Limited Detectability of Network Functional Alterations in a Tauopathy Model Using Mouse Primary Cortical Cultures.

Background: Tauopathies are neurodegenerative disorders characterized by the abnormal hyperphosphorylation and aggregation of the microtubule-associated protein tau, leading to disrupted neuronal connectivity and progressive brain dysfunction. Despite their clinical relevance, most in vitro models have focused primarily on molecular and cellular aspects, with limited emphasis on alterations in network dynamics and functional connectivity. Methods: We developed an in vitro tauopathy model based on mouse primary neuronal cultures, enabling the investigation of network-level alterations under controlled conditions. We compared three experimental groups: untreated control cultures, cultures exposed to extracellular wild-type tau, and cultures treated with pathological tau (pTau) isolated from the sarkosyl-insoluble fraction of P301S (+/-) transgenic mice. To increase susceptibility to tau-induced pathology, all conditions were additionally transduced with adeno-associated viral vectors encoding human P301L tau. To quantify for damage, spontaneous neuronal activity was monitored throughout network maturation-from day in vitro (DIV) 7 to 16-using fluorescence calcium imaging, and multiple metrics describing network dynamics and functional organization were compared at DIV 12. Results: We observed that exposure to pTau did not induce overt cytotoxicity or major disruptions in global network dynamics, although a mild increase in network bursting activity was observed. Longitudinal analysis of network maturation further revealed largely similar developmental trajectories across experimental groups, with only subtle and persistent differences in bursting-related activity in pTau-treated cultures. Conclusions: We propose that the early developmental stage of the cultures, together with the intrinsic bursting and ongoing synaptic plasticity of primary neuronal networks, masks subtle pathological effects. This may limit the sensitivity of two-dimensional in vitro systems to detect network-level dysfunction, suggesting that more mature or structurally complex models, such as brain organoids, may be required to reveal robust functional deficits.