Identification of sleep-promoting neurons in vitro

The neurons responsible for the onset of sleep are thought to be located in the preoptic area1,2,3 and more specifically, in the ventrolateral preoptic nucleus (VLPO)4,5,6. Here we identify sleep-promoting neurons in vitro and show that they represent an homogeneous population of cells that must be inhibited by systems of arousal during the waking state. We find that two-thirds of the VLPO neurons are multipolar triangular cells that show a low-threshold spike. This proportion matches that of cells active during sleep in the same region6. We then show, using single-cell reverse transcriptase followed by polymerase chain reaction, that these neurons probably contain γ-aminobutyric acid (GABA). We also show that these neurons are inhibited by noradrenaline and acetylcholine, both of which are transmitters of wakefulness3,7,8. As most of these cells are also inhibited by serotonin but unaffected by histamine, their overall inhibition by transmitters of wakefulness is in agreement with their relative inactivity during waking with respect to sleep6. We propose that the reciprocal inhibitory interaction of such VLPO neurons with the noradrenergic, serotoninergic and cholinergic waking systems to which they project5,9,10 is a key factor for promoting sleep.