Introduction
The gut and brain form the gut-brain axis through bidirectional nervous, endocrine, and immune communication. A change in one of these systems will most certainly have effects on the other systems. Disorders in the composition and quantity of gut microbiota can affect both the enteric nervous system and the central nervous system [
1]. Specifically, microbiota has the capacity to impact the regular function of the brain, which can in turn affect the composition of microbiota via specific substances. Specific molecules and metabolic pathways in microbiota have been shown to be linked to neural development and neurodegenerative disorders, including Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, schizophrenia, and multiple sclerosis [
1‐
3].
Valproic acid (VPA) is a medication used for epilepsy and mood swings. Children prenatally exposed to VPA have an increased chance of being diagnosed with autism [
4‐
7]. In addition, VPA exposure leads to accelerated or early brain growth which also occurs in some cases of autism [
8]. Most importantly, VPA causes an alteration in the excitation/inhibition the cerebral cortex. Specifically, rats exposed to VPA in utero present with an increased glutamatergic and a decreased GABAergic component in the cortex [
9]. The VPA rat model of autism experiences behavioral, immune, and microbiota changes similar to those described in patients with autism. We recently discovered that specific GABAergic interneuron types, the parvalbumin (PV)+ Chandelier (Ch) and PV+ Baskets cells (Bsk) cells, are decreased in the prefrontal cortex in autism [
10,
11]. We also demonstrated that when VPA is administered via intraperitoneal injection to pregnant rats at a specific day of prenatal development with a specific dose (E (embryonic day) 12.5, 400 mg/kg), the offspring of these rats (“400-E12 VPA rats”) experienced a decrease in the number of PV+ Ch and PV+ Bsk cells in their adult cerebral cortex similar to what we found in humans with autism (under revision). In addition, the 400-E12 VPA rats also experienced behavioral changes similar to those exhibited by patients with autism (under revision).
ASD patients suffer from gastrointestinal problems and experience changes in the gut microbiota, including shifts in levels of
Firmicutes,
Bacteroidetes,
and Proteobacteria with the abundance of
Lactobacillares and
Clostridia [
12,
13]. Other gut commensals found to be altered in autism belong to the genera such as
Bifidobacterium,
Lactobacillus,
Prevotella, and
Ruminococcus [
14]. Microbiome changes have been also described in several mouse models for autism, with one publication in a VPA mouse indicating a decreased abundance for
Bacteroidetes in VPA exposed offspring [
15]. It is not yet clear whether the changes in the microbiome associated to specific disease states are a cause or a consequence of the disease. Recent studies indicate that gut microbiota transplantation can transfer behavioral phenotypes, suggesting that the gut microbiota may be a modifiable factor modulating the development or pathogenesis of neuropsychiatric conditions. In this study, we investigated changes in microbial richness and microbiome composition in rats in response to VPA prenatal administration (400 mg/kg at E12) and found VPA-induced alterations similar to those seen in autism.
Discussion
The gut and brain form the gut-brain axis through bidirectional nervous, endocrine, and immune communications. Mammalian species often contain similar microbiome richness at the level of phylum, but diversity and richness of species are highly variable among individuals [
23]. This variability is determined by many factors, including genetics, environment, diet, disease, stress, and age [
24]. When microbiota composition is altered due to any of these factors, the function of the intestinal mucosal barrier is reduced; and bacterial products such as amyloids and lipopolysaccharides leak, increasing the permeability of the blood brain barrier, which, in turn, affects the central nervous system [
25].
Humans with autism and mice models of autism have shown significant alterations in their microbiota composition. Children with autism present with more GI symptoms than typically developing children, and the severity of their GI symptoms is correlated to the severity of their behavioral symptoms [
26,
27]. These children also demonstrate bacterial dysbiosis, which has been suggested to play a role in autism’s etiology [
28]. While different studies have found changes in specific bacteria are often associated to dysbiosis in autism, it is generally accepted that the gut microbial community of patients with autism displays a higher relative abundance of Lactobacillacease and Clostridia and a reduced incidence of the
Prevotella and other fermenters [
29‐
35].
Studies in mice have allowed to better understand the role of the microbiota in autism [
36]. The lack of microbiota produces changes in behavior. For example, germ-free mice lack a preference for spending time with another mouse over spending time in an empty chamber and deviate from the experimental expectation that they would spend more time exploring a space containing a new mouse rather than a familiar mouse [
37,
38]. Germ-free mice also show a differential gene expression associated with neuronal structure and function in the amygdala [
39]. Germ-free rats present with a social deficit phenotype in the reciprocal social interaction test [
40]. Antibiotic treatment in wildtype and mouse models of autism also affects social behavior [
15,
41,
42]. On the other hand, the use of probiotics ameliorates behavioral deficits [
38,
42]. Together, these data point out a role of microbiota in regulating behavior. The nature of microbiota has been studied in several mouse models for autism. The inbred mouse, BTBR, that presents with the full spectrum of ASD-like behavior, shows an overall decrease in bacterial diversity characterized by an increase in the relative abundance of the genus
Akkermansia and a decrease in abundance of
Bifidobacterium and Clostridiales [
43‐
45]
. In addition, BTBR mice have impaired intestinal integrity and a deficit in the intestinal tight junction proteins
Ocln and
Tjp1 [
46]. Environmental mice models of autism have also produced information about the importance of microbiota in this condition. In the maternal immune activation (MIA) mouse model, the species richness did not differ significantly between control and MIA offspring, but the offspring displayed decreased intestinal barrier integrity, altered gut microbiota, and increased abundance of the families Lachnospiraceae, Porphyromonadaceae, and Prevotellaceae [
47]. In the maternal high-fat diet (MHFD) mouse model for autism, the diversity of the microbiota was decreased compared to the control group, with marked decreased in
Lactobacillus,
Parabacteroides,
Helicobacter, and
B. uniformis. In this study, we demonstrated that species richness in the fecal microbial community in the autistic-like rat model, the 400-E12 VPA rat, was significantly reduced. Using next-generation sequencing technology in a murine autism model, it was reported that the microbiome composition in mice in utero exposed to VPA presented with a decreased of
Bacteroids [
15]. Other gut commensals found to be altered in the VPA mice were
Deltaproteobacteris and
Erysipelotrichales. These changes in VPA mouse microbiota composition were coincident with changes in behaviors linked to autism [
15].
Our 400-E12 VPA rats showed a decrease in microbial diversity (species richness). Specifically, significant increases in the abundance of α-Proteobacteria, Eubateriaceae, Rikenellaceae, and Staphylococcaceae. On the other hand, Enterobacteriaceae was significantly decreased by VPA exposure in utero. At the genus level, we found a significantly higher abundance of the genus Anaerotruncus in the control group and a significantly increased abundance of the genera Allobaculum, Anaerofustis, Proteus, and Staphylococcus in the VPA group.
This is the first time the microbial species richness and microbiome composition have been studied in a rat model for autism, the 400-E12 VPA rat. The decrease in microbial diversity in this rat model was consistent with the observations in human autism and most of the mouse models of autism studied to date. The gut microbial composition was largely similar to that of humans with autism and murine autism-like models. The enteric bacteria, especially the class Clostridia, are known to play an important role in children with autism (Frye et al. 2015). In our study, Clostridia is the most dominant class in the rat fecal microbial community, accounting for more than 60% of all sequence reads, followed by the class Bacteroidia with more than 30% of the sequences. Among the 100 OTU significantly impacted by prenatal VPA administration, the vast majority of them, 94, belonged to Clostridia, suggesting that ecological manipulation via antibiotics or pre- or pro-biotic approaches targeting this class of gut bacteria may prove effective in alleviating autism symptoms. A significant reduction in microbial species richness, such as Chao1, in the 400-E12 VPA rats was consistent with the observation in BTBR T
+Itpr3
tf/J mouse model of autism [
44]. However, biodiversity encompasses both species richness and evenness as well as interactions among species in the ecosystem [
16]. While a marked reduction in species richness was evident in the rats with prenatal VPA exposure, species evenness in the rat gut microbial community did not appear to be impacted. Furthermore, the microbial co-occurrence patterns and microbial interactions in the community appeared to be preserved in the rats with prenatal VPA exposure.
Moreover, our findings provide further evidence of sex-specific alterations of gut microbiome by prenatal VPA administration in rodents [
15]. For example, in male rats, the abundance of the family Coriobacteriaceae as well as the class Coriobacteriia was significantly repressed by VPA. An OTU (GreenGene ID_1113282), belonging to Mollicutes, was significantly increased by VPA. On the other hand, a twofold increase in the relative abundance of the phylum Proteobacteria, from 1.03% in the control rats to 2.17% in the male rats with VPA exposure, was observed. The VPA-induced increase became more evident in the class α-Proteobacteria, from 0.14% in the control male rats to 0.56% in the male rats with prenatal VPA exposure. The Proteobacteria are known to be a marker for an unstable microbial community and a risk factor of human disease [
48,
49]. An elevated Proteobacteria level is frequently associated with metabolic disorders and intestinal inflammation. The pathological relevance of elevated Proteobacteria abundance in autism warrants further investigation. In contrast to male rats, prenatal VPA exposure induced a distinguishingly different set of microbial taxa in female rats. The abundance of the genus Staphylococcus and the family S24-7 was significantly increased by prenatal VPA exposure only in female rats. A significant elevation of Candidatus Arthromitus, which harbors commensal SFB, by VPA was observed only in female rats. Numerous studies have established solid links between SFB colonization and human disease [
50]. As a potent inducer of IgA production and T
H17 immune responses as well as innate immunity, SFB may play a role in the pathogenesis of autism. Indeed, a recent study shows that pregnant mice colonized with SFB were more likely to produce offspring with maternal immune activation (MIA)-associated abnormalities [
41].
The composition of the microbiota is of great importance to the function of the brain. Bacteria can regulate brain function through several mechanisms. Some bacteria, such as
Bifidobacterium and
Lactobacillus, that inhabit in the gut, have the capacity to produce anti-inflammatory cytokines, while other, such as
Clostridium and
Ruminococcus [
51], can produce pro-inflammatory cytokines
. Metabolic products of the gut microbiota, such as short-chain fatty acids, have also been implicated in autism. Gut microbiota has been suggested to regulate many nervous functions including neurogenesis, differentiation, myelination, formation and integrity of the blood-brain barrier, neurotrophin and neurotransmitter release, apoptosis, gap junction modification, and synaptic pruning [
52]. Moreover, several microRNAs participate in signaling networks through the intervention of the gut microbiota [
53]. In addition, gut microbiota release inflammatory cytokines that can act as epigenetic regulators and regulate gene expression being a factor for example in cancer risk and diabetes-associated autoantigens [
54‐
56]. Here, we demonstrated that VPA also alters the metabolite potential of the microbial community in rats. VPA prenatal administration significantly elevated 21 bacterial pathways while repressing 8 pathways. Among them, there was an increase in activation of the bacterial secretion system, DNA replication, DNA repairs, and recombination proteins and a decrease in ABC bacterial transporter pathways. These data indicate a potentially higher activity of those pathways related to bacterial survival and function.
In conclusion, our data on the gut microbial community of the 400-E12 rats in response to prenatal VPA exposure indicate that this model, in addition to demonstrating behavioral and anatomical similarities to autism, also mimics the microbiota features of autism, making it one of the best-suited rodent models for the study of autism.