**5. Brain alterations induced by prenatal exposure to VPA**


medial prefrontal cortex of rats exposed to VPA, with enhanced synaptic plasticity and short-

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Synaptic impairments were already described in autism, which may be related to neuroligins alterations. Neuroligins are a family of proteins which play a central role in synaptic matura‐ tion and were affected in rats after *in utero* exposure to VPA. Neuroligin 3 mRNA expression was decreased in the hippocampus, especially in *cornu ammonis*(CA1) and dentate gyrus [62].

Synaptic plasticity is influenced by brain-derived neurotrophic factor (BDNF), a factor that modulates several neurochemical parameters. High levels of BDNF have been reported in the blood of patients with autism [102]. BDNF acts through TrkB-mediated activation of various signal transduction pathways, including pathways that involve PI3K, mitogen-activated kinase (MAPK), and phospholipase C-γ [103]. Infusion of BDNF in the nucleus *accumbens* of aged rats restored synaptic plasticity and improved cognition [104] and some environmental factors, such social isolation, results in low levels of BDNF in the hippocampus of rats [105]. Animals exposed to VPA *in utero* display decreased cortical BDNF mRNA expression. It is important to notice that altered levels of the transcript will not necessarily mean an altered protein expression [63]. Diminished BDNF may lead to altered synaptic development; once it is known that this neurotrophic factor is involved in development and function of serotonergic

Several hypotheses have arisen to explain the social deficits in autism. One of these proposals points an alteration in opioidergic mechanisms as a likely causative of behavioral impairments in this disorder [107]. Opioid peptides are involved in stress responses and affective states, and blockage of their receptors causes dysphoria in humans. Enkephalins are part of the opioid family and are distributed in brain areas, like the striatum and the nucleus accumbens, involved in processing emotional information, anxiety and fear. Exposure to VPA reduced proenkephalin mRNA expression in both the core and shell of the nucleus *accumbens* and dorsal

The monoamine system is also altered in patients with autism and their relatives. It was demonstrated that children with autism have increased 5-HT (serotonin) synthesis capacity when compared to children with typical development [108]. Besides, it is widely known that sleep disorders are common in autistic children [109]. Interestingly, increased levels of serotonin was found in pre-frontal cortex of rats prenatally exposed to VPA in association with disrupted sleep/awake rhythm. The elevated levels of 5-HT were found during light phase of animals' circadian rhythm [74]. It was proved that serotonergic neurons have a silent firing rate during REM sleep [110], indicating that the sleep disturbance found in the animals may be related to increased levels of 5-HT found in their prefrontal cortex. In addition, higher levels of 5-HT were also reported in the left side of hippocampus and in blood from rats [111]. However, using the whole hippocampus, it was demonstrated 46% decrease in 5-HT levels

Recently, we observed hippocampal reactive astrogliosis in the group of rats exposed *in utero* to VPA (see ref [84]). After 15 postnatal days, hippocampal astrocytes were intensely immunoreactive to the astroglial marker Glial Fibrillary Acidic Protein (GFAP) (Figure 2).

*striatum* of rats concomitantly to anxious-like behavior [91].

from rats exposed to VPA *in utero* [70].

and long-term fear memories [82].

neurons [106].


**Table 7.** Brain alterations induced by *in utero* exposure to VPA

Behavioral outcomes started to be studied, demonstrating a number of anatomic and behav‐ ioral features characteristic of human cases by exposing rodents' embryos to VPA at the time of neural tube closure. One of the affected structures in the brains is the cerebellum. Magnetic resonance imaging showed that patients with autism have reduced size of the cerebellum when compared to controls, displaying smaller vermal lobules VI and VII. This abnormality is probably an outcome of developmental hypoplasia and not likely shrinkage or deterioration after full development had been achieved [100]. Similar alterations were found in brains from rat model of autism induced by prenatal exposure to VPA [9]. Exposed rats showed a reduction in the number of motor neurons of the earliest-forming motor nuclei (V, XII), and had the VI th and III rd cranial nerve nuclei affected. In the same way, another work found diminished number of cells in the posterior lobe of the cerebellum [86]. In this context, cerebellar anatomy alterations in humans might be due to loss of neurons in the cranial nerve motor nuclei, as demonstrated in rats.

The amygdala is likely to be also linked to autism, due to its involvement in social-emotional behavior. Rats exposed to VPA *in utero* had longer lasting and harder to extinguish fear memories, which could be explained by the hyperreactivity and hyperplasticity found in the lateral amygdala [96, 101]. Another work found enhanced long-term potentiation (LTP) in the medial prefrontal cortex of rats exposed to VPA, with enhanced synaptic plasticity and shortand long-term fear memories [82].

**5. Brain alterations induced by prenatal exposure to VPA**

150 Recent Advances in Autism Spectrum Disorders - Volume I

Rat

Mice

demonstrated in rats.

**Table 7.** Brain alterations induced by *in utero* exposure to VPA

Once prenatal exposure to VPA became a reliable tool to model autism, more brain alterations

**Rodent VPA (mg/kg) Findings References**

<sup>350</sup> Reduction in the number of motor neurons from hypoglossal and

<sup>500</sup> Reduction in the number of putative synaptic contacts in connection

<sup>400</sup> Prolonged neuronal progenitor cells proliferation in embrionary

<sup>600</sup> Decreased number of purkinje cells, neuronal degeneration and

<sup>500</sup> Reduction in the number of Parvalbumin -positive inhibitory neurons

<sup>500</sup> Nissl-positive cell loss in the middle and lower layers of the prefrontal

Behavioral outcomes started to be studied, demonstrating a number of anatomic and behav‐ ioral features characteristic of human cases by exposing rodents' embryos to VPA at the time of neural tube closure. One of the affected structures in the brains is the cerebellum. Magnetic resonance imaging showed that patients with autism have reduced size of the cerebellum when compared to controls, displaying smaller vermal lobules VI and VII. This abnormality is probably an outcome of developmental hypoplasia and not likely shrinkage or deterioration after full development had been achieved [100]. Similar alterations were found in brains from rat model of autism induced by prenatal exposure to VPA [9]. Exposed rats showed a reduction in the number of motor neurons of the earliest-forming motor nuclei (V, XII), and had the VI th and III rd cranial nerve nuclei affected. In the same way, another work found diminished number of cells in the posterior lobe of the cerebellum [86]. In this context, cerebellar anatomy alterations in humans might be due to loss of neurons in the cranial nerve motor nuclei, as

The amygdala is likely to be also linked to autism, due to its involvement in social-emotional behavior. Rats exposed to VPA *in utero* had longer lasting and harder to extinguish fear memories, which could be explained by the hyperreactivity and hyperplasticity found in the lateral amygdala [96, 101]. Another work found enhanced long-term potentiation (LTP) in the

500 Altered distribution of 5-HT neurons in the dorsal raphe nucleus. [71]

oculomotor nuclei. [9]

period. [79]

chromatolysis. [98]

in the neocortex. [99]

cortex and in the lower layers of the somatosensory cortex. [65]

between layer 5 pyramidal neurons. [97]

were investigated in rodents exposed to this teratogen, as summarized in Table 7.

Synaptic impairments were already described in autism, which may be related to neuroligins alterations. Neuroligins are a family of proteins which play a central role in synaptic matura‐ tion and were affected in rats after *in utero* exposure to VPA. Neuroligin 3 mRNA expression was decreased in the hippocampus, especially in *cornu ammonis*(CA1) and dentate gyrus [62].

Synaptic plasticity is influenced by brain-derived neurotrophic factor (BDNF), a factor that modulates several neurochemical parameters. High levels of BDNF have been reported in the blood of patients with autism [102]. BDNF acts through TrkB-mediated activation of various signal transduction pathways, including pathways that involve PI3K, mitogen-activated kinase (MAPK), and phospholipase C-γ [103]. Infusion of BDNF in the nucleus *accumbens* of aged rats restored synaptic plasticity and improved cognition [104] and some environmental factors, such social isolation, results in low levels of BDNF in the hippocampus of rats [105]. Animals exposed to VPA *in utero* display decreased cortical BDNF mRNA expression. It is important to notice that altered levels of the transcript will not necessarily mean an altered protein expression [63]. Diminished BDNF may lead to altered synaptic development; once it is known that this neurotrophic factor is involved in development and function of serotonergic neurons [106].

Several hypotheses have arisen to explain the social deficits in autism. One of these proposals points an alteration in opioidergic mechanisms as a likely causative of behavioral impairments in this disorder [107]. Opioid peptides are involved in stress responses and affective states, and blockage of their receptors causes dysphoria in humans. Enkephalins are part of the opioid family and are distributed in brain areas, like the striatum and the nucleus accumbens, involved in processing emotional information, anxiety and fear. Exposure to VPA reduced proenkephalin mRNA expression in both the core and shell of the nucleus *accumbens* and dorsal *striatum* of rats concomitantly to anxious-like behavior [91].

The monoamine system is also altered in patients with autism and their relatives. It was demonstrated that children with autism have increased 5-HT (serotonin) synthesis capacity when compared to children with typical development [108]. Besides, it is widely known that sleep disorders are common in autistic children [109]. Interestingly, increased levels of serotonin was found in pre-frontal cortex of rats prenatally exposed to VPA in association with disrupted sleep/awake rhythm. The elevated levels of 5-HT were found during light phase of animals' circadian rhythm [74]. It was proved that serotonergic neurons have a silent firing rate during REM sleep [110], indicating that the sleep disturbance found in the animals may be related to increased levels of 5-HT found in their prefrontal cortex. In addition, higher levels of 5-HT were also reported in the left side of hippocampus and in blood from rats [111]. However, using the whole hippocampus, it was demonstrated 46% decrease in 5-HT levels from rats exposed to VPA *in utero* [70].

Recently, we observed hippocampal reactive astrogliosis in the group of rats exposed *in utero* to VPA (see ref [84]). After 15 postnatal days, hippocampal astrocytes were intensely immunoreactive to the astroglial marker Glial Fibrillary Acidic Protein (GFAP) (Figure 2). Astrocytes are the major cell type in the central nerve system (CNS) and provide a variety of critical supportive functions that maintain neuronal homeostasis, participating of the synapse and the glutamatergic metabolism [112]. These cells become reactive in VPA group, charac‐ terized by up-regulation of GFAP and apparently show higher number of processes than the control cells as demonstrated by the squares in A and B.

toe-walking, have already been reported [116]. Evaluation of motor cortex neurons of rats exposed to VPA *in utero* showed no changes in length or volume of either basilar or api‐ cal dendrites, but presented greater dendritic arborization in comparison with controls. This data indicates that pruning of neurons is abnormal in animals prenatally exposed to VPA [95]. Evidence suggests that the same may happen in patients with autism, since there are reports of increased brain weight in autopsy cases of autism [117]. However, the involvement of the abnormal pruning in motor cortex neurons with motor disturban‐ ces in autism deserves further investigation. Individuals with autism are more likely to present hearing deficits. In a study with a group of 199 children and adolescents, 3.5%

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The superior olivary complex (SOC) plays different roles in hearing. It is located within the lower brainstem and it is involved in encoding temporal features of sound and descending modulation of the cochlear nucleus and cochlea for listening in background noise. Rats exposed to VPA *in utero* showed reduced number of neurons and disrupted neuronal morphology in the SOC. Neuronal cell bodies were smaller and more round, indicating that these anatomical feature might have a role in the hearing difficulties that are a common in patients with autism [87]. In a study with brains of patients with autism similar morphological alterations were

The cerebellum have been the focus of studies involving active and chronic neuroinflammatory process in autistic patients, demonstrating the presence of proinflammatory chemokines such as MCP-1 as well as antiinflammatory cytokines such as TGF-β1 in this brain structure. These findings support the idea that a chronic state of specific cytokine activation occurs in autism [113]. Because neuroimmune responses are influenced by the genetic background of the host, the role of neuroinflammation in the context of the genetic and other factors that determine

The spectrum of autism comprises a multifactorial group of disorders, with phenotypic diversity related to the symptoms and increasing prevalence. One of the major challenges of cognitive neuroscience is to understand how changes in the structural properties of the brain affect the plasticity exhibited whenever a person develops, ages, learns a new skill, make social interaction or adapts to a disease. In ASD, it is necessary studies in this field attempting to explain and understand the trigger of autism. In this context, it is not easy to find a single animal model able to captures the entire molecular and cellular alterations observed in patients

Studies of *in utero* interventions in the search for animal models of autism, together with the study of potential clinical markers to ASD are innovative and may generate strategies aiming (a) the prevention of autism; (b) the construction of laboratory kits as new tools to improve and anticipate diagnosis; (c) the study of neuroglial plasticity; (d) the search for new clues to unravel the etiology of ASD. Challenged by these complexities, it is necessary to evaluate the

found, including soma size, shape and number of neurons in the SOC [119].

the autism phenotype remains an important issue to be investigated.

**6. Concluding remarks and scientific challenges**

with ASD.

had profound bilateral hearing loss or deafness [118].

Seven Fresh-frozen *post mortem* tissues from individuals with autism and CSF from six living autistic patients were investigated for cytokine protein profiling [113]. This study shows an active neuroinflammatory process in the cerebral cortex, white matter, and notably in the cerebellum. Immunocytochemical studies showed marked activation of microglia and astroglia. The cytokine profiling indicated that the macrophage chemoattractant protein (MCP)-1 and tumor growth factor-beta1, both derived from neuroglia were the most prevalent cytokines in brain tissues. We presumed that microglia/macrophage-derived pro-inflamma‐ tory cytokines regulate the transition of astrocytes into reactive astrogliosis. Nevertheless, the mechanisms which regulate the level of astroglial cell activity in the hippocampus from VPA autism model need to be investigated.

**Figure 2.** Astrocyte immunoreactive to GFAP in hippocampus from rats. A. Representative image from control group, B. Representative image from VPA group. Scale bar = 50 μm

Glutamatergic excitatory synapses are the major type of synapses in the brain and it was found that glutamate metabolism is altered in autistic CNS, particularly the glutamate receptors AMPA, NMDA and mGluR5 [114]. In agreement, rats exposed *in utero* to VPA show impair‐ ments in excitatory/inhibitory brain balance [78]. In this context, impairment in excitatory and inhibitory signaling during certain periods of development is proposed to be involved in the autism pathophysiology [115].

Although social impairments are one of the most important features observed in autism, patients present several other symptoms, including motor disturbances. Motor stereoty‐ pies are part of the so called autism triad of impairments, but hypotonia, motor apraxia, toe-walking, have already been reported [116]. Evaluation of motor cortex neurons of rats exposed to VPA *in utero* showed no changes in length or volume of either basilar or api‐ cal dendrites, but presented greater dendritic arborization in comparison with controls. This data indicates that pruning of neurons is abnormal in animals prenatally exposed to VPA [95]. Evidence suggests that the same may happen in patients with autism, since there are reports of increased brain weight in autopsy cases of autism [117]. However, the involvement of the abnormal pruning in motor cortex neurons with motor disturban‐ ces in autism deserves further investigation. Individuals with autism are more likely to present hearing deficits. In a study with a group of 199 children and adolescents, 3.5% had profound bilateral hearing loss or deafness [118].

The superior olivary complex (SOC) plays different roles in hearing. It is located within the lower brainstem and it is involved in encoding temporal features of sound and descending modulation of the cochlear nucleus and cochlea for listening in background noise. Rats exposed to VPA *in utero* showed reduced number of neurons and disrupted neuronal morphology in the SOC. Neuronal cell bodies were smaller and more round, indicating that these anatomical feature might have a role in the hearing difficulties that are a common in patients with autism [87]. In a study with brains of patients with autism similar morphological alterations were found, including soma size, shape and number of neurons in the SOC [119].

The cerebellum have been the focus of studies involving active and chronic neuroinflammatory process in autistic patients, demonstrating the presence of proinflammatory chemokines such as MCP-1 as well as antiinflammatory cytokines such as TGF-β1 in this brain structure. These findings support the idea that a chronic state of specific cytokine activation occurs in autism [113]. Because neuroimmune responses are influenced by the genetic background of the host, the role of neuroinflammation in the context of the genetic and other factors that determine the autism phenotype remains an important issue to be investigated.
