Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired social interaction, communication difficulties, and restricted/repetitive behaviors. A substantial genetic component contributes to ASD, with chromosomal 16p11.2 deletions being a significant risk factor. Mouse models with this deletion exhibit behavioral abnormalities resembling ASD characteristics. Recent research suggests that impaired neural network integration contributes to ASD, manifesting as reduced gamma oscillations during sensory processing. Previous studies demonstrated that rhythmic 40 Hz light flicker stimulation effectively improves cognitive deficits in animal models of Alzheimer's disease and stroke by entraining regional brain oscillations. This study hypothesized that 40 Hz light flicker could similarly ameliorate behavioral deficits in the 16p11.2 deletion mouse model of ASD by modulating PFC oscillations. Given sex-specific differences in ASD prevalence and neurological behaviors in 16p11.2 deletion mice, this study focused exclusively on female mice. The prefrontal cortex (PFC) plays a crucial role in social behavior, and adenosine, a neuromodulatory molecule, is implicated in PFC-dependent behaviors. Adenosine's four receptor subtypes (A₁R, A₂AR, A₂BR, A₃R) exhibit varying effects on neurotransmission, and its role in ASD is of considerable interest, making it a potential mechanistic link in the light stimulation's effects. This study aimed to investigate whether 40 Hz light flicker improves social novelty deficits in female 16p11.2 deletion mice and to elucidate the underlying mechanisms, focusing on the role of adenosine.

The literature review extensively covered the prevalence and genetic basis of ASD, focusing on the 16p11.2 deletion as a strong genetic risk factor. It highlighted studies demonstrating impaired neural network integration and gamma oscillation dysfunction in ASD, specifically referencing findings in Neuroligin 3 R451C knock-in mice. The efficacy of 40 Hz stimulation in ameliorating cognitive deficits in various neurological disorders (Alzheimer's, stroke, Parkinson's disease) was also reviewed. Existing research on the 16p11.2 deletion mouse model, emphasizing sex-specific differences in behavioral phenotypes, was discussed, justifying the study's focus on female mice. The role of adenosine and its receptor subtypes in regulating neurotransmission and their potential involvement in ASD were discussed extensively, emphasizing the need for further investigation into this neuromodulatory system.

The study used female 16p11.2 deletion mice and their wild-type (WT) littermates. Behavioral tests, including open field, novel object recognition, and three-chamber tests, assessed locomotion, anxiety, sociability, and social novelty preference. Electrophysiological techniques (patch-clamp recordings) measured spontaneous excitatory postsynaptic currents (sEPSCs) and inhibitory postsynaptic currents (sIPSCs) to assess neurotransmission. Local field potentials (LFPs) were recorded from the PFC to evaluate neural network activity. Immunofluorescence staining quantified excitatory synapse density (VGLUT1) and microglial morphology (Iba1). Golgi staining assessed dendritic spine density. Fiber photometry measured adenosine release in the PFC during 40 Hz light flicker stimulation. Microdialysis and HPLC determined adenosine concentrations in PFC dialysates. Pharmacological manipulations (A₁R antagonist DPCPX, A₂AR antagonist SCH58261, adenosine injection) and genetic manipulations (A₁R knockdown using shRNA) were used to investigate the mechanistic role of adenosine. Stereotaxic surgery was performed for viral injections (AAV). Statistical analyses (t-tests, ANOVAs, etc.) compared groups and assessed significance. The ARRIVE guidelines were followed for animal experimentation.

Female 16p11.2 deletion mice showed impaired social novelty preference but normal locomotion, anxiety, and cognitive memory compared to WT littermates. 14 days of 40 Hz light flicker treatment significantly ameliorated the social novelty deficit without affecting other behaviors. 40 Hz light stimulation normalized the elevated LFP power in the PFC of 16p11.2 deletion mice. The treatment reduced the frequency of sEPSCs, indicating reduced excitatory neurotransmission, without altering the amplitude of sEPSCs, the firing rate of PFC pyramidal neurons, or the number of neurons. 40 Hz light stimulation reduced the density of excitatory synapses, as evidenced by VGLUT1 immunostaining and Golgi staining. The 40 Hz light flicker increased adenosine release in the PFC, which was confirmed by fiber photometry and microdialysis. In vitro experiments demonstrated that adenosine suppressed sEPSC frequency via A₁R activation. Blocking A₁R with DPCPX or knocking down A₁R expression abolished the beneficial effects of 40 Hz light stimulation on social novelty preference and synaptic transmission. In contrast, blocking A₂AR did not produce these effects. Intraperitoneal injection of adenosine also alleviated the social novelty deficit. Microglia did not appear to be involved in mediating the therapeutic effects of the light stimulation.

The study's findings demonstrate that 40 Hz light flicker is an effective non-invasive intervention for ameliorating social novelty deficits in a female mouse model of 16p11.2 deletion syndrome. The results highlight the crucial role of adenosine and its A₁ receptor in mediating the therapeutic effects of the light stimulation. The reduction in excessive excitatory neurotransmission, without affecting neuronal excitability or density, suggests a specific and targeted mechanism of action. The lack of microglial involvement suggests that the observed effects primarily occur through modulation of synaptic function rather than synaptic pruning. The sex-specific nature of the findings underscores the importance of considering sex as a variable in ASD research and therapeutic development. The identification of adenosine as a key mediator opens up avenues for developing novel ASD treatments targeting adenosine signaling pathways.

This study provides compelling evidence for the therapeutic potential of non-invasive 40 Hz light flicker stimulation in treating ASD, identifying adenosine and its A₁ receptor as critical mediators of its effects. The results suggest that enhancing adenosine signaling could be a novel therapeutic strategy for alleviating ASD symptoms. Future research should investigate the precise molecular mechanisms of adenosine signaling and the long-term effects of 40 Hz light stimulation, exploring its efficacy in other mouse models of ASD and ultimately its potential translation to human clinical trials. The sex-specific aspects require further studies to unravel the diverse spectrum of ASD mechanisms in different sexes.

This study's findings may not be generalizable to all forms of ASD, as the 16p11.2 deletion accounts for a relatively small percentage of ASD cases. Further research is needed to examine the effects of 40 Hz light stimulation in other mouse models of ASD. The study focused solely on female mice, limiting the generalizability of the findings to male mice and individuals with ASD. The relatively short duration of the light stimulation treatment requires further investigation into the long-term effects and the potential for a maintenance regimen.