2 research outputs found
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.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
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Astrocytes actively support long-range molecular clock synchronization of segregated neuronal populations.
Acknowledgements: The authors would like to thank Marina Nanni and IIT clean-room staff for their technical support in primary cultures preparations and microfabrication, respectively, and the IIT-Neurofacility technical staff for their excellent support. We also thank Alberto Perna for his helping with the Supporting Video, and the Animal Facility staff of IIT central research labs in Genoa for their assistance in animal experiments. This work was supported by intramural funds of Fondazione Istituto Italiano di Tecnologia (IIT) to D.D.P.T. and L.B.; O.B.M. was partly supported by the European Research Executive Agency (REA) through the FP7-PEOPLE-2014-IEF ‘ASTROCLOCK’ (629867); Fondazione CARIPLO research grant (2015-0590) and “Ramon y Cajal” contract (RYC2018-026293-I).In mammals, the suprachiasmatic nucleus of the hypothalamus is the master circadian pacemaker that synchronizes the clocks in the central nervous system 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. In this work, we investigated the implication of two possible pathways: (i) astrocytes-mediated synchronization and (ii) neuronal paracrine factors-mediated synchronization. By taking advantage of a lab-on-a-chip microfluidic device developed in our laboratory, here we report that both pathways are involved. We found the paracrine factors-mediated synchronization of molecular clocks is diffusion-limited and, in our device, effective only in case of a short distance between neuronal populations. Interestingly, interconnecting astrocytes define an active signaling channel that can synchronize molecular clocks of neuronal populations also at longer distances. At mechanism level, we found that astrocytes-mediated synchronization involves both GABA and glutamate, while neuronal paracrine factors-mediated synchronization occurs through GABA signaling. These findings identify a previously unknown role of astrocytes as active cells that might distribute long-range signals to synchronize the brain clocks, thus further strengthening the importance of reciprocal interactions between glial and neuronal cells in the context of circadian circuitry