Changes in Connexin-43 Expression and Neuropil Ultrastructure in the Cerebral Cortex of Bats During the Sleep–Wake Cycle

Sleep maintains central nervous system homeostasis, but many molecular mechanisms of sleep–wake regulation remain poorly understood. Connexin-43 (Cx43), the astrocytic gap junction protein, supports neuroglial communication, yet its role in physiological sleep is little studied. Here, we analyzed Cx43 expression, gap junction organization and neuropil ultrastructure in the motor cortex of common noctule bats (Nyctalus noctula) during sleep and wakefulness using confocal microscopy, transmission electron microscopy, semi-automated image analysis, and molecular dynamics. Sleep was associated with higher Cx43 immunoreactivity and with more and larger astrocytic gap-junction plaques than wakefulness. Across neuropil fields, synaptic, process and mitochondrial counts and total active-zone length were higher during wakefulness, and mean mitochondrial profiles were larger during sleep; after averaging within animals, only total active-zone length remained significantly greater during wakefulness. More perisynaptic astrocytic process (PAP) profiles contacted synapses during wakefulness, whereas individual PAPs were larger during sleep; both contrasts remained significant after animal-level averaging. As a secondary, hypothesis-generating analysis, we simulated histidine protonation of the Cx43 hemichannel as a simplified model of acidic conditions. Cerebral pH was not measured, and the simulations do not explain the sleep-associated Cx43 increase. In silico, protonation reduced conformational flexibility and narrowed the central and extracellular pore regions.