ObjectiveTo explore the distribution and contact pattern of sensory relay nuclei in the brainstem that projecting to the dorsal raphe nucleus (DRN) by using neural circuit tracing technique

so as to investigate the morphological basis of sensory information regulating wakefulness/sleep. MethodsRetrograde transporting tracing fluorescent particles RetroBeads were injected into the DRN of adult male C57BL/6 mice (n=3)

and the expression of RetroBeads was observed in the sensory relay nuclei of the brainstem. Furthermore

AAV-CaMKIIα-EGFP and RetroBeads were injected into the parabrachial nucleus (PBN) of another 3 mice to observe the distribution of the fiber terminals in DRN and the sensory inputs from the brainstem. ResultsBy the injection of RetroBeads into the DRN

we identified the sensory relay nuclei projecting to the DRN. These nuclei included the superior olivary nucleus

rostral solitary tract nucleus

caudal solitary tract nucleus

parvicellular part of medial vestibular nucleus and magnocellular part of medial vestibular nucleus

accounting for 3.11%

2.95%

10.40%

10.45% and 1.75% of the total brainstem inputs of the DRN

respectively. In addition

there were a large number of RetroBeads labeled neurons in the PBN

accounting for 71.33% of the total input of DRN. Meanwhile

AAV-CaMKIIα-EGFP mediated anterograde tracing showed that the PBN neurons sent dense fiber projections to the DRN. PBN received the inputs form the trigeminal sensory nucleus and spinal tract nucleus

which relay pain

temperature and touch sensation of the head and face. They accounted for 6.14% and 49.86% of the total brainstem input of PBN. The rostral solitary tract nucleus

caudal solitary tract nucleus and parvicellular part of medial vestibular nucleus also projected to the PBN

accounting for 11.67%

20.53% and 8.94% of the total brainstem input of PBN

respectively. In addition

a few labeled neurons were found in the inferior olivary nucleus

accounting for 2.14% of the total brainstem input of PBN. ConclusionThe DRN receives the direct inputs from the superior olivary nucleus

solitary tract nucleus

vestibular nucleus group and PBN. At the same time

the DRN also indirectly receives the inputs from the solitary tract nucleus

inferior olivary nucleus

vestibular nucleus group

trigeminal sensory nucleus and trigeminal spinal nucleus through the PBN.