Despite many studies on dopamine changes in autism, specific alterations in midbrain dopamine neurons projecting to the striatum and cortex remain unclear. Mouse models with diverse SH3 domain and ankyrin repeat containing protein 3 (Shank3) deficiencies are used for investigating autistic symptoms and underlying neurobiological mechanisms. SHANK3 belongs to postsynaptic proteins crucial for synapse formation during development, and disruptions in SHANK3 structure could lead to impaired neurite outgrowth and altered dendritic arborization and morphology. Therefore, we aimed to investigate whether Shank3 deficiency (Shank3B) leads to changes in the morphology of primary neuronal cell cultures from dopaminergic brain regions of neonatal mouse pups and whether it results in alterations in synaptic proteins in dopaminergic nerve pathway projection areas (striatum, frontal cortex). Significantly reduced neurite outgrowth was observed in primary dopaminergic neurons from the midbrain and striatum of Shank3-deficient compared to WT mice. A decrease in Synapsin I immunofluorescence signal in the cortical neurons isolated from Shank3-deficient mice was found, although neurite arborization changes were less severe. Importantly, the deficit in the length of the longest neurite was confirmed in primary cortical neurons isolated from Shank3-deficient mice. No changes in the gene expression of synaptic proteins were observed in the striatum and frontal cortex of Shank3-deficient mice, but an altered gene expression profile of dopaminergic receptors was found. These results show structural changes of dopaminergic neurons, which may explain autistic symptomatology in the used model and provide a basis for understanding the long-term development of autistic symptoms.

Changes in hippocampal neurons are known to play a critical role in social memory deficits associated with autism spectrum disorders (ASD). Although the theory of excitatory-inhibitory imbalance in autism pathogenesis is well established, early developmental alterations in the hippocampus remain insufficiently characterized. Alterations in gamma-aminobutyric acid (GABA)ergic neurons and their markers are thought to underlie synaptic changes in inhibitory circuits. Therefore, this study was designed to: (1) quantify glutamatergic and GABAergic neuron populations in the hippocampus; (2) characterize inhibitory postsynaptic currents (IPSCs) in primary hippocampal neurons; and (3) assess gene expression of selected GABAergic markers in two autism-like animal models, namely Shank3 -deficient mice and prenatally valproate (VPA)-exposed rats. A reduced proportion of GABAergic neurons was observed in the hippocampus of both models. An increase in the number of glutamatergic neurons was found only in the hippocampus of prenatally VPA-exposed rats. This was accompanied by a decrease in IPSC frequency in primary hippocampal neurons from prenatally VPA-exposed rats, while no significant changes were found in Shank3 -deficient mice. Altered temporal dynamics of inhibitory synaptic transmission were demonstrated in both models by a decreased cumulative probability of inter-event intervals for inhibitory currents. Furthermore, reduced gene expression levels of Gabarap and Gabarapl1 were detected in Shank3 -deficient mice, whereas decreased Gat1 expression level was found in prenatally VPA-exposed rats at postnatal day 5. These findings strongly support the excitatory-inhibitory imbalance hypothesis in ASD. Thus, genetic or environmentally induced GABAergic changes in the hippocampus may underlie hippocampus-dependent social memory alterations in ASD.

Abnormalities in gamma-aminobutyric acid (GABA)ergic neurotransmission play a role in the pathogenesis of autism, although the mechanisms responsible for alterations in specific brain regions remain unclear. Deficits in social motivation and interactions are core symptoms of autism, likely due to defects in dopaminergic neural pathways. Therefore, investigating the morphology and functional roles of GABAergic neurons within dopaminergic projection areas could elucidate the underlying etiology of autism. The aim of this study was to (1) compare the morphology and arborization of glutamate decarboxylase (GAD)-positive neurons from the midbrain tegmentum; (2) evaluate synaptic activity in primary neurons from the striatum; and (3) assess GABAergic postsynaptic puncta in the ventral striatum of wild-type (WT) and Shank3-deficient mice. We found a significant decrease in the number of short neurites in GAD positive primary neurons from the midbrain tegmentum in Shank3-deficient mice. The application of a specific blocker of GABA_A receptors (GABA_AR) revealed significantly increased frequency of spontaneous postsynaptic currents (sPSCs) in Shank3-deficient striatal neurons compared to their WT counterparts. The mean absolute amplitude of the events was significantly higher in striatal neurons from Shank3-deficient compared to WT mice. We also observed a significant reduction in gephyrin/GABA_AR γ2 colocalization in the striatum of adult male Shank3-deficient mice. The gene expression of collybistin was significantly lower in the nucleus accumbens while gephyrin and GABA_AR γ2 were lower in the ventral tegmental area (VTA) in male Shank3-deficient compared to WT mice. In conclusion, Shank3 deficiency leads to alterations in GABAergic neurons and impaired GABAergic function in dopaminergic brain areas. These changes may underlie autistic symptoms, and potential interventions modulating GABAergic activity in dopaminergic pathways may represent new treatment modality.

Alterations in dopaminergic pathways are implicated in the pathogenesis of neuropsychiatric disorders, including autism spectrum disorder (ASD). However, changes in morphology and neurite outgrowth within the dopaminergic system under autism-relevant conditions remain poorly understood. Prenatal valproate (VPA) administration is a well-established animal model for studying autism-related brain changes in offspring. Therefore, the aim of this study was to examine the effects of prenatal VPA exposure on (1) the morphology of primary neurons isolated from dopaminergic brain regions, and (2) the expression of molecular motors and neurite outgrowth-related proteins, along with (3) the levels of dopamine and dopamine receptors in the midbrain and striatum, analysed at postnatal day 30 (P30). Neurons from the ventral tegmental area (VTA) of rats prenatally exposed to VPA showed a significant reduction in both the number and length of neurites. Similar, but to a lesser extent, changes were also found in primary striatal tyrosine hydroxylase (TH)-positive neurons, compared to controls. A significant increase in total dopamine levels was observed in the midbrain of rats prenatally exposed to VPA, regardless of sex, while no significant changes were detected in the striatum at P30. TH levels were unchanged in dopaminergic regions at P30, while dopamine receptor type 4 gene expression was significantly increased in the VTA of prenatally VPA-exposed male rats, with no other significant gene expression changes. These results demonstrate that prenatal VPA exposure induces region-specific neuronal and dopaminergic changes in the midbrain and striatum, enhancing our understanding of the neurodevelopmental mechanisms potentially underlying ASD-like symptomatology.