Wakefulness enhances somatic Ca²⁺ activity of microglia and astrocytes in the auditory cortex via distinct neuronal...

Astrocytes and microglia maintain cortical homeostasis via dynamic intracellular calcium (Ca²⁺) signaling, but their state-dependent activity and neuronal dependencies in the auditory cortex remain poorly understood. Here, using in vivo two-photon imaging, combined with cell-type-specific viral expression of GCaMP sensors, we monitored somatic Ca²⁺ transients in awake and anesthetized mice. Wakefulness significantly enhanced the active cell fraction, frequency, and amplitude of somatic Ca²⁺ events in both glial populations compared to the anesthetized state. Local application of tetrodotoxin (TTX) completely abolished microglial Ca²⁺ activity, revealing an obligatory dependence on neuronal firing. In contrast, astrocytic Ca²⁺ events persisted at similar frequencies but exhibited significantly reduced peak amplitudes, indicating partial autonomy coupled with neuronal modulation. Together, our findings demonstrate that wakefulness coordinately boosts glial Ca²⁺ activity, while microglia and astrocytes rely on fundamentally distinct relationships with local neuronal networks.