Trisomy 21 impairs synchronized activity and connectivity in developing human down syndrome cortical excitatory neuron networks

Down syndrome (DS) is the most common genetic cause of intellectual disability, affecting one in 700 live births worldwide, and is caused by trisomy of the human chromosome 21 (Hsa21), yet the cellular and molecular mechanisms driving neuronal network dysfunction remain poorly understood. Here, we investigated whether trisomy 21 (TS21) alters the spontaneous synchronised activity of excitatory neuron networks, potentially contributing to the neurodevelopmental phenotypes of DS. By modelling human cortical development in vitro with TS21 and matched control human iPSCs from two DS donors, we investigated the impact of Hsa21 triplication on neural network activity and connectivity. Calcium imaging revealed an early and pronounced reduction in neuronal activity in TS21 cortical neurons, including a marked loss of synchronised bursting. These deficits persisted up to 80 days in vitro and for over 5 months in vivo following transplantation into the mouse forebrain, as shown by multiphoton calcium imaging through a cranial window. Viral trans-synaptic tracing identified significant reduction of neuronal connectivity in TS21 neuronal networks in vitro, suggesting that reduced network connectivity contributes to the dramatic reduction of synchronised bursting. Furthermore, TS21 neurons displayed significantly reduced expression of voltage-gated potassium channels, with single-neuron recordings confirming a reduction of hyperpolarization-activated currents. Together, these findings demonstrate long-lasting impairments in human cortical excitatory neuron network function associated with Trisomy 21.