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.Three-dimensional (3D) brain models hold great potential for the generation of functional in vitro models to advance studies on human brain development, diseases and possible therapies. The routine exploitation of such models, however, is hindered by the lack of technologies to chronically monitor the activity of neural aggregates in three dimensions. A promising new approach consists in growing bio-artificial 3D brain model systems with seamless tissue-integrated biosensing artificial microdevices. Such devices could provide a platform for in-tissue sensing of diverse biologically relevant parameters. To date there is very little information on how to control the extracellular integration of such microscale devices into neuronal 3D cell aggregates.
In this direction, in the present work I contributed to investigated the growth of hybrid neurospheroids obtained by the aggregation of silicon sham microchips (100x100x50\u3bcm3) with primary cortical cells. Interestingly, by coating microchips with different adhesion-promoting molecules, we reveal that surface functionalization can tune the integration and final 3D location of self-standing microdevices into neurospheroids. Morphological and functional characterization suggests that the presence of an integrated microdevice does not alter spheroid growth, cellular composition, nor network activity and maturation. Finally, we also demonstrate the feasibility of separating cells and microchips from formed hybrid neurospheroids for further single-cell analysis, and quantifications confirm an unaltered ratio of neurons and glia.
These results uncover the potential of surface-engineered self-standing microdevices to grow untethered three-dimensional brain-tissue models with inbuilt bioelectronic sensors at predefined sites
