Neuropeptide S (NPS) neurons: Parabrachial identity and novel distributions

Abstract

Neuropeptide S (NPS) increases wakefulness. A small number of neurons in the brainstem express Nps. These neurons are located in or near the parabrachial nucleus (PB), but we know very little about their ontogeny, connectivity, and function. To identify Nps-expressing neurons within the molecular framework of the PB region, we used in situ hybridization, immunofluorescence, and Cre-reporter labeling in mice.The primary concentration of Nps-expressing neurons borders the lateral lemniscus at far-rostral levels of the lateral PB. Caudal to this main cluster, Nps-expressing neurons scatter through the PB and form a secondary concentration medial to the locus coeruleus (LC). Most Nps-expressing neurons in the PB region are Atoh1-derived, Foxp2-expressing, and mutually exclusive with neurons expressing Calca or Lmx1b.Among Foxp2-expressing PB neurons, those expressing Nps are distinct from intermingled subsets expressing Cck or Pdyn. Examining Nps Cre-reporter expression throughout the brain identified novel populations of neurons in the nucleus incertus, anterior hypothalamus, and lateral habenula. This information will help focus experimental questions about the connectivity and function of NPS neurons. INTRODUCTIONWake-promoting stimulants like caffeine and amphetamines are inherently anxiogenic. Despite the benefits of enhanced wakefulness and attention, their anxiogenic propensity limits therapeutic potential in patients with inattention or hypersomnolence. Conversely, sedative effects of most anxiolytic drugs limit their use in patients with insomnia or anxiety. These complementary limitations frustrate the treatment of many patients with cognitive, affective, and sleep disorders. A nonanxiogenic stimulant medication or nonsedative anxiolytic medication would be enormously beneficial. For these reasons, the discovery of neuropeptide S (NPS) was an exciting development. NPS was identified Dake Huang and Richie Zhang contributed equally.

Molecular and anatomical characterization of parabrachial neurons and their axonal projections

“…Fn1 and Pappa were originally chosen to represent N4 , but the transcripts were either exclusively in vasculature ( Fn1 ) or too weak and dispersed to see obvious positive cells ( Pappa ). Our HiPlex experiment (Bregma 5.0–5.4 mm) did not include rostral PBN-associated cells that are labeled as PPN and NLL by the AMBA ( Huang et al, 2022 ). Thus, we examined expression of Ntsr1 and Cck as representatives of N4 by RNAScope in rostral sections (Bregma -4.8 mm).…”

“…N9 was also predicted to express Gal and Nps . Because Nps is in the far rostral region of PBN ( Huang et al, 2022 ), we used RNAscope to show Nps -positive cells that co-express Gal reside ventral to the N4 population of Ntsr1/Cck neurons along the border PPN and NLL shown in AMBA. There is also a small population of Th -positive neurons medial to the Nps/Gal cells ( Figure 5—figure supplement 3 ).…”

“…The five coronal sections used for the HiPlex experiments did not include the most rostral region of the PBN, which explains our difficulty in locating clusters N4 and N9 . We did not include probes for some genes that are known to be expressed in the PBN and detected by scRNA-Seq ( Table 1 ), e.g., Penk ( Engström et al, 2001 ), Grp ( Karthik et al, 2022 ), Nmb ( Huang et al, 2022 ), and Mc4r ( Paues et al, 2006 ). It will be important to determine whether any of them are co-localized with the in situ probes that we used.…”

Efferent projections ofNps-expressing neurons in the parabrachial region

“…In Nps Cre-reporter mice, we identified additional NPS neurons extending rostrally alongside the lateral lemniscus, as well as novel populations in the nucleus incertus, lateral habenula, and anterior hypothalamus (Huang et al, 2022).…”

“…The Atoh1 macropopulation includes a subpopulation of neurons that express Nps and produce neuropeptide S (NPS; Huang et al, 2022). The PB region in mice contains two clusters of NPS neurons -a rostral group near the lateral lemniscus and a caudal group near the locus coeruleus (Clark et al, 2011;Huang et al, 2022;Liu et al, 2011) -and this pattern is similar in the human brainstem (Adori et al, 2015).…”