Advanced microstructured platforms for neuroscience: from lab-on-chips for circadian clock studies to next generation bionic 3D brain tissue models

In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is considered the master circadian pacemaker which coordinates circadian rhythms in the central nervous system (CNS) and across the entire body. The SCN receives light input from the eyes through the retinohypothalamic tract and then it synchronizes other clocks in the CNS and periphery, thus orchestrating rhythms throughout the body. However, little is known about how so many cellular clocks within and across brain circuits can be effectively synchronized to entrain the coordinated expression of clock genes in cells distributed all over the brain. In this work I investigated the possible implication of two possible pathways: i) paracrine factors-mediated synchronization and ii) astrocytes-mediated synchronization. To study these pathways, I adopted an in vitro research model that I developed based on a lab-on-a-chip microfluidic device designed and realized in our laboratory. This device allows growing and compartmentalizing distinct neural populations connected through a network of astrocytes or through a cell-free channel in which the diffusion of paracrine factors is allowed. By taking advantage of this device, upon its validation, I synchronized neural clocks in one compartment and analyzed, in different experimental conditions, the induced expression of clock genes in a distant neural network grown in the second compartment. Results show that both pathways can be involved, but might have different roles. Neurons release factors that can diffuse to synchronize a neuronal population. The same factors can also synchronize astrocytes that, in turn, can transmit astrocyte-mediated molecular clocks to more distant neuronal populations. This is supported by experimental data obtained using microfluidic devices featuring different channel lengths. I found that paracrine factors-mediated synchronization occurs only in the case of a short distance between neuronal populations. On the contrary, interconnecting astrocytes define an active channel that can transfer molecular clocks to neural populations also at long distances. The study of possibly involved signaling factors indicate that paracrine factors-mediated synchronization occurs through GABA signaling, while astrocytes-mediated synchronization involves both GABA and glutamate. These findings strength the importance of the synergic regulation of clock genes among neurons and astrocytes, and identify a previously unknown role of astrocytes as active cells in distributing signals to regulate the expression of clock genes in the brain. Preliminary results also show a correlation between astrocyte reactivity and local alterations in neuronal synchronization, thus opening a new scenario for future studies in which disease-induced astrocyte reactivity might be linked to alterations in clock gene expression.