve-ments without fully normalizing the outcomes is related to treatment duration and timing. The treatment param-eters for the KD were chosen based upon their known ef-ficacy for treating intractable epilepsy in rodent models [16] . Owing to the complexity of ASD and the develop-mental nature of the disorder, KD treatment may need to be maintained for a longer duration or provided at an earlier time point. As the KD for the treatment of ASD has only recently been recognized, there is little research to support either explanation, and both possibilities re-quire further examination. This study did, however, clearly demonstrate that the KD was able to modify com-plex social behaviors and mitochondrial respiration in the prenatal VPA model of ASD.

Acknowledgment

The authors would like to thank Professor Bin Hu and Taylor Chomiak, PhD (University of Calgary and the Hotchkiss Brain In-stitute), for helping to establish the VPA model in our hands and for guidance and thoughtful input on this project. This study was supported by the Alberta Children’s Hospital Research Institute for Child and Maternal Health as well as the Alberta Children’s Hospital Foundation.

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