EXPLORING HOW CHRONIC OPTOGENETIC INHIBITION INFLUENCES IN VITRO NEURONAL NETWORK MATURATION

In-vivo, neuronal networks develop under the constant influence of afferent input, with sensory-evoked activity sculpting connectivity and driving maturation of neuronal circuits. By contrast, in-vitro models such as dissociated cultures and organoids lack structured input, leading to immature dynamics dominated by global bursting. Chronic stimulation has been shown to accelerate maturation and promote physiological oscillations, largely relying on electrical stimulation, which lacks cell-type specificity and requires close neuron-electrode coupling.
We expressed the inhibitory opsin Jaws in excitatory neurons in postnatal rat cortical cultures and recorded activity using 120-channel multielectrode arrays, characterizing its network-level effects and exploring chronic optogenetic stimulation to guide network maturation.
Jaws activation in excitatory neurons is more complex than simple inhibition: it elicits a post-illumination “rebound” network burst, whose presence and properties depend on stimulation intensity and the previous states of the network. Additionally, from DIV15-20, cultures were daily opto-stimulated. Chronic stimulation resulted in minor reduction in the proportion of spikes within bursts and pronounced increase in inter-burst interval, suggesting altered firing dynamics and decreased synchronous network excitability. Importantly, inter-spike interval distributions revealed an emergent rhythmic component matching the stimulation period, indicating entrainment of intrinsic dynamics.
These findings show that long-term optical stimulation can reshape neuronal network activity, providing a powerful and non-invasive strategy to drive maturation bypassing limitations of electrical stimulation. By combining excitatory and inhibitory optogenetic actuators with adaptive stimulation patterns, this approach can lead to more physiologically relevant developmental trajectories, enhancing the translational value of in-vitro models for disease modeling and neuromodulation research.