Locomotor activity as expressed by horizontal, vertical, and repetitive measures across infusion days was used to assess abnormal animal behavior. Assessment of locomotor activity indicates that PPA infusions induced hyperactivity and stereotypy in rodents consistent with the findings from previous studies by our group [7–12]. These behaviors bear some resemblance to the hyperactive and repetitive behaviors which are recognized as core symptoms of ASD . PPA and other short chain fatty acids are known to increase intracellular neuronal and glial acidification and calcium proportions, thereby producing widespread effects on neurotransmitter release including glutamate, dopamine, norepinepherine, and serotonin, each of which play a role in elicitation of locomotor activity, as seen in the present study [42–44]. In addition, PPA has been shown to increase glutamatergic transmission, leading to excitability in brain regions linked to locomotor activity [45, 46]. Collectively, these observations are consistent with an emerging theory of ASD as a disruption of excitatory/inhibitory neuronal activity . The fact that PPA infusions consistently induce elevations in locomotor activity in rodents indicates that this model may be a useful tool for studying the neurological mechanisms involved in the behavioral disturbances seen in ASD.
Alterations in phospholipid molecular species
The phospholipid profiles observed in control animals are consistent with those reported in the literature for rat brain ([48, 49]with the exception of SM molecular species. Typically, molecular species with sphingosine base (C18:1) make up the largest proportion of the SM profile in mammals. In this study, molecular species with sphinganine bases (C18:0) accounted for the largest proportion of rat brain SM profile. At this time, we have no suitable/conclusive explanation for this observed variation in brain SM profile following careful perusal of the data to ensure mass spectral interpretation, data normalization, and calculations are correct to the best of our knowledge. However, a similar SM profile with saturated base predominating has been reported previously in human retina .
Phospholipids are the major structural components of neuronal and other cellular membranes, and include PC, PE, PS, PI, and SM [50, 51]. All of these phospholipid classes were observed to have altered molecular species distribution following PPA infusion. It has been suggested that common neurodevelopmental disorders such as ASD could be associated with functional deficiencies or imbalances in fatty acid synthesis/metabolism [5, 27, 29, 36]. Most of these former studies evaluated the fatty acid composition following hydrolysis of either total or individual phospholipid classes. Alterations in specific phospholipid molecular species could not only contribute more specific clinical criteria, but could also provide a basis for mechanistic interpretations. However, to date, only one study  has evaluated the intact phospholipid molecular species in the blood of ASD patients. Further, these authors only analyzed PE phospholipid molecular species. In the present study, we evaluated SM, PC, PE, PS, and PI molecular species following infusion with PPA and the induction of abnormal (ASD-like) behaviors. Alterations were observed in 21 brain and 30 blood phospholipid molecular species. Pastural et al. observed elevations in the relative proportions of plasmalogen PE, saturated, and polyunsaturated PE molecular species in the plasma of ASD patients. In our plasma analyses we also observed elevations in the relative proportions of polyunsaturated PE species, but the relative reduction in plasmalogen PE was in contrast with their findings. In addition, plasma from PPA-infused rats demonstrated elevations in the relative proportions of some saturated SM and PC molecular species, diacyl and plasmalogen monounsaturated PC species, PS monounsaturated species, and some polyunsaturated PI molecular species (36:3, 40:4, and 40:6). Elevations were also observed in the proportions of some brain PC polyunsaturated molecular species, PS monounsaturated, and PE plasmalogen species in the present study.
Analysis of SM, PC, PI, PE, PS, and PC phospholipid classes were conducted with both brain and blood samples. In many cases, the same phospholipid molecular species were altered in both blood and brain, but the direction and the relative proportions of these alterations were not consistent between both sample types. Much of the published work demonstrating alterations in lipid metabolism in autism has been done using plasma obtained from ASD patients [5, 27, 32, 36]. The findings from these studies include elevations in saturated fatty acids [27, 29, 30], accompanied by a decline in plasmalogens [5, 29, 30], mono and polyunsaturated (ω3 + ω6) fatty acids [5, 29, 30, 32, 36, 52]. Conversely, others have reported elevations in the proportions of mono and poly unsaturated fatty acids [5, 27, 30, 53]. However, the origin of these altered fatty acids and plasmalogens are unknown, because the structures of the phospholipids were destroyed by hydrolysis during sample analysis. This paper is the most comprehensive study to date which examines intact PL molecular species alteration in relation to autism. Currently, there is no clear uniform mechanism governing the etiology and early detection of ASD, and no accepted biomarkers are available. Although this study is descriptive in nature, it provides considerably more information than is currently available, and as such provides a foundation for defining which intact phospholipid molecular species can be altered in relation to ASD. This could provide a framework for future studies to elucidate the mechanisms associated with the observed lipid alterations and their relationship to behavioral changes in ASD.
The use of an animal model allowed comparison of brain and plasma PL during the period of PPA-induced ASD-like behaviors. The observations that small amounts (1.04 micromole/infusion) of PPA into brain can influence plasma lipid composition are considered intriguing. However, the plasma alterations noted did not correlate directly with those in brain. It could be that this difference merely reflects the nature of the PPA rodent model where treatment is limited to 8 days. Furthermore, ASD likely involves genetic, metabolic, and environmental factors which could result in systemic as well as CNS effects.
It is evident from the data presented in this study that PPA infusion produced small but significant alterations in the composition of brain and plasma phospholipid species. These alterations occurred independent of diet. The most notable alterations were observed in the composition of brain SM, diacyl mono and polyunsaturated PC, PI, PS, PE, and plasmalogen PC and PE molecular species. These observations are considered interesting because alterations in brain lipid composition, particularly during development can potentially have serious consequences on CNS function.
Potential physiological consequences of altered phospholipid molecular species and their relation to ASD
Lipid mediated signaling and neuroinflammation in ASD
The pathological consequences of disturbances in phospholipid metabolism could include alterations in signal transduction involving the generation of second messengers derived from docosahexaenoic (C22:6n3) and arachidonic (C20:4n6) acids . The observation that PPA infusion increased the proportions of brain PI and PC molecular species containing arachidonic acid and decreased the proportions of PS and PE molecular species with docosahexaenoic acid, suggests that PPA could influence the innate neuroinflammatory process observed in autism. Metabolism of arachidonic and docosahexaenoic acids released from the sn-2 position of the glycerol moiety by phospholipase A2 results in the formation of eicosanoids and docosanoids, respectively . Both eicosanoids and docosanoids are potent modulators of the inflammatory response system. Eicosanoids are inflammatory mediators that induce the formation of proinflammatory cytokines such as tumor necrosis factor (TNF), interleukin 1 (IL-1), and interleukin 6 (IL-6). Elevated levels of TNF, IL-1, and IL-6 have been reported in plasma of ASD patients . Docosanoids on the other hand are antiflammatory and include protectins and resolvins that have known neuroprotective effects . There is now emerging evidence that autism may be accompanied by abnormalities in the inflammatory response system , and that this abnormality may be related to the increases in oxidative stress [55–57], innate neuroinflammation , and altered lipid profiles [5, 27, 30, 32, 53] reported in ASD.
The increased accumulation of brain molecular species with the eicosanoids precursor (arachidonic acid), and the reduced proportions of the molecular species containing the docosanoids precursor (docosahexaenoic acid) observed in this study are interesting, considering the increased innate neuroinflammation (reactive astrogliosis and activated microglia) previously observed with this model  and reported for the brain of ASD patients at autopsy .
Several of the studies analyzing the hydrolyzed fatty acid components obtained from the blood of ASD patients also report alterations in the proportions of arachidonic and docosahexaenoic acids [27, 29, 32, 36]. This could indicate aberrations in fatty acid elongation and desaturation may occur in the etiology of ASD. Arachidonic and docosahexaenoic acids are cleaved from the sn-2 position of the glycerol moiety by phospholipase A2. Genetic sites linked to autism on chromosome 8q22 are in the proximity of the gene (8q24) for secretory phospholipase A2 . Collectively, these findings suggest possible involvements of arachidonate, docosahexaenoate, and phospholipase A2 in the signal cascade associated with the innate neuroinflammation observed in ASD.
Membrane fluidity and stability
Biological membranes are predominantly bilayers in which the inner and outer leaflets have different phospholipid compositions. In contrast, blood plasma phospholipids are present as monolayers, sourrounding lipoproteins, and the effect of fluidity on lipoprotein function is not well understood. Here we will refer to both cellular bilayers and blood lipoprotein monolayers collectively as ‘membranes’. Phospholipid molecular species distribution influences bilayer physical properties such as fluidity and this affects membrane protein function. Monolayer molecular species composition also affects physical properties. Organisms can adjust the order or fluidity of their cellular membranes in response to changes in their physiological environment by altering their lipid composition . Alterations in brain and blood phospholipid composition consistent with an adjustment in the order or fluidity of the membrane in these tissues in response to PPA infusions were observed in this study. This adjustment was reflected by changes in the relative proportions of unsaturated, diacyl, and/or plasmalogen species. Alterations in desaturation can have profound changes on membrane fluidity because increased carbon-carbon double bonds make unsaturated fatty acids more mobile, flexible, and fluid . Changes in the relative proportions and the composition of diacyl and plasmalogen forms of phospholipids can also impact the fluidity of the membrane. For example, plasmalogen species facilitate membrane fusion six times faster than diacyl species .
Phosphatidylcholine, which has a large polar head group does not pack closely in membrane bilayers and tends to be more fluid compared to PE, which has a small head group and packs more closely in membranes, making them less fluid at physiological temperatures. The PC/PE balance in cell membranes is thought to regulate membrane fluidity and stabilize the membrane [50, 60]. In both brain and blood membranes, PPA infusions alter the PC and PE composition (both diacyl and plasmalogen forms) possibly disrupting this balance. Alterations in brain membrane lipid composition affecting fluidity have been found to be associated with a number of behavioral abnormalities, as well as neurological and psychiatric disorders in both adults and children . Alterations in membrane fluidity affect membrane properties, which in turn can affect the functions of integral membrane proteins, ion channels and the permeability of solutes across the membrane . It is unclear at this preliminary stage whether or not any of these processes are affected by PPA infusion in this model, or if they are related to the etiology of ASD. These are the subjects of further studies in our laboratory.
Lipid oxidation/peroxisomal function
Plasmalogens are vinyl ether lipids found in PE, PC and PS. Several studies have reported increases  or declines [5, 29, 30] in plasmalogens obtain from the bloods of ASD patients. Typically plasmalogens have docosahexaenoic or arachidonic acids esterified in the sn-2 position of the glycerol moiety, and are essential for normal brain development and functions. Reduced plasmalogens and docosahexaenoic acid levels are characteristic of peroxisomal-associated neurological disorders such as infantile Refsum disease, adrenoleukodystrophy, adrenomyeloneuropathy, and Zellweger’s syndrome [5, 27, 59]. The only study we know that analyzed the intact PE molecular species of blood obtained from ASD patients observed an overall increase in PE plasmalogens species, and this increase was accompanied by an increase in docosahexaenoic acid containing molecular species . In our rat model, we observed an overall reduction in the relative proportions of PE plasmalogens in plasma, inclusive of species with docosahexaenoic acid at the sn-2 position of the glycerol moiety. However, consistent with , elevated proportions of PE polyunsaturated diacyl species were observed in plasma, inclusive of docosahexaenoate containing species.
In brain, a reduction in the relative proportions of diacyl and plasmalogen PE molecular species containing docosahexaenoic acid was observed in this study following PPA infusion. This was accompanied by a relative increase in several other PE plasmalogen molecular species lacking docosahexaenoic acid in the sn-2 position of the glycerol moiety. Plasmalogens act as a reservoir for docosahexaenoic acid, and both compounds have synthetic steps that occur in the peroxisome, providing a biochemical link . Peroxisomal disorders are characterized by abnormal peroxisomal biogenesis associated with altered functionality of the two rate limiting enzymes in plasmalogen synthesis, acyl-coenzyme A (CoA): dihydroxyacetonephosphate acyltransferase and alkyldihydoxyacetonephosphate synthase [36, 59]. It appears from the findings presented in this study and those from previous reports [5, 27, 29, 30, 52] that aberrations in plasmalogen metabolism may occur in ASD, implying that peroxisomal dysfunction could be involved.
Plasmalogens are considered endogenous antioxidants, because their vinyl ether bonds are efficient neutralizers of reactive oxygen species, which damage the polyunsaturated fatty acids present in the sn-2 position of plasmalogen phospholipids . The alterations observed in plasmalogen molecular species in relation to the alterations in brain and blood polyunsaturated PE and PC molecular species, may be a response to increase oxidation following PPA infusions. This is very interesting because oxidative damage of lipids has been suggested to play a part in the pathogenesis of many neurological diseases including autism [26, 55]. In addition, increased oxidative stress, and decreased antioxidant capacity have been previously reported in this rodent model [7, 8], and also found to be present in ASD patients [55–57]. Interestingly, oxidative damage has been shown to uncouple the gap junctions in astrocytes [63, 64]. Arachidonic and docosahexaenoic acids which are very susceptible to oxidative damage have been shown to modulate the coupling capacity of gap junctions [65–67]. In this study, PPA infusion led to alterations in the proportions of brain arachidonic and docosahexaenoic acids containing molecular species.
Collectively, the findings presented in this study, along with those observed in the blood of ASD patients; indicate that alterations in peroxisomal associated lipid metabolism and increased oxidative stress, possibly predisposing polyunsaturated fatty acids to oxidative damage may be associated with the pathogenesis of ASD.