Calcium ions (Ca2+) are essential for many cellular signaling mechanisms and enter the cytosol mostly through voltage-gated calcium channels. Here, using high-speed Ca2+ imaging up to 20 kHz in the rat layer five pyramidal neuron axon we found that activity-dependent intracellular calcium concentration ([Ca2+]i) in the axonal initial segment was only partially dependent on voltage-gated calcium channels. Instead, [Ca2+]i changes were sensitive to the specific voltage-gated sodium (NaV) channel blocker tetrodotoxin. Consistent with the conjecture that Ca2+ enters through the NaV channel pore, the optically resolved ICa in the axon initial segment overlapped with the activation kinetics of NaV channels and heterologous expression of NaV1.2 in HEK-293 cells revealed a tetrodotoxin-sensitive [Ca2+]i rise. Finally, computational simulations predicted that axonal [Ca2+]i transients reflect a 0.4% Ca2+ conductivity of NaV channels. The findings indicate that Ca2+ permeation through NaV channels provides a submillisecond rapid entry route in NaV-enriched domains of mammalian axons.

Highlights d Drug-induced increases in neuronal activity are followed by rebound sleep d Galn-expressing neurons are active during rebound sleep d galn expression is increased after neuronal activity and sleep deprivation (SD) d Galn neuropeptide is required for rebound sleep after high neuronal activity and SD

When faced with predatorial threats, escaping towards shelter is an adaptive action that offers long-term protection against the attacker. From crustaceans to mammals, animals rely on knowledge of safe locations in the environment to rapidly execute shelter-directed escape actions1–3. While previous work has identified neural mechanisms of instinctive escape4–9, it is not known how the escape circuit incorporates spatial information to execute rapid and accurate flights to safety. Here we show that mouse retrosplenial cortex (RSP) and superior colliculus (SC) form a monosynaptic circuit that continuously encodes the shelter direction. Inactivation of SC-projecting RSP neurons decreases SC shelter-direction tuning while preserving SC motor function. Moreover, specific inactivation of RSP input onto SC neurons disrupts orientation and subsequent escapes to shelter, but not orientation accuracy to a sensory cue. We conclude that the RSC-SC circuit supports an egocentric representation of shelter direction and is necessary for optimal shelter-directed escapes. This cortical-subcortical interface may be a general blueprint for increasing the sophistication and flexibility of instinctive behaviours.

Sleep pressure homeostatically increases during wake and dissipates during sleep, but the molecular signals and neuronal substrates that measure homeostatic sleep pressure remain poorly understood. We present a pharmacological assay in larval zebrafish that generates acute, short-term increases in wakefulness followed by sustained rebound sleep after washout. The intensity of global neuronal activity during drug-induced wakefulness predicted the amount of subsequent rebound sleep. Whole brain mapping with the neuronal activity marker phosphorylated extracellular signal-regulated kinase (pERK) identified preoptic Galanin-expressing neurons as selectively active during rebound sleep, and the relative induction of galanin transcripts was predictive of total rebound sleep time. Galanin is required for sleep homeostasis, as galanin mutants almost completely lacked rebound sleep following both pharmacologically induced neuronal activity and physical sleep deprivation. These results suggest that Galanin plays a key role in responding to sleep pressure signals derived from neuronal activity and functions as an output arm of the vertebrate sleep homeostat.