Autism spectrum disorder (ASD) is a group of complex neurodevelopmental disorders characterized by the presence of multiple and persistent deficits in social communication and social interaction associated with restricted interests and stereotyped and repetitive behaviors (American Psychiatric Association, 2013; Grzadzinski et al., 2013). The prevalence of ASD is on the rise, as documented in recent surveys (Baio et al., 2018). There are more males than females with ASD (a 4:1 ratio), and there are relevant clinical differences between the sexes (Tillmann et al., 2018).

The etiology of ASD is still poorly understood, but it appears highly genetically heritable. Indeed, there are identified genetic mutations—although not fully penetrant—in approximately 15% of the cases. Non-specific environmental risks factors, such as parental age, fetal exposure to alcohol or valproate, and premature birth/low birth weight, have also been identified (Murdoch and State, 2013; De Rubeis and Buxbaum, 2015; Sestan and State, 2018; Han et al., 2021a,b). The most frequent comorbidities and associated symptoms are learning/intellectual disabilities, sleep disturbances, epilepsy, attention deficit with hyperactivity, irritability (including self-injuries behavior, and temper tantrums), anxiety, and depression. Frequently used medications include methylphenidate and atomoxetine for attention disorders, melatonin for sleep disorders, and selective serotonin reuptake inhibitors (SSRIs) for anxious-depressive symptoms. Second-generation antipsychotics, namely risperidone, and aripiprazole have demonstrated clinical efficacy in behavioral disorders of ASD, and the United States Food and Drug Administration (FDA) has approved them for this use (Accordino et al., 2016). The presence and the severity of these symptoms are very heterogeneous among patients. Usual care includes psychosocial interventions as well as educational measures, patient and family support, and behavioral therapies such as applied behavioral analysis.

The considerable heterogeneity in the expression and severity of the core and associated symptoms have been an obstacle toward understanding ASD. Variability in the social domain ranges from a near absence of interest in interacting with others to more subtle difficulties managing complex social interactions that require an understanding of other people’s goals and intentions and social context cues (American Psychiatric Association, 2013; Grzadzinski et al., 2013). Similarly, repetitive behaviors range from simple motor stereotypies and/or a preference for sameness to much more complex and elaborate rituals accompanied by emotional dysregulation or tantrums when these rituals are interrupted. Some individuals with ASD lack basic speech abilities, while others can have language deficits that are mild and limited to language pragmatics. While a majority of individuals with ASD exhibit some level of intellectual impairment, intelligence quotients vary from the severe and profoundly impaired to above-average (Frye, 2018).

A theory of excitatory/inhibitory imbalance in ASD was proposed by Rubenstein and Merzenich (2003). This neurobiological model is based on the theory that autism and related disorders might reflect an increase in the ratio between excitation and inhibition leading to hyper-excitability of cortical circuits. The theory was attractive because it provided a potential explanation for the frequent observation of reduced GABAergic signaling in ASD (Cellot and Cherubini, 2014; Brondino et al., 2016). In addition, since inhibition was known or believed to contribute to sharpening the selectivity of excitatory responses in many brain areas, the loss of inhibition could lead to enhanced “noise” and imprecision in learning and cognition (Rubenstein and Merzenich, 2003).

Since this initial formulation, however, other studies have suggested a nearly opposite hypothesis; namely, that at least some ASDs might be characterized by a reduction in the ratio between excitation and inhibition (Vignoli et al., 2010; Spooren et al., 2012). An excitatory/inhibitory (E/I) neurotransmission imbalance in cortical neurons, using dysfunctional glutamatergic and GABAergic neurotransmission, has been reported in multiple studies, and altered glutamine levels and/or abnormal GABA/creatine levels in brain tissue or plasma are the relevant findings (Vignoli et al., 2010; Carlson, 2012; Spooren et al., 2012; Gao and Penzes, 2015; Dickinson et al., 2016; Lee et al., 2017; Al-Otaish et al., 2018; Goel and Portera-Cailliau, 2019; Port et al., 2019; Bruining et al., 2020; Culotta and Penzes, 2020). Moreover, a recent study showed that high glutamate/glutamine levels have also been associated with altered functional connectivity in key brain regions for ASD, such as the dorsal anterior cingulate cortex, and insular, limbic, and parietal regions (Siegel-Ramsay et al., 2021).

An E/I imbalance might be the consequence of an increased glutamatergic activity alongside with a decrease in the GABAergic signaling. Previous studies found that genes involved in the regulation of the neurogenesis or the synaptogenesis may be linked to the E/I imbalance (Vignoli et al., 2010). In neurodevelopmental disorders, an E/I imbalance could arise directly through alterations in genes coding for glutamatergic receptors or synaptic proteins (Lee et al., 2017; Culotta and Penzes, 2020). The synapse organizers, neurexins and their binding neuroligins, are implicated in the formation and maintenance of excitatory and inhibitory synapses. Heterozygous deletions eliminating exons of the neurexin-1α gene in patients with ASD have been detected, and the functional significance of this recurrent deletion is still unclear (Frye, 2018). However, the role of neurexins/neuroligins in the pathogenesis of neurodevelopmental disorders has been demonstrated (Dickinson et al., 2016; Al-Otaish et al., 2018; Wang et al., 2018; Goel and Portera-Cailliau, 2019; Port et al., 2019; Siegel-Ramsay et al., 2021). The final effect of changes in glutamatergic and GABAergic systems in ASD may be an overall increase in the ratio of excitation to inhibition (E/I) (Howell and Smith, 2019). The E-I ratio in neocortical structures is determined by pyramidal glutamatergic neurons and inhibitory GABAergic parvalbumin (PV)-positive interneurons that are modulated by minicolumns, i.e., groups of functionally autonomous neurons whose afferent and efferent connections influence the functioning of microcircuits), which are abnormal in ASD (Nelson and Valakh, 2015; Uzunova et al., 2016). There are several candidate mechanisms for glutamatergic hyperexcitability. Neuroligins (NL1-4) and neurexins (Nrxns 1–3) have been linked with ASD via point mutations and truncations, as well as chromosomal rearrangements that have been identified in the region of interest (Snijders et al., 2013; Port et al., 2014; Dickinson et al., 2016). SHANK1, SHANK2, and SHANK3 are scaffolding proteins that influence the postsynaptic density of glutamatergic synapses and are of primary importance in ASD. SHANK3 is reported to be involved in Phelan-McDermid syndrome (PMS) a form of ASD associated with moderate to severe intellectual disability (ID). Regarding SHANK2 and SHANK1, they were found altered in ASD associated with mild ID as well as in high functioning individuals (Zikopoulos and Barbas, 2013; Cochran et al., 2015; Howell and Smith, 2019; Bruining et al., 2020; Culotta and Penzes, 2020; Siegel-Ramsay et al., 2021). As to the mechanisms of GABAergic inhibitory dysfunction, the link with core ASD symptoms in humans is still under investigation. The deficit in binocular rivalry, a visual function that is thought to rely on the balance of excitation/inhibition in the visual cortex, has been observed in ASD individuals (Dunn and Jones, 2020). The link between GABA and binocular rivalry dynamics was found specifically absent in ASD pointing to an insufficient GABA inhibitory function (Dunn and Jones, 2020).

Based on these findings, research efforts have been directed to identify agents that have the potential to reverse this abnormal signaling, namely, excitation to inhibition (E/I) imbalance from preclinical models to clinical trials.

Dysfunctional glutamatergic transmission occurs through different pathways of altered excitatory transmission: downregulation of AMPA receptors, abnormalities in NMDA-receptor-mediated plasticity, and altered mGluR signal transduction. Research has implicated glutamate hyperactivation and GABAergic system hypofunction in the pathogenesis of ASD. In neurons and glia, glutamate is involved in intracellular transduction mechanisms at NMDA and mGlu receptors (Uzunova et al., 2014). AMPA receptors are thought to be implicated in behavioral disorders associated with the core symptoms in children and adolescents with ASD (Lipton, 2006; Uzunova et al., 2014).

Glutathione and N-Acetylcysteine (NAC) are antioxidants that minimize oxidative stress and the downstream negative effects thought to be associated with oxidative stress. Based on magnetic resonance imaging studies, NAC modulates brain glutamate and can thus potentially modulate the E/I balance (Deepmala et al., 2015; Minarini et al., 2017).

In Hardan’s et al. 12-week double-blind, placebo-controlled randomized study of children with ASD (n = 33), 14 subjects in the NAC group and 15 in the placebo group completed the trial. Oral NAC was well tolerated with limited side effects (Hardan et al., 2012). Wink et al. (2016) reported no statistically significant difference between the NAC and placebo groups on the CGI-Improvement (P > 0.69), but glutathione was significantly higher in the NAC group (P < 0.05). NAC treatment was well tolerated but had no significant impact on social impairment in youth with ASD (Wink et al., 2016). Dean et al. (2017) examined a total of 102 children; 98 (79 males and 19 females; age range: 3.1–9.9 years) formed the study group. There were no differences between the NAC and placebo groups on any of the outcome measures for either primary or secondary endpoints. In a double-blind, placebo-controlled add-on study to risperidone (n = 40), 600 mg of NAC twice a day significantly decreased ABC-Irritability but did not change ABC-Social Withdrawal (Nikoo et al., 2015).

The limitations of the reviewed studies include the potential lack of sensitivity of the scales used to assess real improvement and behavioral changes, the absence of biological markers that would allow correlation of any clinical improvement with biological changes, and the heterogeneity of ASD symptoms. Additional randomized double-blind, placebo-controlled trials with the presented E/I modulators are required to draw firm conclusions.

Novel modulators of E-I imbalance are under study, and currently there is a modified form of memantine—nitrosynapsin—that holds promise for this purpose. Nytrosynapsin has been tested in preclinical models of ASD and demonstrated a neuroprotective action that attenuates E/I imbalance and has the potential of modifying the core defects of ASD (Ghatak et al., 2021). In addition, oxytocin might be a promising E/I modulator agent for social impairment as the oxytocinergic system is related to GABA-mediated E/I control within the dynamic interplay of social interaction. The involvement of OXT includes both endogenous and exogenous release in a still poorly known mechanism (Lopatina et al., 2018).

However, the central role of OXT in the social brain has been further demonstrated, and future studies are anticipated to evaluate the novel modifiers of E/I imbalance in ASD.

Transcranial magnetic stimulation (TMS) can be applied as a therapeutic modality in ASD to restore the imbalance of excitation and inhibition, and modulate synaptic plasticity and gamma oscillations (Oberman et al., 2016; Uzunova et al., 2016; Khaleghi et al., 2020; Casanova et al., 2021). A recent meta-analysis showed a moderate though significant effect of TMS on repetitive behaviors and core social dysfunction in ASD (Barahona-Corrêa et al., 2018). It is worth noting that TMS may be preferred in patients who cannot take medications or are not responsive to therapies that may be combined with medications, but further studies are needed.

The lack of preclinical data from animal models in this study is a major drawback and would have been informative, but this was out of scope since we focused on current clinical trials in ASD. Another limitation that needs to be mentioned is that the selection of the studies was based on subjective criteria based on availability of clinical trials in ASD according to our criteria that are overall in low number and need further studies.