Behavioural Brain Research 278 (2015) 542–548

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Research report

Intracerebroventricular injection of propionic acid, an enteric metabolite implicated in autism, induces social abnormalities that do not differ between seizure-prone (FAST) and seizure-resistant (SLOW) rats Sandy R. Shultz a,∗ , Noor A.B. Aziz a , Li Yang a,b , Mujun Sun a , Derrick F. MacFabe c , Terence J. O’Brien a a

Melbourne Brain Centre, Department of Medicine, The Royal Melbourne Hospital, The University of Melbourne, Parkville, VIC, Australia Department of Histology and Embryology, Kunming Medical University, Kunming, Yunnan, China c The Kilee Patchell-Evans Autism Research Group, Department of Psychology and Psychiatry, University of Western Ontario, London, ON, Canada b

h i g h l i g h t s • • • •

Examined interactive effects of PPA and FAST models of ASD on rat social behavior. PPA induced social abnormalities and astrogliosis, regardless of FAST or SLOW strain. FAST rats not treated with PPA did not have social deficits compared to SLOW rats. FAST rats were hyperactive and had increased astrogliosis compared to SLOW rats.

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Article history: Received 29 July 2014 Received in revised form 27 October 2014 Accepted 30 October 2014 Available online 6 November 2014 Keywords: Autism Animal model Short chain fatty acid Astrogliosis Attention deficit hyperactivity disorder

a b s t r a c t Autism is a complex neurodevelopmental disorder that is characterized by social abnormalities. Genetic, dietary and gut-related factors are implicated in autism, however the causal properties of these factors and how they may interact are unclear. Propionic acid (PPA) is a product of gut microbiota and a food preservative. PPA has been linked to autism, and PPA administration to rats is an animal model of the condition. Seizure-prone (FAST) and seizure-resistant (SLOW) rats were initially developed to investigate differential vulnerability to developing epilepsy. However, FAST rats also display autistic-like features, and have been proposed as a genetic model of autism. Here we examined the effects of PPA on social behavior in FAST and SLOW rats. A single intracerebroventricular injection of PPA, or phosphate-buffered saline (PBS), was administered to young-adult male FAST and SLOW rats. Immediately after treatment, rats were placed in same-treatment and same-strain pairs, and underwent social behavior testing. PPA induced social abnormalities in both FAST and SLOW rat strains. While there was no evidence of social impairment in FAST rats that were not treated with PPA, these rats were hyperactive relative to SLOW rats. Post-mortem immunofluorescence analysis of brain tissue indicated that PPA treatment resulted in increased astrogliosis in the corpus callosum and cortex compared to PBS treatment. FAST rats had increased astrogliosis in the cortex compared to SLOW rats. Together these findings support the use of PPA as a rat model of autism, but indicate there are no interactive effects between the PPA and FAST models. © 2014 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: FAST, seizure prone rats; SLOW, seizure resistant rats; PPA, Propionic acid; ADHD, Attention deficit hyperactivity disorder; SCFA, Short chain fatty acid; ICV, intracerebroventricular; PBS, Phosphate buffered saline; GFAP, Glial fibrillary acid protein; PFA, Paraformaldehyde; ANOVA, Analysis of variance. ∗ Corresponding author. Tel.: +61 3 90356522; fax: +61 3 9347 1863. E-mail address: [email protected] (S.R. Shultz). http://dx.doi.org/10.1016/j.bbr.2014.10.050 0166-4328/© 2014 Elsevier B.V. All rights reserved.

Autism spectrum disorders (ASD) are a class of neurodevelopmental conditions that affects approximately 1 in 88 individuals [1]. The hallmark clinical features of ASD include social abnormalities, though other cognitive, sensory, and motor deficits are common [2,3]. ASD has a high comorbidity with other neurological conditions including epilepsy and attention deficit hyperactivity disorder

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(ADHD; [2,4]). While the causal factors of ASD remain unknown, there is strong evidence for a multi-genetic role in ASD [2]. However, research also indicates that environmental and gut-related factors may be important, and that autism may be a multisystem disorder affecting metabolic, immune and gastrointestinal systems [3,5–8]. As it is difficult to address questions surrounding causality and interactions between genetic and environmental factors in the ASD patient setting, the use of animal models may provide insight into these potential relationships. Propionic acid (PPA) is a short chain fatty acid (SCFA) that has been implicated as a possible gut-derived environmental factor in ASD [5,9,10]. PPA is produced as a fermentation product by many autism associated gut bacteria, and is also a common preservative in food products that may exacerbate ASD symptoms [9,10]. Moreover, PPA can readily cross both the gut-blood and bloodbrain barriers, and can have neuroactive effects similar to those implicated in ASD including intracellular acidification, activation of specific G-coupled receptors, alteration of neurotransmitter release and synthesis, gap junction gating, neuroinflammation, free radical formation, altered lipid profiles, mitochondrial dysfunction, and alteration of gene expression [9–11]. Studies administering intracerebroventricular PPA to rodents report social abnormalities, cognitive impairments, and sensorimotor dysfunction [12–14]. Examination of brain tissue from PPA treated rats has revealed reactive astrogliosis, activated microglia, oxidative stress, glutathione depletion, mitochondrial dysfunction, and alteration of phospholipid/acylcarnitine profiles, all of which are consistent with the findings in ASD patients ([10,12–16], [5]). Taken together, PPA may be involved in clinical ASD, and administration of PPA and related SCFA to rodents represents a valid and useful model to investigate the potential role of environmental and/or gut-related factors in the pathogenesis of the condition. Autism is often co-morbid with seizure disorders, and they may share similar etiologies [17]. Initially developed to provide insight into epilepsy, seizure-prone (FAST) and seizure-resistant (SLOW) rat strains were produced through selective breeding based on seizure susceptibility [18]. Additionally, co-morbid with their increased susceptibility to epileptogenesis, FAST rats have been reported to display ASD-like features including learning and attention deficits, impulsivity, hyperactivity, delays in social development, repetitive behavior, and numerous physiological abnormalities similar to those in ASD, and have therefore been proposed as a unique genetic model of ASD [17,19,20]. Here we aimed to explore how central exposure to the enteric bacterial metabolite, PPA, would affect social behavior in FAST rats with a genetic predisposition to the condition. FAST and SLOW rats were treated with PPA or PBS-vehicle via intracerebroventricular (ICV) injection. Following treatment, rats underwent social behavior testing and then post-mortem immunofluorescence analysis of brain tissue for reactive astrogliosis. The results demonstrated that PPA treatment in both FAST and SLOW rats strains resulted in social abnormalities, consistent with clinical features seen in ASD, as well as reactive astrogliosis in the corpus callosum and cortex. Of note, FAST rats treated with PBS-vehicle did not show social abnormalities relative to SLOW rats treated with PBS-vehicle, which is not consistent with their use as a model of ASD. However, FAST rats did display baseline hyperactivity, as well as astrogliosis, compared to SLOW rats, which may have implications regarding their use as a model of ADHD and their enhanced seizure susceptibility. 2. Material and methods 2.1. Subjects The FAST and SLOW rat strains used in this experiment were originally developed at McMaster University, Hamilton, Ontario

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[18]. The FAST and SLOW rats used in current experiments were offspring from the 43rd and 45th breeding generations, and were bred in the Melbourne Brain Centre animal housing facilities. A total of 50 young-adult male rats were used in this study. Rats were 8 weeks old, weighed 200–250 g, and were naïve to all experimental procedures at the time of surgery. After surgery, rats were individually housed in standard acrylic cages (26 cm × 48 cm × 21 cm), kept under a 12:12 light/dark cycle (lights on 7:00 h) at a controlled temperature (21 ± 1 ◦ C), and had ad libitum access to food and water for the duration of the experiment. All procedures were in accordance with Australian Code of Practice for the care and use of animals in scientific purposes by The Florey Animal Ethics Committee (12077-UM). 2.2. ICV cannula surgery As previously described [12,13], rats were anaesthetized in a sealed Plexiglas box with 4% isoflurane and 2 L/min oxygen flow. After induction of anesthetic, rats were given a subcutaneous injection of analgesic (carprofen, 5 mg/kg), and were placed in a standard stereotaxic device equipped with a nose cover to maintain gas anesthesia (2% isoflurane and 0.5 L/min oxygen) throughout the surgery. Under sterile conditions, rats were implanted with a 23-gauge cannula in the right lateral ventricle, with the tip of the cannula (PlasticsOne, USA) at the following coordinates with reference to Bregma: anterior/posterior −1.4 mm; medial/lateral 1.8 mm; dorsal/ventral −3.0 mm. Four stainless steel screws were inserted into the skull around the cannula to provide an anchor for dental acrylic that affixed the cannula to the skull. The guide cannula was sealed with a removable plug (Plastics One, USA) before and after injection. 2.3. Pre-treatment open field testing Following a one-week recovery after the cannula surgery, and one day prior to receiving PPA or PBS treatment, each individual rat was placed in the center of the open field and given a 10 min period to freely explore the open field/social behavior testing apparatus [12,21]. The apparatus consisted of a large circular open-field (90 cm diameter, 40 cm high walls) with sawdust bedding covering the floor of the arena. A CD camera was positioned above the center of the arena and the room was equipped with auto-adjustable operating room light. The camera was connected to a computer for recording and analysis using the TopScan Behavior Analyzing System (CleverSys, USA). This software objectively tracks rat behavior and computes quantitative variables. Activity in the open field is commonly used to assess motor function and exploratory behavior in rats. Furthermore, as rats prefer a sheltered environment when placed into a novel setting, the amount of time spent and the number of entries into the middle of the open field, which represents a vulnerable and unshielded environment, is used as an indicator of anxiety-like behavior [22]. As such, the following activity and anxiety-related behavior measures were calculated here [21,23]: 1. Distance travelled (cm) by each individual rat; 2. Time spent in the middle of the arena (66 cm diameter); and 3. Number of entries into the middle of the arena. 2.4. Experimental groups and ICV injections Following a one-week recovery from cannula surgery and pretreatment open field testing, rats were randomly assigned to one of four experimental groups: FAST + PPA (4 ␮L, 0.26 M; n = 12), SLOW + PPA (n = 14), FAST + phosphate buffered saline vehicle (PBS) (4 ␮L; n = 12) and SLOW + PBS (n = 14). Solutions were buffered to physiological pH 7.5 before injection. Doses were determined from previous dose-response studies [10,12–14].

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As previously described [10,12–14], ICV injections were delivered using a 30-gauge injection cannula (Plastics One, USA) connected to a syringe pump (World Precision Instruments, USA) via PE10 tubing (Plastics One, USA). The tip of the injection cannula protruded 0.5 mm beyond the tip of the guide cannula. Each injection delivered 4 ␮L of solution over a one-minute duration. To ensure that the entire volume was delivered, the injection cannula was allowed to stay in place for an additional 60 s before being removed. Rats were placed in the open field for social behavior testing immediately after the injection cannula was removed [12]. 2.5. Social behavior testing As previously described [12], pair-based rat social testing was conducted in a large circular open-field with sawdust bedding. A CD camera was positioned above the center of the arena and connected to a computer for recording and analysis using the TopScan Behavior Analyzing System (CleverSys, USA). Prior to injections, the posterior surface of one rat from each pair was dyed black to ensure that TopScan could differentiate and track each rat individually [12]. Immediately after receiving their assigned PPA or PBS injections, same-strain and same-treated rat pairs were placed in the middle of the testing apparatus and allowed to freely explore and interact for 30 min. TopScan objectively calculated the following social behavior and activity measures for each 10 min of test time [12]: 1. The mean distance (cm) apart between the rat pair; 2. Percentage of time rat pairs spent within a 5 cm proximity of each other; 3. The number of social approaches each individual rat initiated; and 4. Distance travelled (cm) by each individual rat. 2.6. Immunofluorescence staining and analysis 24 h after social testing, rats (8/group) were deeply anaesthetized with sodium pentobarbital (270 mg/mL) and transcardially perfused with ice cold PBS (0.1 M) followed by 4% paraformaldehyde (PFA). Brains were removed, post-fixed in paraformaldehyde for 24 h at 4 ◦ C, then immersed in 70% ethanol until undergoing a 24 h paraffin-embedding cycle [24]. Once embedded in paraffin wax, brains were cut using a paraffin microtome into 8 ␮m sections and mounted onto slides. For immunofluorescence staining, the sections were dewaxed in 100% xylene for 5 min and hydrated in decreasing graded ethanol to water (100%, 96%, 70% ethanol, water). Antigen retrieval was performed in a temperature- and pressure-controlled Decloaking Chamber Plus (BioCare Medical, USA) in TRIS-EDTA buffer (10 mM TRIS-BASE, 1mMEDTA, pH 9) at 125 ◦ C for 10 min, followed by cooling under tap water for 10 min. Sections were then equilibrated to room temperature in PBS (pH 7.4) for 5 min and were blocked in PBS blocking buffer (3% heat inactivated goat serum with 2% heat inactivated BSA, PBS at pH 7.4) for 1 h at room temperature. The sections were then incubated with rabbit polyclonal primary antibody for glial fibrillary acid protein (GFAP; astrocyte marker, 1:500; DakoCytomation, Denmark) overnight at 4 ◦ C. Sections were then washed with PBS and incubated with antirabbit alexa-Fluor 350-conjugated phalloidin (1:1000; Invitrogen, USA) for 1 h at room temperature. The sections were then washed with PBS and coverslips were mounted with fluorescent mounting medium (DakoCytomation, Denmark; [24]). The stained sections were examined with an upright fluorescence microscope (Zeiss, Germany). All images were captured using identical camera settings (e.g. time of exposure, brightness, contrast and sharpness). For analysis of GFAP immunoreactivity in cortex, two images were captured adjacent to the injection site at

10x field of view. For analysis of GFAP immunoreactivity in corpus callosum adjacent to the injection site, the images were captured at 20x field of view. The total area of positive GFAP immunoreactivity was quantified for each image using Image-Pro Plus 6.0 (Media Cybernetics, USA; [12,24,25]). A researcher blinded to the experimental conditions completed all immunofluorescence staining and analysis. 2.7. Statistical analyses SPSS 21.0 (IBM Corp, USA) software was used for all statistical analysis, and statistical significance was set at p < 0.05. Pre-treatment open field outcomes and social behavior testing outcomes were analyzed using repeated measures analysis of variance (ANOVA), with treatment and strain as the between-subjects variables and time as the within-subjects factors. GFAP immunoreactivity was analyzed with two-way ANOVAs, with treatment and strain as the between-subjects variables. Bonferroni post hoc comparisons were carried out when appropriate. 3. Results 3.1. Pre-treatment open field As indicated by a significant strain effect (F1,42 = 11.329, p < 0.005; FAST rats > SLOW rats; Fig. 1A), FAST rats exhibited an increase in distance travelled compared to SLOW rats during pretreatment open field testing. There were no significant effects for treatment or significant interactions between strain and treatment (p > .05). There were also no significant differences on the anxietylike behavior measures of time spent and number of entries in the middle of the open field. These findings indicate that FAST rats displayed increased locomotor activity prior to receiving their assigned treatments. 3.2. Social behavior Rats pairs treated with PPA exhibited greater mean distance apart compared to rat pairs treated with PBS (F1,20 = 6.109, p < 0.05; PPA > PBS; Fig. 2A). Rats treated with PPA also spent less time within a 5 cm proximity (F1,20 = 8.644, p < 0.05; PPA < PBS; Fig. 2B) and made fewer social approaches (F1,44 = 28.925, p < 0.001; PPA < PBS; Fig. 2C) compared to rats treated with PBS during social behavior testing. Taken together, findings suggest that PPA induces social deficits, but that these deficits do not differ between FAST and SLOW rats. There was also a significant effect for time (F2,88 = 26.117, p < 0.001; 10 min > 20 and 30 min, p < 0.001), indicating more social approaches during the first 10 min of social testing. 3.3. Activity during social behavior testing During social behavior testing, locomotor activity was also assessed. FAST-PBS rats travelled a greater distance compared to all other groups during social testing (Fig. 3). ANOVA and post-hoc comparisons confirmed this impression with the finding of a significant strain × treatment interaction (F1,44 = 9.524, p < 0.005; FASTPBS > all other groups, p < 0.001). There were also significant main effects found for time (F2,88 = 44.587, p < 0.001; 10 > 20 > 30 min, p < 0.001), strain (F1,44 = 10.574, p < 0.005; FAST > SLOW), and treatment (F1,44 = 27.942, p < 0.001; PBS > PPA). 3.4. Astrogliosis FAST rats had increased astrogliosis in the cortex, as evidenced by increased GFAP immunoreactivity, compared to SLOW rats (significant effect for strain: F1,28 = 24.049, p < 0.001; FAST > SLOW;

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Fig. 1. Pre-treatment open field behavior. Prior to receiving their assigned treatments, FAST rats were hyperactive compared to SLOW rats, as indicated by greater distance travelled in an open field (A). There were no significant differences between FAST and SLOW strains on the anxiety-like behavior measures of time spent in the middle of the open field (B) or the number of entries into the middle of the open field (C). Histogram bars represent means of data during the 10 min open field testing, and error bars represent ± SEM. * = FAST rats > SLOW rats, p < 0.05. See Results for additional statistical details.

Fig. 2. Social behavior testing in FAST and SLOW rats treated with PPA or PBS. Regardless of strain, rat pairs treated with PPA displayed social abnormalities compared to PBS-treated pairs, as evidenced by a greater mean distance apart (A) and less time spent within a 5 cm proximity (B). Rats treated with PPA also made fewer social approaches compared to their PBS-treated counterparts (C). Histogram bars represent means of data during the 30 min social behavior testing, data points represent means of data collected during each of the 10 min segments of social behavior testing, and error bars represent ± SEM. * = PPA-treated rats significantly different than PBS-treated rats, p < 0.05. See Results for additional statistical details.

see Fig. 4A). Furthermore, rats treated with PPA had increased astrogliosis compared to rats treated with PBS in both the corpus callosum (significant effect for treatment: F1,28 = 28.210, p < 0.001; PPA < PBS; see Fig. 4A), and cortex (significant effect for treatment: F1,28 = 4.556, p < 0.05; PPA < PBS; see Fig. 4B).

Fig. 3. Distance travelled during social behavior testing. FAST rats treated with PBS display increased activity during social behavior testing, as evidenced by greater distance travelled in the open field compared to all other groups. Data points represent means of data collected during each 10 min segment of social behavior testing, and error bars represent ± SEM. *** = FAST + PBS > all other groups, p < 0.005. See Results for additional statistical details.

4. Discussion 4.1. Further support for the PPA model of ASD Here we found that a single ICV injection of PPA, regardless of FAST or SLOW strain, resulted in social deficits as evidenced by increased mean distance apart, reduced proximity, and fewer social approaches compared to rat-pairs treated with PBS-vehicle. These findings are consistent with those from previous studies indicating similar social impairments in adult [12] and adolescent [14] male Long-Evans rats treated with PPA. As PPA treated rats did not display a significant decrease in distance travelled during social testing compared to the SLOW + PBS rats, gross motor impairments do not appear to be a confounding factor in social measures. However, it should be noted that previous studies have reported fine motor abnormalities after PPA injections [10,13], and it is interesting to speculate whether these more subtle motor abnormalities might affect social interactions. The current study also found that a single ICV injection of PPA resulted in reactive astrogliosis, as indicated by increased GFAP immunoreactivity in the corpus callosum and cortex. This PPA-induced astrogliosis is also consistent with previous findings from rats treated with PPA [10,12–14] and in humans with ASD [26–28], and may indicate a neuroinflammatory response [12,14,27]. It is interesting to speculate whether this response may have contributed to the social impairments observed [28], however, considering the rapid onset of the social impairments it seems unlikely that the PPA-induced astrogliosis was the mechanism responsible for initiating the social deficits. Rather, they are likely to

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Fig. 4. Immunofluorescence GFAP analysis. Representative images of GFAP immunoreactivity in cortex (A-D; 10X field of view) and corpus callosum (F-I; 20X field of view) from SLOW and FAST rats treated with PBS or PPA. Rats treated with PPA had increased astrogliosis in the cortex (E) and corpus callosum (J) than rats treated with PBS, as evidenced by an increased area of GFAP immunoreactivity. FAST rats had increased astrogliosis in the cortex (E) than SLOW rats, as evidenced by an increased area of GFAP immunoreactivity. Histogram bars represent means of data, and error bars represent ± SEM. * = PPA > PBS, p < 0.005; # = FAST > SLOW, p < 0.005. Scale bars = 50 ␮m. See Results for additional statistical details.

be explained by one or more of a number of the rapid-acting neural effects of PPA, including activation of specific G coupled receptors, intracellular acidification, alteration of neurotransmitter systems, oxidative stress, and mitochondrial dysfunction [10,12,29–33]. As the actions of PPA and related SCFA on biological systems are complex ([34], [9]), further research is required to determine the exact causal mechanisms underlying PPA-induced behavioral abnormalities. Related to the causal mechanism, whether or not the effects of PPA on social behavior are acute or chronic also remains to be studied. Nonetheless, the changes reported here in FAST and SLOW rats treated with PPA bear resemblance to the behavioral and neuropathological abnormalities that occur in humans with ASD, and further support PPA as a relevant model of ASD. 4.2. Do FAST rats model ASD? Previous studies have reported that FAST rats exhibit a range of behavioral deviations, including social abnormalities [35,36]. Based on these collective past findings, FAST rats have now been presented as a potential model of ASD ([19]; Gilby et al., 2008; [17]). Unexpectedly, here we found no differences between FAST and SLOW rats treated with control PBS-vehicle on our social behavior measures. Furthermore, the effects of PPA on social behavior and astrogliosis did not differentially affect FAST or SLOW strains, suggesting a lack of interaction between these proposed environmental/gut-related (PPA) and inherited (FAST) models of ASD. Considering that social impairments are a hallmark feature

of ASD, the apparent absence of social deficits in FAST rats treated with PBS-vehicle raises questions as to whether FAST rats are an appropriate model of ASD. While no social deficits were observed in the FAST + PBS rats, they were found to be hyperactive both prior to treatment and after PBS-vehicle treatment compared to SLOW rats. These findings are consistent with previous findings indicating a hyperactive and impulsive phenotype in FAST rats [37–39]. FAST rats have also been proposed as an animal model relevant to ADHD (Gilby et al., 2008), another condition that is associated with epilepsy [40]. Given that FAST rats failed to exhibit the hallmark social deficits of ASD while displaying the hallmark hyperactive symptom of ADHD, the FAST behavioral phenotype may be more representative of ADHD than ASD. As FAST rats had reactive astrogliosis in the cortex compared to SLOW rats, it is possible that reactive astrocytes or other neuroinflammatory mediators may contribute to the behavioral phenotype in FAST rats [41,42]. Furthermore, the heightened neuroinflammation may have important implications regarding the FAST rats increased sensitivity to seizures and epilepsy [43–47]. Interestingly, FAST rats treated with PPA failed to display a hyperactive phenotype during social behavior testing. Although ICV injections of PPA can induce a hyperactive phenotype in rats when they are tested individually [10], PPA-induced hyperactivity does not occur when rats are placed in a social environment with another rat [12]. This lack of hyperactivity may be due to the PPAinduced social impairments, as activity/exploration may decrease in an environment shared with a rat they are attempting to avoid

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[12]. As such, the decreased activity in the FAST-PPA rats relative to the FAST-PBS rats may be due to the PPA-induced social deficits.

Melbourne Hospital Neuroscience Foundation grant to TOB, and a Canadian Institute of Health Research fellowship to SRS.

5. Limitations

References

Previous studies have reported social abnormalities in FAST rats [35,36]. Specifically, FAST rats were found to initiate more playful attacks, and were more likely to defend an attack, compared to SLOW rats [35]. FAST rats were also found to display more evasive defense tactics that reduced the likelihood of prolonged interactions [35]. Here we found no differences between FAST and SLOW rats that were treated with PBS-vehicle on the social behavior measures of mean distance apart, proximity, and social approaches, which raises questions regarding whether FAST rats are an appropriate model of ASD. However, it is important to consider that the lack of social deficits in FAST + PBS rats observed here may be due to a number of methodological factors. For example, previous studies of social behavior in FAST rats have used different housing conditions, juvenile rats, and different social measures than those used here [35,36]. As such, it is possible that FAST rats do exhibit social deficits similar to those in ASD when examined under different experimental conditions. Similarly, the current finding that PPA does not differentially affect FAST and SLOW strains may also be related to methodological reasons including the dose, duration, timing, and route of PPA exposure, as well as factors such as the age and sex of the rats [48–50]. Here we studied the acute effects of a single dose of PPA delivered directly to the brain of an adult male rat via ICV injection. Although this approach is appropriate for an initial proof of concept study, future studies should explore the use of different doses and treatment schedules of PPA, as well as using an administration method more representative of what might occur in the human setting. Notably, recent studies have found that and preand neonatal exposure to PPA alters development and behavior in adolescent rats [48–50]. As such, it remains possible that PPA may have an interactive effect in FAST rats under different experimental settings. Overall, future studies are still necessary to permit more concrete conclusions as to whether FAST rats represent a valid model of ASD and if PPA affects FAST and SLOW rats differently. 6. Conclusions A single ICV injection of PPA was found to impair social behavior and induce astrogliosis in both FAST and SLOW rat strains. These findings are consistent with previous studies investigating the effects of PPA administration in other rat strains, and resemble those from ASD patients, providing further support for the robustness of the PPA model of ASD. There were no differences between FAST and SLOW rats treated with PBS on any social measures. As social impairments are a hallmark symptom of ASD, the apparent lack of social abnormalities in vehicle-treated FAST rats raises questions regarding their use as a model of ASD. Although further studies are required to assess the usefulness of FAST rats in the ASD context, the current findings of hyperactivity in untreated and vehicle-treated FAST rats suggest that they may be a more appropriate model of ADHD. Findings of increased astrogliosis in the cortex of FAST rats may contribute to their behavioral phenotype, as well as other FAST-related abnormalities (e.g. seizure susceptibility), and warrants further investigation. Acknowledgements The authors thank Dr. Krista Gilby for providing access to the FAST and SLOW rat strains used in this study. This study was funded by grants from the University of Melbourne to SRS, a Royal

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