lthough it has been known since the early years of this century that lesions of the preoptic area could cause insomnia, and that many neurons in this area have sleep-active activity profiles, the mechanism by which these neurons regulate sleep has not been clearly defined. We began by examining the inputs to the tuberomammillary nucleus, a histaminergic cell group in the posterior lateral hypothalamus that has been implicated in arousal. We found that inputs to the core of the nucleus (cell bodies and proximal dendrites) arise almost exclusively from a collection of neurons in the ventrolateral preoptic area (VLPO). We further traced the efferents from the VLPO into the brainstem, where they innervated other monoamine cell groups (dopaminergic ventral tegmental area; serotoninergic dorsal, median, and medullary raphe nuclei; noradrenergic locus coeruleus; and cholinergic pedunculopontine tegmental nucleus). This connectivity suggested that the VLPO neurons may selectively regulate the ascending arousal systems in the brain. We therefore used the expression of Fos as a marker for cellular activation to examine the activity of VLPO neurons across the wake-sleep cycle. We found that many neuronal systems in the brain are active during waking, and almost all become quiescent very soon after the onset of sleep, with two exceptions. First, the circadian circuitry in the suprachiasmatic nucleus and the intergeniculate leaflet expresses Fos during the light cycle, in sleeping rats (which are nocturnal animals). In addition, the VLPO neurons consistently showed activation, with numbers of Fos-positive neurons in the VLPO correlating tightly with the amount of sleep in the previous hour. When animals were sleep deprived, so that they slept during the night, only VLPO showed Fos activation during sleep, indicating that this is not a circadian phenomenon. Combining this Fos staining with retrograde transport from the tuberomammillary nucleus, we demonstrated that up to 70% of the retrogradely labeled neurons in the VLPO are Fos-positive during sleep. Finally, we combined retrograde transport from the tuberomammillary nucleus with immunocytochemistry for different neurotransmitters. We found that about 80% of retrogradely labeled cells were immunoreactive for both galanin and GABA, indicating that this is an inhibitory pathway. Our findings suggest that the VLPO may act during sleep to profoundly inhibit the entire ascending arousal system, and hence may play a causative role in the induction of sleep.
