**3. Glutamate**

is taken to the inside cell furtherly degrades by the transaminase or succinate semialde‐

GABA has a key role in the regulation of early developmental stages of cell migration, neuronal differentiation and stages of maturation [11]. Besides, formation of GABAergic system has a critical role in migration of GABAergic neurons and formation of glutamer‐ gic system mediated excitatory processes that regulate cortical inhibitory system [12]. Therefore, it is not suprising that especially in ASD and in many neurodevelopmental disorders GABAergic system is the main responsible [13, 14]. In addition, the high prevalence of epilepsy in patients with autism have made it worth to investigate GABA

Neurochemical abnormality that postulated to be associated with pathophysiology of ASD is the reduction in the expression of GAD65 and GAD67 which cause suppression of GABAergic inhibition [16]. Fatemi and his colleagues [17], in the cerebellum and parietal cortex of patients has shown significantly decrease in two isoforms of the rate-limiting enzyme which are responsible for the conversion of glutamate to GABA. Detection of low platelet GABA levels in children with ASD [18] and postmortem studies that illustrtaed significant reduction in GABAA and GABAB receptor subunit in various brain regions [19, 20] support the widespread dysfunction of GABAergic system in patients with ASD. Reduced production or signaling of GABA cause hyperexcitability state and leads to cognitive dysfunction [21]. Deletional mutations of genes encoded by chromosome 15q11-q13 which is some of the GABAA receptor subtype unites (GABRB3, GABRA5 and GABRG3) might be cause of reduction in GABAergic transmission, and these mutations have been suggested to be a risk factor ASD [14]. Also, many of the candidate genes associated with ASD are expressed in interneurons [22]. Antiepileptic agents, especially benzodiazepines has been used in ASD and epilepsy coexisted patients and they have shown to improve socialization and communication skills, though, in some cases, they lead to increased anxiety and aggression, because of this, the information mentioned above is not clear yet [23,24]. Lemonier and Ben-Ari [25] sugeested that the inhibition of Na / K / Cl transporter (NKCC1) lead intracellular increased Cl levels, so the GABAergic transmis‐ sion will change depolarization to the hyperpolarization and in five ASD cases they get positive results after the treatment with NKCC1 inihbitor bumetanide. Then they carried out double blind randomized controlled clinical trial of bumetanide for treatment of ASD for 3 months of period in 54 patients, the results has shown to provide a significant improvement of ASD symptoms [26]. In utero exposure to valproate in mice model, has caused dissappearance of swicth between GABA excitation / inhibition and this problem has shown to lead the devel‐ opment of chronic chlorine deficits and autistic-like behavior [27]. Ion channels mutated mouse model which led to the reduced GABAergic transmission, and the corelation between ASD symptoms and reduced GABAergic transmission level and with benzodiazepine treatment

As a result of animal model publications and studies conducted in patients with ASD has confirmed the hypothesis of "decreased GABAergic transmission in ASD patients". In future studies, to develop a new therapeutic agents, and to even prevent the disease focus should be

hide dehidrogenase enzymes [9].

16 Autism Spectrum Disorder - Recent Advances

neurotransmitter system in individuals who has ASD [15].

autistic-like behavior to has shown to decrease [28].

directed on the GABA neurotransmitter system.

Glutamate is essential excitatory neurotransmitter of the central nervous system. It is synthe‐ sized from glutamine via glutaminase enzyme. There are two types which are iyontropic and metabotropic receptors. Metabotropic receptors (mGluR) are coupled with G protein and within the cell according to signaling pathways they divided 3 into subtypes: Group I (mGluR1 and mGluR5), group II (mGluR2 and mGluR3), Group III (mGluR4 and mGluR6-8). Group I works through activation of phospholipase C whereas Group II and Group III works through decreasing cyclic AMP level [29]. Ionotropic receptors which are coupled with ion-channel, have 3 sub-types: N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole‐ propionic acid (AMPA) and kainate receptors. Kainat receptors located presynaptically at the hippocampus, stimulation of them reduce glutamatergic transmission [30]. Induction of AMPA receptors, these are associated with learning and memory, lead to the long-term potentiatio (LTP) and long-term depressio of (LTD) [31]. High levels of glutamate leading to overstimulation of NMDA receptors and cause a high amount of calcium influx, which is main responsible for excitotoxicity lead to the neuronal damage. Therefore, optimization of the level of glutamate in the synaptic cleft is critical. To protect post-synaptic neurons from excitotoxic effect the neuronal glutamate transporters which reside at the presynaptic membrane take back glutamate into cell from synaptic cleft. In final stage, glutamate is destroyed with GAD [1]. Balance between excitation / inhibition is crucial for synaptogenesis and plasticity, especially in first 3 years of life [32]. Blockade of NMDA receptors in the prenatal period initiates apoptosis in neurons [1].

From this point, glutamate plays a central role in shaping the architecture of the brain. Cell migration, maturation and developmental stages, such as synaptogenesis and neuroplastisic‐ ity is accomplished with the optimum glutamat transmission level [33, 34]. At the same time it is directly associated with cognitive processes such as memory and learning [35].

Glutamate receptors associated with ASD are highly expressed in the hippocampus and cerebellum [36]. For these reasons, the role of glutamatergic system in patiets with ASD has been substantially investigated, two opposite hypotheses regarding the role of this system have been proposed [37]. First hypotheses of ASD has been proposed hypoglutamatergic state [38, 39, 40], the second postulated the depletion of GABAergic inhibition excitation / inhibition rate which eventually lead to the hyperglutamatergic state [41, 42, 43]. Consistent with the hypothesis suggested that ASD is hypoglutamatergic disorder, in 1998 Carlsson has postulated decrease in glutamate signaling lead to activation of receptors at the cortical GABA interneur‐ ons and this state cause significant depression in excitator glutamate circuit [38, 44].

Other supportive evidence is hypoglutamatergic state in mouse models caused similiar presentation to ASD including inability to change behavior paradigm, limitation in habits and behavior [45] In a postmortem study patients with ASD has shown significant decrease in AMPA type 2 and 3 in cerebellum tissue [40].

Another hypothesis that might be surrogate to explain ASD is hypoglutamatergic state and associated cortical tissue hyperexcitability in spesific cortical areas. Some studies has demon‐

stratedhigher serumglutamate levels inindividualswithautism[46].Increasedglutamate level probably connected with diminished GAD enzyme level [47, 48, 49]. This diminish also explain reduction in GABA transmission [50]. First study was done by Shimmura has illustrated higher serumglutamate levels andlower glutamine levels [51]. Secondly Shimmura et al.[52]hasdone another study they researchedbrain tissue from 7 postmortem ASDpatients,they found higher levels of glutamate and glutamine levels at anterior cingulate cortex, interstingly levlels of glutaminase, glutamine synthase, and GAD were normal. As mentioned above ASD patients have high incidence of epilepsy, this is due to increase in glutamatergic activity [53, 54].

Animal models and conducted clinical studies in ASD subjects support hyperglutamatergic hypothesis. Silverman et al. [55] is conducted a study on ASD core symptoms observed mice model and found that GRN-529 (allosteric modulators of mGluR5 receptor) ameliorated all core symptoms of ASD. Another study conducted with AMPA receptor agonist (Ampakin) relieved symptoms of respiratory system on mice model with Rett syndrome [56]. Lamotrigine, which reduce glutamate transmission, has improved communication skills, socialization and behavior problems in 28 children diagnosed with ASD [57]. Ketamine, an NMDA receptor antagonist, has been shown to have a positive impact on focused attention in ASD cases [58].

Another NMDA receptor antagonist, memantine, significant improvement was observed on learning, language skills and in the areas of socialization in patients with ASD [59]. Recently, a randomized controlled study carried out, the memantine and risperidone receiving group were compared to placebo and risperidone receiving group, at the 10th week of treatment, memantine and risperidone received group better recovered compared to only risperidone received group in terms of the irritability, stereotypies and hyperactivity symptoms [60]. Recently, non-invasive brain imaging techniques such as magnetic resonance spectroscopy has enabled measurment of glutamate levels in brain tissue. Since first study was published in 2006 to date there were 15 studies done and conflicting results have been obtained [37]. In some studies, the anterior cingulate cortex [61] and auditory cortex [62] areas glutamate levels was increased compared to healthy controls, while in others there was no difference, and in the rest lower glutamate levels was observed [63, 64].

Some researchers thought these two hypotheses related to glutamatergic system are not completely opposite, some spesific cortical areas has increased excitatory / inhibitory ratio whereas in other regions, this ratio could turn opposite [44].

As a result, it is not clear yet whether the ASD individuals hyper or hypoglutamatergic, but it is clear that there is dysfunction in the glutamatergic system. New investigations has focused more in hyper-glutamatergic state and efforts are directed at glutamate receptor antagonismin order to develop new therapeutic agents. A better understanding of the glutamatergic system agents in the future will contribute to enlight ASD pathogenesis.
