Lastly, we tested for associations between individual z-scores of subjects with autism for the replicated whole-brain LCOR-neurotransmitter co-localization findings and different autism symptom domains as measured with the Autism Diagnostic Observation Schedule (ADOS)32 (Supplementary Table S16). In ABIDE1, no significant associations were found with the ADOS total score nor the three subscores (communication, social interaction, stereotyped behavior and restricted interests [SBRI]). A significant but weak negative association was observed in ABIDE2 between SBRI and the strength of LCOR-GABAa co-localizations (r(236) = -0.133, p = 0.040, 95% CI [-0.255, -0.006]) and a positive with the strength of LCOR-VAChT co-localizations (r(236) = 0.141, p = 0.030, 95% CI [0.014, 0.264]) (Supplementary Fig. S6). Both findings did not survive a correction for multiple comparisons.

Here, we provide evidence for consistent local functional activity (LCOR) decreases in individuals with autism. These alterations co-localized with in-vivo derived distributions of specific receptors and transporters covering dopaminergic, glutamatergic, GABAergic and cholinergic neurotransmission. The LCOR pattern observed in autism across both cohorts was similar to the effect of NMDA-antagonist ketamine but not to the effect of the GABAergic medication midazolam. Ketamine reduced LCOR in regions exhibiting decreases in autism. At the neurochemical level, the LCOR-neurotransmitter co-localization profile of ketamine was similar to changes observed in autism.

Previous studies investigating functional alterations in autism have often yielded inconsistent findings, potentially due to small sample sizes, the phenotypic heterogeneity of autism, or varying methodological approaches. Our results support the notion of consistent group-level local activity decreases as measured using LCOR in brain regions implicated in self-referential processing, social cognition, cognitive and emotional regulation. These findings align with recent rs-fMRI meta-analyses indicating local hypoactivity within the PCC, precuneus and right temporal gyri and reinforce the role of functional abnormalities of the DMN in the pathophysiology of autism. Moreover, other studies utilizing ABIDE or other datasets have reported comparable alterations in regional homogeneity (ReHo), a metric closely related to LCOR. The central role of the DMN in the pathophysiology of autism has been further emphasized by recent efforts to identify neurobiological subtypes of autism, which have observed converging abnormalities in both the DMN and frontoparietal networks across subtypes. Additionally, task-based fMRI studies have shown reduced activation in DMN regions during tasks requiring self-related versus other-related judgements. Taken together, these findings suggest that decreased functional activity within DMN nodes may be related to the challenges individuals with autism face in self-referential processing and theory of mind. Conversely, we did not observe consistent LCOR increases across both ABIDE datasets.

Most previous studies were restricted to the investigation of macroscale brain networks based on hemodynamic signals of rs-fMRI. To gain a deeper understanding of the neurobiological mechanisms underlying the emergent functional activity alterations in autism, it is crucial to integrate data from molecular neuroimaging. This approach could extend the brain connectivity framework by incorporating a molecular perspective. Adopting this approach, we found LCOR alterations observed across both ABIDE datasets to be spatially related to dopaminergic, glutamatergic, GABAergic and cholinergic neurotransmission. Specifically, all co-localizations were negative with stronger LCOR reductions in autism being associated with increased availability of respective receptors and transporters in health. In line with the observed clinical heterogeneity and recently suggested existence of autism subtypes, these findings support the notion that multiple neurotransmitter systems may be involved in the neurobiological underpinnings of autism.

We observed the strongest similarity of autism patterns with glutamatergic NMDA and mGluR5 receptor distributions, which represents the main excitatory neurotransmitter system. Glutamate acts in a homeostatic relationship with the inhibitory GABA system balancing neuronal excitability. The glutamatergic system plays an important role in brain development and neuroplasticity. A balanced E/I ratio is considered to be crucial for maintaining mental health, with better overall cognitive performance in healthy children associated with a lower E/I ratio, particularly in higher-order association cortices such as the DMN nodes. Conversely, an increased E/I ratio has been frequently implicated in the autism phenotype. For instance, elevated glutamate levels, which can be found in autism in peripheral blood serum and within the central nervous system, have been shown to have neurotoxic effects potentially leading to neuronal cell death and volumetric reductions as shown in structural MRI studies. A previous multimodal imaging study observed a negative association between increased glutamate concentrations in the dorsal ACC and reduced functional connectivity between the dorsal ACC and insular, limbic and parietal regions in male subjects with autism. The dorsal ACC plays an important role in top-down cognitive control and adaptive, flexible behavior with decreased functional connectivity in this region linked to deficient response inhibition and more repetitive behavior in autism. Consistently, higher mGluR5 densities can be observed in male adults with autism.

The crucial role of glutamate dysregulation in the neurobiology of autism has been highlighted by pharmacological studies manipulating the glutamatergic system. Although the results are inconsistent, D-Cycloserine, a NMDA receptor agonist, showed positive effects in treatment of social difficulties and stereotypies in subjects with autism. Conversely, the uncompetitive NMDA receptor antagonist ketamine led to reduced mentalizing performance in a social cognition task associated with increased neural activity in the superior temporal sulcus and anterior precuneus as well as increased psychotic symptoms in healthy adults. In accordance, early postnatal ketamine administration in mice increased stereotyped behavior and social difficulties in later adulthood combined with elevated glutamate and reduced GABA levels in the amygdala and hippocampus. Fragile X syndrome, the most commonly known single-gene cause of autism, is also linked to dysregulation of mGluR5 signaling, and pharmacological mGluR5 antagonists have demonstrated promising effects in preclinical and early clinical studies. However, in clinical trials, intranasal ketamine administration in adolescents and young adults with autism did not lead to significant improvements in core autistic symptoms. Conceptually, one would expect a successful treatment to normalize brain functional patterns associated with a specific clinical condition. Our own findings are therefore in line with the negative outcome of the ketamine trial, showing that ketamine actually induces autism-like brain patterns by reducing LCOR in regions where it is also reduced in autism. Corroborating this finding, the neurochemical profile induced by ketamine significantly correlates with the neurochemical profile found in autism across all tested neurotransmitter systems. Specifically, the effect of ketamine on LCOR co-localized with the distribution of D1, NMDA and GABAa receptors suggesting that ketamine-induced manipulation of the E/I balance may lead to brain activity patterns that resemble those observed in autism. Future pharmacological studies should investigate the effectiveness of drugs manipulating this balance opposing the effects of ketamine in relieving autistic symptoms.

Optogenetic studies demonstrated that experimentally elevating the E/I ratio within the rodent medial prefrontal cortex, an important DMN hub, caused altered information processing and social divergence. Notably, compensatory elevation of inhibitory neurons alleviated social divergence by balancing the E/I ratio. Potentially, an atypical expression of GABAa within the DMN contributes to autism symptom severity. For instance, Oblak et al. (2010) found reduced GABAa receptors and benzodiazepine binding sites in the PCC and fusiform gyrus post mortem in subjects with autism. Additionally, meta-analyses accumulate evidence for a negative association between local GABA concentrations and functional activation of the medial prefrontal cortex and ACC during emotion processing tasks. Within our study, pharmacologically increased inhibition by GABAa-potentiator midazolam in healthy volunteers did not affect LCOR in the DMN regions where LCOR was decreased in autism. Conversely, midazolam significantly elevated LCOR in regions where LCOR was increased in autism, indicating a partial induction of autism-like brain patterns and complementing the findings observed with ketamine. At the neurochemical level, midazolam effects did not co-localize with the neurochemical signature of autism (Fig. 2B, and Supplementary Fig. S3). To date, there is modest evidence of the effectiveness of GABA modulators such as arbaclofen and acamprosate in treating autistic symptoms. Noteworthy, distinct biological alterations, such as elevated glutamate neurotransmission or disrupted GABAergic signaling, can independently contribute to an increased E/I ratio in the brains of individuals with autism. These diverse mechanisms may ultimately converge to be associated with similar autistic phenotypes, complicating the interpretation of the underlying neurobiological pathways driving our observed results.

Several studies support the notion of altered dopamine and serotonin neurotransmission in autism, a hypothesis further reinforced by clinical observations indicating a small positive effect of the atypical antipsychotics aripiprazole and risperidone, as well as selective serotonin reuptake inhibitors, on alleviating stereotypies and aggressive behavior in some autism subgroups. For instance, an overexpression of the dopaminergic system has been implicated in the modulation of stereotyped behavior in autism animal models. The dopamine hypothesis of autism suggests that social difficulties may result from dysfunction in the mesocorticolimbic system, while stereotypies are thought to arise from abnormalities within the nigrostriatal circuitry. We observed that LCOR reductions in autism were negatively co-localized with dopaminergic D1/D2 receptors and the dopamine transporter. These co-localizations did not yield significant correlations with specific symptom domains. Similarly, we did not find consistent in-vivo co-localization between LCOR and serotonergic neurotransmission across the ABIDE cohorts. Elevated blood serotonin levels have been reported in only a subset of autistic children (~25%), and much of the supporting evidence comes from animal models. For both of these analyses, our restriction to whole-brain analyses and disregard of potential subtypes may have limited the ability to detect some regionally and subtype specific co-localization patterns.

In that regard it is also important to note that the included multi-site datasets are characterized by considerable heterogeneity with respect to the availability of demographic, medication status and clinical information (e.g., seizure history) as well as in terms of image quality. This variability may obscure more nuanced insights into the neurobiological mechanisms underlying autism. Moreover, the cross-sectional and group-averaging nature of our study design precludes any causal interpretation of our findings. Future research should aim to conduct large-scale, standardized, and longitudinal investigations to enable the identification of distinct autism subtypes and their associated neurobiological profiles.

Finally, it should be emphasized that the autistic brain may exhibit altered responses to pharmacological agents due to differences in neurotransmitter system responsivity. This potential variability limits the generalizability of our pharmacological dataset, which was further constrained by the inclusion of only male participants and the impossibility to systematically account for the potential influence of prescribed medication on our findings. Furthermore, the generalizability of our findings is limited by the exclusion of autistic individuals with intellectual disability.

We find LCOR alterations in subjects with autism to negatively co-localize with the in-vivo derived distribution of dopaminergic, glutamatergic, GABAergic and cholinergic neurotransmitter systems in two large independent cohorts. This pattern of LCOR alterations in autism was similar to the effect induced by the NMDA-antagonist ketamine supporting in-vivo evidence for disturbed E/I ratio. These findings advance understanding of the pathophysiology of autism-related functional alterations and may guide new hypotheses for pharmacological interventions. Future neuro-subtyping studies with balanced gender representation aimed at disentangling the heterogeneity within autism will be crucial for identifying biologically and clinically distinct subgroups that allow for precision psychiatry approaches in autism.