Developmental effect of RASopathy mutations on neuronal network activity on a chip

RASopathies are a group of genetic disorders caused by mutations in genes encoding components and regulators of the RAS/MAPK signaling pathway, resulting in overactivation of signaling. RASopathy patients exhibit distinctive facial features, cardiopathies, growth and skeletal abnormalities, and varying degrees of developmental delay, neurocognitive impairment, intellectual disabilities, and attention deficits. At present, it is unclear how RASopathy mutations cause neurocognitive impairment and what their neuron-specific cellular and network phenotypes are. Such knowledge would be crucial for the development of specific therapies, which are still lacking for the treatment of the neurocognitive and behavioral symptoms in RASopathies. Here, we investigated the effect of RASopathy mutations on the establishment and functional maturation of neuronal networks. We isolated cortical neurons from RASopathy mouse models and cultured them for several weeks in multiwell plates with implanted multielectrode arrays. The arrays were used for longitudinal recordings of spontaneous activity in developing networks as well as for recordings of evoked responses in mature neurons. To facilitate the analysis of large and complex data sets resulting from long-term multielectrode recordings, we developed MATLAB-based tools for data processing, analysis and statistical evaluation. Longitudinal analysis of spontaneous network activity revealed a convergent developmental phenotype in neurons carrying the gain-of-function RASopathy mutations Ptpn11 D61Y and Kras V14l. The phenotype was more pronounced at early time points and faded out over time, suggesting compensatory effects during network maturation. Nevertheless, persistent differences in excitatory/inhibitory balance and network excitability were observed in mature networks. This study improves the understanding of the complex relationship between genetic mutations and clinical manifestations in RASopathies by adding insights into functional network processes as an additional piece of the puzzle. ### Competing Interest Statement The authors have declared no competing interest. * AxIS : Axion Integrated Studio bic : bicuculline CFC : cardio-facio-cutaneous syndrome CS : costello syndrome DIV : days in vitro eact : currently active number of active electrodes emax : maximum number of active electrodes E/I : excitation and inhibition FCS : fetal calf serum GUI : graphical user interphase ICC : Immunocytochemistry IFI : immunofluorescence intensity JMML : juvenile myelomonocytic leukemia MAPK : mitogen-activated protein kinase MBR : mean bursting rate MEA : multielectrode array MFR : mean firing rate mwMEA : multiwell MEA NB : network burst NBA : NeurobasalTM -A media NS : Noonan syndrome PC : principle component PCA : Principal component analysis PCR : polymerase chain reaction ROI : region of interest RT : room temperature SA : spontaneous activity SD : standard deviation STIM : test stimulation STTC : spike time tiling coefficient TET : tetanus stimulation VGAT : vesicular GABA transporter VGLUT1 : vesicular glutamate transporter wMBR : weighted mean bursting rate wtMFR : mean firing rate weighted to total number of electrodes