Study Objectives The regulation of sleep-wake cycles is crucial for the brain’s health and cognitive skills. Among the various substances known to control behavioral states, intraventricular injection of neuropeptide S (NPS) has already been shown to promote wakefulness. However, the NPS signaling pathway remains elusive. In this study, we characterized the effects of NPS in the ventrolateral preoptic nucleus (VLPO) of the hypothalamus, one of the major brain structures regulating non-rapid eye movement (NREM) sleep. Methods We combined polysomnographic recordings, vascular reactivity, and patch-clamp recordings in mice VLPO to determine the NPS mode of action. Results We demonstrated that a local infusion of NPS bilaterally into the anterior hypothalamus (which includes the VLPO) significantly increases awakening and specifically decreases NREM sleep. Furthermore, we established that NPS application on acute brain slices induces strong and reversible tetrodotoxin (TTX)-sensitive constriction of blood vessels in the VLPO. This effect strongly suggests that the local neuronal network is downregulated in the presence of NPS. At the cellular level, we revealed by electrophysiological recordings and in situ hybridization that NPSR mRNAs are only expressed by non-Gal local GABAergic neurons, which are depolarized by the application of NPS. Simultaneously, we showed that NPS hyperpolarizes sleep-promoting neurons, which is associated with an increased frequency in their spontaneous IPSC inputs. Conclusion Altogether, our data reveal that NPS controls local neuronal activity in the VLPO. Following the depolarization of local GABAergic neurons, NPS indirectly provokes feed-forward inhibition onto sleep-promoting neurons, which translates into a decrease in NREM sleep to favor arousal.

Astrocytes play important roles in brain function via dynamic structural and functional interactions with neurons. Yet the underlying mechanisms remain poorly defined. A typical feature of astrocytes is the high expression of connexins, which mediate their extensive intercellular communication and regulate their structural properties. In particular, connexin 30 (Cx30), one of the two connexins abundantly expressed by astrocytes, was recently shown to be a critical regulator of excitatory synaptic transmission by controlling the astroglial coverage of synapses. However, the role of Cx30 in the regulation of inhibitory synaptic transmission and excitatory/inhibitory balance remains elusive. Here, we investigated the role of astroglial Cx30 on the electrophysiological and morphological properties of five classes of hippocampal CA1 stratum oriens and pyramidale neurons, defined by the unsupervised Ward's clustering. Using Cx30 knockout mice, we found that Cx30 alters specific properties of some subsets of CA1 interneurons, such as resting membrane potential and sag ratio, while other parameters, such as action potential threshold and saturation frequency, were more frequently altered among the different classes of neurons. The excitation‐inhibition balance was also differentially and selectively modulated among the different neuron subtypes. Only slight morphological differences were observed on reconstructed neurons. Altogether, these data indicate that Cx30 differentially alters the electrophysiological and morphological properties of hippocampal cell populations, and modulates both their excitatory and inhibitory inputs. Astrocytes, via Cx30, are thus active modulators of both excitatory and inhibitory synapses in the hippocampus.