A multi-electrode array and immunofluorescence workflow to characterize impacts of HHV-6 infection in induced pluripotent stems cells differentiated to mixed neuronal endpoints

Roseoloviruses, including HHV-6A and HHV-6B, are known to be neurotropic and have been linked to various neurological disorders. However, the impact of these infections on neuronal signaling and network activity remains poorly understood, largely due to the challenges in monitoring electrical activity in individual neurons during viral infection.

To address this, researchers have developed a protocol utilizing induced pluripotent stem cell (iPSC)-derived neuronal cultures, employing multi-electrode array (MEA) recordings and immunofluorescence staining to investigate neurophysiological changes during roseolovirus infection.

Two specific culture platforms were established: one using NGN2-induced forebrain neurons and the other utilizing progenitor cell-derived neuron-astrocyte mixed cultures. Cell composition in these cultures was confirmed through immunofluorescence staining with neuronal, glial, and viral markers. Functional activity was evaluated using extracellular recordings from the MEA2100 system, examining parameters such as mean firing rate, inter-spike interval, single-electrode bursting, and network burst activity in both roseolovirus-infected and control (uninfected) states.

Additionally, pharmacological treatments with bicuculline, gabazine, and nicotine were applied to verify the functional responsiveness of defined neuronal neurotransmitter chemotypes.

The findings indicate that HHV-6A infection alters neuronal firing patterns compared to uninfected controls. The outlined methods and workflows offer a systematic approach to study how viral infections affect neuronal excitability, with potential adaptation for comparing the effects of various viruses on nerve cell function, infection trajectories, multiplicity of infection (MOI), and therapeutic interventions.