We propose a multi-fiber solution that can simultaneously record activities of astrocyte-neuronal networks across multiple regions during behaviors. Approach: We employed cell-specific dual-color genetically encoded calcium indicators (GECIs) and multi-fiber photometry to simultaneously measure astrocytic and neuronal Ca2+ transients across multiple brain regions in freely behaving animals.

Multi-fiber photometry, combined with cell-specific dual-color GECIs, represents a powerful approach for investigating astrocytic and neuronal activities across different brain regions during behaviors. This technique serves as a versatile tool for analyzing the multi-regional functional connectivity map of astrocyte-neuronal networks associated with specific behaviors.

Our findings demonstrate that both movements and sensory stimuli induce synchronized and highly correlated Ca2+ transients in astrocytes and neurons of freely behaving mice. In addition, we recorded astrocytic and neuronal Ca2+ transients from multiple brain regions during mouse behaviors. Our observations reveal heightened synchronization of astrocytic and neuronal Ca2+ transients across different brain regions during movements or sensory stimuli, indicating enhanced functional connectivity within brain-wide astrocyte-neuronal networks. Conclusions: Multi-fiber photometry, combined with cell-specific dual-color GECIs, represents a powerful approach for investigating astrocytic and neuronal activities across different brain regions during behaviors. This technique serves as a versatile tool for analyzing the multi-regional functional connectivity map of astrocyte-neuronal networks associated with specific behaviors.

Diverse behaviors rely on coordinated activity and multi-regional functional connectivity within astrocyte-neuronal networks. However, current techniques for simultaneously measuring astrocytic and neuronal activities across multiple brain regions during behaviors remain limited. Aim: We propose a multi-fiber solution that can simultaneously record activities of astrocyte-neuronal networks across multiple regions during behaviors. Approach: We employed cell-specific dual-color genetically encoded calcium indicators (GECIs) and multi-fiber photometry to simultaneously measure astrocytic and neuronal Ca2+ transients across multiple brain regions in freely behaving animals. Results: Our findings demonstrate that both movements and sensory stimuli induce synchronized and highly correlated Ca2+ transients in astrocytes and neurons of freely behaving mice. In addition, we recorded astrocytic and neuronal Ca2+ transients from multiple brain regions during mouse behaviors. Our observations reveal heightened synchronization of astrocytic and neuronal Ca2+ transients across different brain regions during movements or sensory stimuli, indicating enhanced functional connectivity within brain-wide astrocyte-neuronal networks. Conclusions: Multi-fiber photometry, combined with cell-specific dual-color GECIs, represents a powerful approach for investigating astrocytic and neuronal activities across different brain regions during behaviors. This technique serves as a versatile tool for analyzing the multi-regional functional connectivity map of astrocyte-neuronal networks associated with specific behaviors.