Sleep pressure modulates single-neuron synapse number in zebrafish.

Sleep is a nearly universal behaviour with unclear functions1. The synaptic homeostasis hypothesis proposes that sleep is required to renormalize the increases in synaptic number and strength that occur during wakefulness2. Some studies examining either large neuronal populations3 or small patches of dendrites4 have found evidence consistent with the synaptic homeostasis hypothesis, but whether sleep merely functions as a permissive state or actively promotes synaptic downregulation at the scale of whole neurons is unclear. Here, by repeatedly imaging all excitatory synapses on single neurons across sleep-wake states of zebrafish larvae, we show that synapses are gained during periods of wake (either spontaneous or forced) and lost during sleep in a neuron-subtype-dependent manner. However, synapse loss is greatest during sleep associated with high sleep pressure after prolonged wakefulness, and lowest in the latter half of an undisrupted night. Conversely, sleep induced pharmacologically during periods of low sleep pressure is insufficient to trigger synapse loss unless adenosine levels are boosted while noradrenergic tone is inhibited. We conclude that sleep-dependent synapse loss is regulated by sleep pressure at the level of the single neuron and that not all sleep periods are equally capable of fulfilling the functions of synaptic homeostasis.

Noradrenergic tone is not required for neuronal activity-induced rebound sleep in zebrafish.

Sleep pressure builds during wakefulness, but the mechanisms underlying this homeostatic process are poorly understood. One zebrafish model suggests that sleep pressure increases as a function of global neuronal activity, such as during sleep deprivation or acute exposure to drugs that induce widespread brain activation. Given that the arousal-promoting noradrenergic system is important for maintaining heightened neuronal activity during wakefulness, we hypothesised that genetic and pharmacological reduction of noradrenergic tone during drug-induced neuronal activation would dampen subsequent rebound sleep in zebrafish larvae. During stimulant drug treatment, dampening noradrenergic tone with the α2-adrenoceptor agonist clonidine unexpectedly enhanced subsequent rebound sleep, whereas enhancing noradrenergic signalling with a cocktail of α1- and ß-adrenoceptor agonists did not enhance rebound sleep. Similarly, CRISPR/Cas9-mediated elimination of the dopamine ß-hydroxylase (dbh) gene, which encodes an enzyme required for noradrenalin synthesis, enhanced baseline sleep in larvae but did not prevent additional rebound sleep following acute induction of neuronal activity. Across all drug conditions, c-fos expression immediately after drug exposure correlated strongly with the amount of induced rebound sleep, but was inversely related to the strength of noradrenergic modulatory tone. These results are consistent with a model in which increases in neuronal activity, as reflected by brain-wide levels of c-fos induction, drive a sleep pressure signal that promotes rebound sleep independently of noradrenergic tone.

Sleep Circuits and Physiology in Non-Mammalian Systems.

Research over the last 20 years has firmly established the existence of sleep states across the animal kingdom. Work in non-mammalian animal models such as nematodes, fruit flies, and zebrafish has now uncovered many evolutionarily conserved aspects of sleep physiology and regulation, including shared circuit architecture, homeostatic and circadian control elements, and principles linking sleep physiology to function. Non-mammalian sleep research is now shedding light on fundamental aspects of the genetic and neuronal circuit regulation of sleep, with direct implications for the understanding of how sleep is regulated in mammals.

Mapping oxygen concentration in the awake mouse brain.

Although critical for brain function, the physiological values of cerebral oxygen concentration have remained elusive because high-resolution measurements have only been performed during anesthesia, which affects two major parameters modulating tissue oxygenation: neuronal activity and blood flow. Using measurements of capillary erythrocyte-associated transients, fluctuations of oxygen partial pressure (Po2) associated with individual erythrocytes, to infer Po2 in the nearby neuropil, we report the first non-invasive micron-scale mapping of cerebral Po2 in awake, resting mice. Interstitial Po2 has similar values in the olfactory bulb glomerular layer and the somatosensory cortex, whereas there are large capillary hematocrit and erythrocyte flux differences. Awake tissue Po2 is about half that under isoflurane anesthesia, and within the cortex, vascular and interstitial Po2 values display layer-specific differences which dramatically contrast with those recorded under anesthesia. Our findings emphasize the importance of measuring energy parameters non-invasively in physiological conditions to precisely quantify and model brain metabolism.

Calcium dynamics in astrocyte processes during neurovascular coupling.

Enhanced neuronal activity in the brain triggers a local increase in blood flow, termed functional hyperemia, via several mechanisms, including calcium (Ca(2+)) signaling in astrocytes. However, recent in vivo studies have questioned the role of astrocytes in functional hyperemia because of the slow and sparse dynamics of their somatic Ca(2+) signals and the absence of glutamate metabotropic receptor 5 in adults. Here, we reexamined their role in neurovascular coupling by selectively expressing a genetically encoded Ca(2+) sensor in astrocytes of the olfactory bulb. We show that in anesthetized mice, the physiological activation of olfactory sensory neuron (OSN) terminals reliably triggers Ca(2+) increases in astrocyte processes but not in somata. These Ca(2+) increases systematically precede the onset of functional hyperemia by 1-2 s, reestablishing astrocytes as potential regulators of neurovascular coupling.

The psychostimulant modafinil facilitates water maze performance and augments synaptic potentiation in dentate gyrus.

Modafinil is a psychostimulant drug used widely for the treatment of narcolepsy, which also has additional positive effects on cognition. Here, we investigate the effects of modafinil on behavioural performance and synaptic plasticity in rats. Improved acquisition in the water maze task was observed in animals that underwent chronic treatment with modafinil. We found that the distance traveled and escape latency were reduced after the first day in chronically-treated rats, compared to controls. Importantly, swim velocity was similar for both groups, excluding pharmacological effects on motor skills. We also found that modafinil increases synaptic plasticity in the dentate gyrus of urethane-anaesthetized rats; modafinil induced a robust augmentation of the population spike, evident after application of 2 bursts of 200 Hz high-frequency stimulation. Furthermore, the modafinil-dependent enhancement of postsynaptic potentials correlated selectively with theta rhythm augmentation. We propose that modafinil may facilitate hippocampal-associated spatial representation via increased theta-related hippocampal plasticity.