A cluster of neuropeptide S neurons regulates breathing and arousal

Christopher Caleb Angelakos; Kasey S Girven; Yin Liu; Oscar C Gonzalez; Keith R Murphy; Kim J Jennings; William J Giardino; Larry S Zweifel; Azra Suko; Richard D Palmiter; Stewart D Clark; Mark A Krasnow; Michael R Bruchas; Luis de Lecea

doi:10.1016/j.cub.2023.11.018

. Author manuscript; available in PMC: 2024 Dec 18.

Published in final edited form as: Curr Biol. 2023 Dec 5;33(24):5439–5455.e7. doi: 10.1016/j.cub.2023.11.018

A cluster of neuropeptide S neurons regulates breathing and arousal

Christopher Caleb Angelakos ¹, Kasey S Girven ², Yin Liu ³, Oscar C Gonzalez ¹, Keith R Murphy ¹, Kim J Jennings ¹, William J Giardino ¹, Larry S Zweifel ², Azra Suko ², Richard D Palmiter ⁴, Stewart D Clark ⁵, Mark A Krasnow ³, Michael R Bruchas ², Luis de Lecea ^1,^6,^*

PMCID: PMC10842921 NIHMSID: NIHMS1950328 PMID: 38056461

Summary

Neuropeptide S (NPS) is a highly conserved peptide found in all tetrapods that functions in the brain to promote heightened arousal; however, the subpopulations mediating these phenomena remain unknown. We generated mice expressing Cre recombinase from the Nps gene locus (Nps^Cre) and examined populations of NPS+ neurons in the lateral parabrachial area (LPBA), the peri-locus coeruleus (peri-LC) region of the pons, and the dorsomedial thalamus (DMT). We performed brain-wide mapping of input and output regions of NPS+ clusters and characterized expression patterns of the NPS receptor (NPSR1). While the activity of all three NPS+ subpopulations tracked with vigilance state, only NPS+ neurons of the LPBA exhibited both increased activity prior to wakefulness and decreased activity during REM sleep, similar to the behavioral phenotype observed upon NPSR1 activation. Accordingly, we found that activation of LPBA, but not peri-LC NPS+ neurons increased wake and reduced REM sleep. Furthermore, given the extended role of the LPBA in respiration, and the link between behavioral arousal and breathing rate, we demonstrated that LPBA-, but not peri-LC-NPS+ neuronal activation increased respiratory rate. Together, our data suggests that NPS+ neurons of the LPBA represent an unexplored subpopulation regulating breathing and they are sufficient to recapitulate the sleep/wake phenotypes observed with broad NPS system activation.

Keywords: Neuropeptide S, Neuropeptide S Receptor, Sleep, Arousal, Fiber Photometry, Lateral Parabrachial, Breathing, Respiration

eTOC Blurb

The Neuropeptide S (NPS) system is an important regulator of wakefulness and breathing. Angelakos et al. investigate the major NPS cell populations in the mouse brain and show that a cluster of NPS-expressing neurons in the lateral parabrachial area are sufficient for enhancing behavioral arousal and regulating respiration.

Introduction

Sleep disorders are deeply connected with neuropsychiatric diseases¹. Among the neuronal systems that modulate vigilance states, neuropeptides have emerged as critical players in maintaining boundaries between sleep and wakefulness^2–8. Neuropeptide S (NPS), named after its terminal serine residue, is conserved in all tetrapods⁹ and was identified as an important regulator of arousal and anxiety¹⁰. Subsequent studies infusing the NPS peptide intracerebroventricularly (ICV)¹¹ or into the ventrolateral preoptic nuclei¹² recapitulated arousal findings, while antagonism of the sole NPS receptor (NPSR1; formerly called GPR154) increased NREM sleep and decreased wakefulness¹³. In humans, single nucleotide polymorphisms resulting in overactivation of the NPSR1 have been associated with shorter sleep duration^14,15.

Given the health and cognitive consequences associated with insufficient sleep¹⁶, elucidating the neurobiological underpinnings of sleep and arousal are paramount. Although NPS is believed to be one of the sparsest neuropeptides, labeling between only a few hundred^17,18 and a few thousand neurons in the mouse brain¹⁹, and an estimated 20,000 – 25,000 neurons in the human pons²⁰, its receptor is broadly expressed in critical regions regulating sleep and behavior^17,21. Previous studies implementing peptide infusion, mouse knockouts, and pharmacological manipulation of the NPSR1 lack the spatial resolution necessary to understand the role of NPS on arousal.

The NPSR1 was first functionally described as an asthma-associated gene, at the time called GPR154 or GPRA (G protein-coupled receptor for asthma susceptibility)²². Although direct evidence linking NPSR1 to asthma is lacking, several studies have reported an association between NPSR1 and asthma or elevated serum immunoglobulin E^23–31, which was summarized by Zhang and Tao³². In situ hybridization studies suggest that NPS cell bodies are prominently expressed in and around brainstem regions known to regulate respiration, including a parabrachial cluster, which contains the preponderance of NPS+ cells (84%) in the human pons^17,20,21. Furthermore, ICV NPS increases arousal and decreases anxiety-like behavior¹⁰, two phenotypes that are bidirectionally related to breathing rate. Despite these early purported links between the NPSR1 and asthma, the role of the NPS system on respiration has scarcely been investigated in vivo, with the lone exception of a single study showing that ICV NPS infusion in mice increases breathing frequency and decreases tidal volume. This effect was dependent on the presence of the NPSR1³³. However, these experiments did not specifically interrogate quiet periods indicative of normal breathing and were incapable of manipulating the NPS system in a brain-region or circuit-specific-manner.

Thus, we generated Neuropeptide S-IRES-Cre (Nps^Cre) (Figure S1A–C) and NPSR1-IRES-Cre (Npsr1^Cre) (Figure S1D–E) knock-in mouse lines to characterize and map the functional circuitry of the NPS system on sleep, wakefulness, and respiration. Whole organism NPS system inactivation and activation recapitulated and clarified previous sleep/wake findings obtained via NPS infusion and receptor agonism/antagonism. Reporter mice identified NPS cell clusters, and viral-assisted monosynaptic tracing experiments identified inputs to, and outputs of, NPS+ neurons. Polysomnography was performed concomitant with in vivo Calcium recordings, which highlighted endogenous local NPS+ neuron activity during sleep/wake behavioral state transitions. Guided by the results of these studies, we manipulated a single cluster of NPS neurons and altered sleep/wake and several respiratory parameters.

Results

Chemogenetic manipulation of NPS system reveals a role in arousal and REM sleep suppression

Nps^Cre knock-in mice were generated (Figure S1A–C) and crossed with transgenic mice expressing either the Cre-inducible inhibitory G-protein coupled DREADD Gi (Nps^Cre∷Rosa26 ^LSL-hM4Di), or the Cre-inducible excitatory DREADD Gq (Nps^Cre∷Rosa26^LSL-hM3Dq). Adult bitransgenic mice were injected on separate days with 1.0 mg/kg CNO or saline during either the light (ZT2) or dark cycle (ZT14), in a counterbalanced design, and sleep/wake was recorded and staged from EEG/EMG recordings. Nps^Cre∷Rosa26 ^LSL-hM4Di mice spent significantly less time awake (CNO: 57.91 ± 5.02% vs Saline: 76.03 ± 3.82%) and significantly more time in NREM sleep (CNO: 37.79 ± 4.51% vs Saline: 21.23 ± 3.82%) in the immediate 3 hours following CNO administration in the dark cycle (Figure 1A–F). Analysis of bout count and durations revealed significantly more NREM bouts in the CNO-treatment group (Figure S2A,B). Nps^Cre∷Rosa26^LSL-hM3Dq mice, on the other hand, did not show increased wakefulness upon CNO treatment in the light cycle (Figure 1J–K, Figure S2F–H). However, REM sleep time was significantly reduced by 47.7% in the 3 hours following CNO administration in the light cycle, in comparison to saline (CNO: 6.02 ± 0.82% vs Saline: 11.51 ± 1.43%), owing mostly to a decrease in the quantity of REM bouts rather than an alteration of REM bout duration (Figure 1J,M,N,O, Figure S2F–H).

Figure 1. — A-I) Sleep/wake time and expression of *Nps*^Cre∷*Rosa26*^LSL-hM4Di mice.

A) Total wake, NREM, and REM sleep time expressed as a percentage of total time for the 3 hours following 1.0 mg/kg CNO or saline injection at ZT14. n = 9 mice (3 males, 6 females). [Wake: Student’s paired t-test, t(8) = 2.792, p = 0.023; NREM: Student’s paired t-test, t(8) = −2.799, p = 0.023; REM: Student’s paired t-test, t(8) = −1.776, p = 0.114].

B-D) Percentage of time in B) wake, C) NREM, and D) REM sleep expressed in half hour bins following CNO or saline injection at ZT14. n = 9 mice (3 males, 6 females). [Wake: repeated-measures ANOVA, main effect of drug: F(1,16) = 6.600, p = 0.021, drug × time interaction: F(5,80) = 0.526, p = 0.756; NREM: repeated-measures ANOVA, main effect of drug: F(1,16) = 6.889, p = 0.018, drug × time interaction: F(5,80) = 0.527, p = 0.755; REM: repeated-measures ANOVA, main effect of drug: F(1,16) = 1.745, p = 0.205, drug × time interaction: F(5,80) = 0.616, p = 0.688]. E) Individual animals time spent in REM (green), NREM (red), and wake (blue), stacked and expressed as a percentage of total time for the 3 hours following CNO or saline injection at ZT14. The top plot represents saline treated animals, while bottom plot represents the same animals treated with CNO.

F) Sleep stage hypnograms for individual animals. Each 4 s epoch is plotted temporally and marked by a green (REM), red (NREM), or blue (wake) bar for the 3 hours following saline (top plot) or CNO (bottom plot) injection at ZT14. White line at 30 min represents the approximate time that CNO should begin inactivating NPS+ cells in the brain.

G-I) Representative images of mCitrine expression (green) marking Gi expression in NPS+ cells of the G) peri-LC, H) LPBA, and I) DMT. The signal was amplified with an eGFP antibody.

J-Q) Sleep/wake time and expression of *Nps*^Cre∷*Rosa26* ^LSL-hM3Dq mice.

J) Total wake, NREM, and REM sleep time expressed as a percentage of total time for the 3 hours following 1.0 mg/kg CNO or saline injection at ZT2. n = 9 mice (6 males, 3 females). [Wake: Student’s paired t-test, t(8) = −0.680, p = 0.516; NREM: Student’s paired t-test, t(8) = −0.974, p = 0.358; REM: Student’s paired t-test, t(8) = 4.162, p = 0.003].

K-M) Percentage of time in K) wake, L) NREM, and M) REM sleep expressed in half hour bins following CNO or saline injection at ZT2. n = 9 mice (6 males, 3 females). [Wake: repeated-measures ANOVA, main effect of drug: F(1,16) = 0.285, p = 0.601, drug × time interaction: F(2.740,43.834) = 1.075, p = 0.366; NREM: repeated-measures ANOVA, main effect of drug: F(1,16) = 0.744, p = 0.401, drug × time interaction: F(2.879,46.062) = 1.320, p = 0.279; REM: repeated-measures ANOVA, main effect of drug: F(1,16) = 8.966, p = 0.009, drug × time interaction: F(2.839,45.425) = 1.793, p = 0.164].

N) Individual animals time spent in REM (green), NREM (red), and wake (blue), stacked and expressed as a percentage of total time for the 3 hours following CNO or saline injection at ZT2. The top plot represents saline treated animals, while bottom plot represents the same animals treated with CNO.

O) Sleep stage hypnograms for individual animals. Each 4 s epoch is plotted temporally and marked by a green (REM), red (NREM), or blue (wake) bar for the 3 hours following saline (top plot) or CNO (bottom plot) injection at ZT2. White line at 30 min represents the approximate time that CNO should begin activating NPS+ cells in the brain.

P,Q) Representative images of mCitrine expression (green) marking Gq expression in NPS+ cells of the P) peri-LC and Q) DMT/PVT. Signal was amplified with an eGFP antibody.

R-Ab) Sleep/wake time and expression of *Npsr1*^Cre∷*Rosa26* ^LSL-hM3Dq mice.

R) Total wake, NREM, and REM sleep time expressed as a percentage of total time for the 3 hours following 1.0 mg/kg CNO or saline injection at ZT2. n = 12 mice (5 males, 7 females). [Wake: Student’s paired t-test, t(11) = −2.847, p = 0.016; NREM: Student’s paired t-test, t(11) = 2.671, p = 0.022; REM: Student’s paired t-test, t(11) = 2.738, p = 0.019].

S-U) Percentage of time in S) wake, T) NREM, and U) REM sleep expressed in half hour bins following CNO or saline injection at ZT2. n = 12 mice (5 males, 7 females). [Wake: repeated-measures ANOVA, main effect of drug: F(1,22) = 6.814, p = 0.016, drug × time interaction: F(2,321,51.071) = 0.516, p = 0.627; NREM: repeated-measures ANOVA, main effect of drug: F(1,22) = 4.963, p = 0.036, drug × time interaction: F(2.385,52.476) = 0.453, p = 0.672; REM: repeated-measures ANOVA, main effect of drug: F(1,22) = 4.457, p = 0.046, drug × time interaction: F(2.126,46.767) = 0.834, p = 0.447].

V) Individual animals time spent in REM (green), NREM (red), and wake (blue), stacked and expressed as a percentage of total time for the 3 hours following CNO or saline injection at ZT2. The top plot represents saline treated animals, while bottom plot represents the same animals treated with CNO.

W) Sleep stage hypnograms for individual animals. Each 4 s epoch is plotted temporally and marked by a green (REM), red (NREM), or blue (wake) bar for the 3 hours following saline (top plot) or CNO (bottom plot) injection at ZT2. White line at 30 min represents the approximate time that CNO should begin activating NPS+ cells in the brain.

X-Ab) Representative images of mCitrine expression (green) marking Gq expression in NPSR1+ cells of the X) basolateral amygdala, Y) endopiriform cortex, Z) laterodorsal thalamus, ventrolateral nucleus, Aa) retrosplenial cortex, and Ab) subiculum. The signal was amplified with an eGFP antibody.

Sleep state was staged in 4 s epochs and discerned from EEG/EMG recordings.

All graphs are expressed as mean ± s.e.m. * represents p<0.05, ** p<0.01, ***<0.001

See also Figure S1 and Figure S2.

We also assessed the effects of chemogenetically activating the NPSR1-expressing neurons directly. A line of NPSR1-IRES-Cre (Npsr1^Cre) knock-in mice (Figure S1D,E) was generated and crossed to the same hM3Dq transgenic line as above. Npsr1^Cre∷Rosa26^LSL-hM3Dq mice exhibited significantly more wake (CNO: 50.60 ± 3.10% vs Saline: 43.44 ± 1.83%) and significantly less REM sleep time (CNO: 6.40 ± 0.90% vs Saline: 8.07 ± 0.49%) upon CNO activation in the light phase (Figure 1R–W, Figure S2K–M). Analysis of bouts of sleep and wakefulness revealed that Npsr1^Cre∷Rosa26 ^LSL-hM3Dq mice had significantly more bouts of wakefulness and significantly shorter duration REM bouts following CNO activation, in comparison to saline. There were no differences in the EEG power spectra between saline and CNO treatment for Nps^Cre∷Rosa26^LSL-hM4Di, Nps^Cre∷Rosa26^LSL-hM3Dq, or Npsr1^Cre∷Rosa26^LSL-hM3Dq mice (Figure S2D–E,I–J,N–O, respectively). Collectively, our chemogenetic data recapitulate the arousal-promoting effects of the NPS system observed in previous studies^10–13.

Nps^Cre is expressed in distinct neuronal clusters with broad afferent and efferent projections

Nps^Cre mice were then crossed with Rosa26^LSL-tdTomato reporter mice to characterize the anatomy of NPS in the mouse brain. Whole-brain microscopy analysis of adult brains revealed prominent NPS+ cell clusters in the lateral parabrachial area (LPBA), medial to the locus coeruleus in the pons (peri-LC), and in the dorsomedial/paraventricular thalamus (DMT/PVT) (Figure 2A–D). Sparse NPS cell labeling in the amygdala, arcuate nucleus of the hypothalamus (Arc), and preoptic area (POA) was also observed, while NPS+ fibers were observed in several areas associated with sleep/wake regulation, including the nucleus accumbens (NAc), ventral tegmental area (VTA), and reticular formation (Table S1, Figure S3A–L). In alignment with other studies of NPS expression in the mouse^17–19, and in contrast to the rat¹⁰, we did not observe any NPS expression in trigeminal principle sensory 5 nucleus.

Figure 2. — A-D) tdTomato reporter expression representing NPS+ cells (red). NPS cells are highly clustered in the A) lateral parabrachial area (LPBA), B) medial to the locus coeruleus (peri-LC), C) dorsomedial thalamus (DMT), D) paraventricular thalamus (PVT). In (B), tyrosine hydroxylase staining (green) marks the LC. n = 2 mice (1 male, 1 female).

E-S) Rabies tracing and quantification of monosynaptic inputs to NPS+ cells in the LPBA (E-I), peri-LC (J-N), and habenula/PVT/DMT (O-S).

E) Representative image of NPS+ starter cells in the LPBA (yellow), expressing both Flex-TVA / Rabies G helper virus (red) and EnvA G-deleted Rabies virus (green). Green cells represent upstream inputs to NPS+ starter cells.

F-H) Representative image of cell populations in the F) inferior colliculus, G) bed nucleus of stria terminalis (BNST), and H) central amygdala that project to NPS+ cells in the LPBA.

I) Quantification of monosynaptic inputs to LPBA NPS+ cells, expressed as a percentage of total. n = 2 mice (1 male, 1 female). Schematic of injection protocol and LPBA target (I inset).

J) Representative image of NPS+ starter cells in the peri-LC (yellow), expressing both Flex-TVA / Rabies G helper virus (red) and EnvA G-deleted Rabies virus (green).

K-M) Representative image of cell populations in the K) median raphe, L) interpeduncular nuclei, and M) zona incerta that project to NPS+ cells in the peri-LC.

N) Quantification of monosynaptic inputs to peri-LC NPS+ cells, expressed as a percentage of total. n = 2 mice (1 male, 1 female). Schematic of injection protocol and peri-LC target (N inset).

O) Representative image of NPS+ starter cells in the habenula (yellow), expressing both Flex-TVA / Rabies G helper virus (red) and EnvA G-deleted Rabies virus (green).

P-R) Representative image of cell populations in the P) PVT and Q-R) DMT that project to NPS+ cells in the habenula.

S) Quantification of monosynaptic inputs to habenula NPS+ cells, expressed as a percentage of total. n = 4 mice (3 male, 1 female). Schematic of injection protocol and DMT target (S inset).

All brain regions with >1% of total monosynaptic inputs to target NPS+ region(s) are plotted.

See also Figure S1, Figure S3, and Table S1.

To ascertain the monosynaptic inputs into NPS-Cre+ cells, we employed Cre-dependent modified-Rabies virus tracing^34,35 in the three main NPS+ cellular clusters of the LPBA, peri-LC, and DMT/PVT. NPS+ cells of the LPBA receive strong inputs from anxiety-associated regions (bed nucleus of stria terminalis [BNST], central amygdala), auditory-processing regions (inferior colliculus, ventral cochlear nucleus), and REM sleep-associated regions (pontine reticular nucleus, periaqueductal gray [PAG]) (Figure 2E–I). Peri-LC NPS+ neurons receive strong inputs from several sleep-associated regions (pontine tegmental area, interpeduncular nucleus, pontine reticular formation, laterodorsal tegmental nuclei, PAG), the arousal- and anxiety-associated median raphe, and the feeding behavior-associated zona incerta (Figure 2J–N). NPS+ cells in the DMT/PVT were difficult to label, with the only double-labeled starter cells consistently appearing in the habenula. From these, we found sparse inputs mostly locally in the DMT and PVT (Figure 2O–S).

To examine the outputs of NPS+ cell populations, we mixed AAV-DJ-EF1α-DIO-YPet-2A-mGFP with AAV-DJ-hSyn-DIO-Synaptophysin-mRuby and injected them into the LPBA, peri-LC, or DMT of Nps^Cre mice (Figure 3A–D). Synaptophysin is a presynaptic vesicle marker that aggregates in axon terminals and can be used as a proxy for synaptic terminals, as previously described³⁶. Whole-brain assessment of mRuby labeling revealed LPBA NPS+ outputs to the POA, PVT, peduncular lateral hypothalamus (PLH), PAG, retrorubral field (RRF), and solitary tract nucleus (NTS) (Figure 3E). Several other regions displayed mRuby puncta more sparsely (Table S1, column 7). Peri-LC-directed injections resulted in the strongest labeling of the Inferior Olive cap of Kooy. Additionally, mRuby puncta were observed in the BNST, PLH, PVT, posterior subthalamic nucleus (PSTH), VTA/parabrachial pigmented nucleus (VTA/PBP), pontine reticular nucleus (PR), and NTS (Figure 3F), with sparse terminal labeling in other areas (Table S1, column 8). As with the modified-Rabies virus infusions, anterograde tracer virus failed to effectively label NPS neurons in the DMT/PVT. Unable to virally target the DMT/PVT with our retrograde and anterograde viruses, we performed RNAscope for NPS, Cre, and DAPI mRNA in the DMT/PVT and LPBA of an adult Nps^Cre mouse. In contrast to the LPBA, where 25.7% of neurons imaged contained NPS mRNA (Figure S3M–O), no NPS mRNA was detected in the DMT/PVT in adulthood (Figure S3P–Q).

Figure 3. — A) LPBA injection site of Cre-dependent Ypet-2a-mGFP (green) / synaptophysin-mRuby (red) in *Nps*^Cre mouse shown at 63x magnification.

B,C) Peri-LC injection site of Cre-dependent Ypet-2a-mGFP (green) / synaptophysin-mRuby (red) in *Nps*^Cre mouse. Shown at 63x magnification (B) and 20x magnification (C).

D) Nucleus incertus / pontine central gray injection site of Cre-dependent Ypet-2a-mGFP (green) / synaptophysin-mRuby (red) in *Nps*^Cre mouse shown at 20x magnification.

E) Schematic summarizing the most prominent outputs of (red) and inputs to (blue) NPS-expressing cells of the LPBA. N = 2 mice (1 male, 1 female). Left panel is a sagittal section, 1.3 mm lateral from midline, with the LPBA in-plane. Right panel is a coronal section of the LPBA injection area (−5.02 mm from Bregma, indicated by black bar in left panel, rotated 90° and displayed coronally).

F) Schematic summarizing the most prominent outputs of (red) and inputs to (blue) NPS-expressing cells of the peri-LC / NI. N = 2 mice (1 male, 1 female). Left panel is a sagittal section, 0.6 mm lateral from midline, with the peri-LC in-plane. The right panel is a coronal section of the peri-LC injection area (−5.52 mm from Bregma, indicated by black bar in left panel, rotated 90° and displayed coronally). Note: pontine central gray (PCG) and laterodorsal tegmental nucleus (LDTg) input, and pontine reticular (PnR) output regions are omitted from the sagittal image (left panel) for viewability. These regions are included in the coronal section (right panel).

White scale bars (lower right in A-D) represent 20 μm in (A) and (B), 50m in (C), and 25um in (D).

E and F) Blue brain regions represent inputs to NPS-expressing cells. Red brain regions represent efferent targets of NPS-expressing cells. Solid lines outlining brain regions represent regions that are in-plane. Dotted lines outlining brain regions represent regions that are out-of-plane. For both schematics, all inputs comprising >3% of total inputs are included, and all outputs with relatively more synaptophysin-mRuby puncta (Table S1, columns 7–8) are included.

Abbreviations: BNST = bed nucleus of stria terminalis, CeM = central amygdala, DMTg = dorsomedial tegmental area, DPGi = dorsal paragigantocellular nucleus, IC = inferior colliculus, IOK = inferior olive cap of Kooy, IP = interpeduncular nuclei, LDTg = laterodorsal tegmental nucleus, LH = lateral hypothalamus, LPBA = lateral parabrachial area, LPO = lateral preoptic area, MnR = median raphe, NTS = solitary tract nucleus, PAG = periaqueductal gray, peri-LC = peri-locus coeruleus, PCG = pontine central gray, PnR = pontine reticular nucleus, PSTH = posterior subthalamic nucleus, PVT = paraventricular thalamus, RRF = retrorubral field, VCA = ventral cochlear area, Ve = vestibular nucleus, VLL = ventral lateral lemniscus, VTA = ventral tegmental area.

See also Figure S3 and Table S1.

Npsr1^Cre is expressed in areas critical for sensory processing, behavior, and arousal

After mapping inputs and outputs from the prominent LPBA and peri-LC NPS+ clusters, we sought to ascertain the distribution of the NPSR1-expressing neurons. Npsr1^Cre mice were crossed to Rosa26^LSL-tdTomato reporter mice and brains were scanned from rostral olfactory bulb to caudal brain stem for tdTomato expression. Dense labeling (>30% of area expressing strong fluorescence) was observed in several regions (Figure 4I, Table S1, column 9), including the anterior olfactory nucleus (AOL) and tenia tecta (Figure 4A), olfactory bulb, orbitofrontal cortex (Figure 4B), frontal association cortex, BNST (Figure 4D), basolateral amygdala and adjacent endopiriform cortex (Figure 4F), and subiculum (Figure 4G). Numerous other regions, which either project to, or receive output from, the LPBA and/or peri-LC NPS+ clusters display more moderate (between 5 – 30% of area exhibiting strong fluorescence) NPSR1 expression (Figure 4I, Table S1, column 9). Notably, the nucleus accumbens shell (Figure 4C), paraventricular nucleus of the hypothalamus (Figure 4E), parabrachial nucleus (Figure 4H), central amygdala, and superior colliculus (quantified in Figure 4I) all contain sizeable populations of NPSR1-expressing neurons and may be involved in NPS-mediated arousal.

Figure 4. — A-H) Representative images of tdTomato and DAPI staining in *Npsr1*^Cre mice. Dense tdTomato expression was observed in the A) anterior olfactory nucleus, B) orbitofrontal cortex, C) nucleus accumbens shell, D) dorsal bed nucleus of stria terminalis, E) paraventricular nucleus of the hypothalamus, F) basolateral amygdala and endopiriform nucleus, G) subiculum, and H) parabrachial nucleus.

I) Quantification of tdTomato expression depicted as a percentage of fluorescence per neuroanatomical region. N = 3 mice (1 male, 2 females). All brain regions with >1% of area expressing strong fluorescence are plotted.

J) Representative image of defined neuroanatomical regions utilized for HALO software processing and quantification of NPSR1 fluorescence.

White scale bars (lower right) represent 100 μm in all panels.

Abbreviations: 3V=3^rd ventricle, AO/AOL = anterior olfactory nucleus, AOB = accessory olfactory bulb, BLA = basolateral amygdala, dBNST = dorsal bed nucleus of stria terminalis, dEN = dorsal endopiriform nucleus, FrA = frontal association cortex, GrO = olfactory bulb, LO = lateral orbital cortex, NacS = nucleus accumbens shell, lOFC = lateral orbitofrontal cortex, vOFC = ventral orbitofrontal cortex, PBN = parabrachial nucleus, PVT = paraventricular nucleus of the hypothalamus. Scp = superior cerebellar peduncle

See also Figure S1 and Table S1.

NPS-expressing cells increase activity preceding transitions to wake and suppress activity during REM sleep

Given the distinct connections of each NPS-expressing cluster, we questioned whether the individual subregions may have disparate functional roles on arousal and REM sleep suppression. Thus, we recorded endogenous intracellular NPS+ calcium activity (GCaMP fluorescence) during sleep/wake behavior by employing combined fiber photometry and polysomnography recordings in each of the three main NPS+ cell clusters (Figure 5). We found that NPS+ cells in the LPBA transiently increase their activity leading up to, and peaking at, transitions to wakefulness. This rise was immediately followed by a relative decline in activity (Figure 5D). In the peri-LC and DMT, a similar trend is observed preceding and following NREM to wake transitions; however, this data did not achieve statistical significance (Figure 5K,R). In combination with our chemogenetic data showing that NPSR1+ activation increases wake bout counts, but not duration (Figure S2K,L), our data supports the notion that NPS is involved in the induction, but not the maintenance, of wakefulness.

We observed a significant and prolonged decline in calcium activity in the LPBA and DMT following the onset of REM sleep (Figure 5E,S). In the peri-LC region, however, there were no noticeable alterations in calcium activity over the course of REM sleep, in comparison to the preceding NREM sleep (Figure 5L). These data suggest that the peri-LC may not be involved in the strong REM-suppressing effects of NPS.

In comparison to asynchronous resting state activity, bulk averages of calcium signals do not fully capture synchronous network activity, which may occur during ethologically relevant sleep states. Transient Ca2+ spiking, which reflects highly synchronous firing events, was detected and aligned to sleep/wake state, similar to what has been described^37,38. Analyses of transient amplitude, within-bout instantaneous transient frequency, and transient rate (transient number per second of sleep stage) were quantified for the LPBA, peri-LC, and DMT/PVT brain regions. In the peri-LC and DMT/PVT, there were significantly more Ca2+ transients per second of wake, compared to the transient rate during both NREM and REM sleep (Figure S4F,I). In the LPBA, however, there were no significant differences in transient rate between wake, NREM, and REM sleep Figure S4C). Moreover, the mean transient amplitude was decreased in REM sleep compared to wake only in the DMT/PVT (Figure S4A,D,G), while the within-bout instantaneous frequency of NPS+ Ca2+ transients was not significantly altered between sleep states for any of the regions investigated (Figure S4B,E,H). Together, these data suggest that neuronal synchrony is heightened during wake in NPS+ neurons of the peri-LC and DMT/PVT, but not in NPS+ neurons of the LPBA.

Lateral parabrachial area-restricted NPS+ activation augments wake and suppresses REM sleep

We then employed subregion-specific manipulation of NPS+ cells. The LPBA cluster provided an ideal target for several reasons. In human pons, the vast majority of NPS+ expression (84%) is in the parabrachial region, while the peri-LC and pontine central gray make up only 5% and 11% of NPS+ expression, respectively²⁰. In addition to this translational rationale, pharmacologic lesions of the LPBA result in significantly less wake time and significantly more REM sleep³⁹, and excitatory neurons of the LPBA mediate awakening under hypoxic conditions^40,41. Finally, our tracing data showed strong inputs into the LPBA from anxiety centers including the central amygdala and BNST (Figure 2E–I). Anxiety regulation appears to be a critical function of the NPS system^10,42–47, and anxiety and arousal are closely linked^48–50.

To examine this link in the context of NPS, we infused adeno-associated virus (AAV) carrying Cre-dependent hM3Dq bilaterally into the LPBA of Nps^Cre mice (Figure 6A). Analysis of sleep and wake revealed a robust decrease in light-cycle REM sleep in the 3 hours following LPBA NPS+ activation (CNO: 5.74 ± 0.58% vs Saline: 9.16 ± 0.56%), including a 76.5% reduction in REM sleep in the hour following CNO activation (ZT2.5 - ZT3.5; CNO: 10.83 ± 1.01% of total time vs Saline: 2.54 ± 0.58% of total time), recapitulating the phenotype observed with whole-brain transgenic hM3Dq activation (Figure 6B–D,H). LPBA-restricted chemogenetic augmentation of wakefulness trended towards significance over the 3-hour post-CNO interval (p=0.075) and significantly enhanced wake in the hour following CNO activation (ZT2.5 - ZT3.5; p=0.037) (Figure 6B,D,F), like the phenotype observed upon whole-brain NPSR1 activation (Figure 1R–W). The REM sleep time reductions appear to be largely driven by a substantial decrease in the number of REM bouts (CNO: 7.25 ± 0.88% vs Saline: 12.33 ± 0.66%), resulting in a compensatory slight increase in average REM duration upon CNO treatment, compared to saline (Figure S5A,B). Additionally, the REM power spectra, but not the wake nor NREM power spectra, was significantly altered following CNO administration in Nps^Cre mice with hM3Dq restricted to the LPBA, in comparison to saline. Specifically, LPBA-restricted NPS+ activation resulted in a decrease in high delta power (1.5 – 4 Hz) and an increase in alpha power (8 – 12 Hz), which appears to be caused by an ~0.5 Hz rightward shift of the REM power spectra (Figure S5D,E). Even though there was no alteration of NREM sleep time, (Figure 6G; p=0.54), CNO-treated Nps^Cre mice with hM3Dq targeted to the LPBA displayed less NREM bout counts and NREM to REM transitions, and more NREM to wake transitions than saline (Figure S5A,C). Altogether, LPBA-restricted NPS activation strongly recapitulates the REM sleep inhibition and arousal-promotion observed upon NPSR1 activation.

Figure 6. — A) Schematic of injection protocol and Cre-dependent hM3Dq targeted to the LPBA.

B-H) Sleep/wake time and expression of LPBA-restricted hM3Dq *Nps*^Cre mice. (11 males, 1 female).

B) Total wake, NREM, and REM sleep time expressed as a percentage of total time for the 3 hours following CNO or saline injection at ZT2. [Wake: Student’s paired t-test, t(11) = −1.97, p = 0.075; NREM: Student’s paired t-test, t(11) = 0.637, p = 0.537; REM: Student’s paired t-test, t(11) = 6.676, p = 0.000035)].

C) Individual animals time spent in REM (green), NREM (red), and wake (blue), stacked and expressed as a percentage of total time for the 3 hours following CNO or saline injection at ZT2. The top plot represents saline treated animals, while bottom plot represents the same animals treated with CNO.

D) Sleep stage hypnograms for individual animals. Each 4 s epoch is plotted temporally and marked by a green (REM), red (NREM), or blue (wake) bar for the 3 hours following saline (top plot) or CNO (bottom plot) injection at ZT2. White line at 30 min represents the approximate time that CNO should begin activating NPS+ cells in the brain.

E) Representative images of NPS+ cells in the LPBA expressing either AAV-hM3Dq (top) or AAV-mCherry control (bottom) at 10x (left) and 20x (right) magnification.

F-H) Percentage of time in F) wake, G) NREM, and H) REM sleep expressed in half hour bins following CNO or saline injection at ZT2. [Wake: repeated-measures ANOVA, main effect of drug: F(1,22) = 2.605, p = 0.121, drug × time interaction: F(5,110) = 1.570, p = 0.174; NREM: repeated-measures ANOVA, main effect of drug: F(1,22) = 0.314, p = 0.581, drug × time interaction: F(5,110) = 1.404, p = 0.228; REM: repeated-measures ANOVA, main effect of drug: F(1,22) = 17.813, p = 0.0004, drug × time interaction: F(2,848,62.655) = 2.747, p = 0.053].

# indicates statistically significant (p = 0.037) if analyzed only over the 1-hour post-CNO onset (ZT2.5 - ZT3.5).

I-N) Plethysmography outputs of various breathing parameters. n = 21 mice (AAV-mCherry control: 7 males, 2 females; AAV-hM3Dq: 11 males, 1 female). Mice expressing hM3Dq exclusively in NPS+ cells of the LPBA exhibited I) significantly more breaths per minute, J) significantly shorter inspiratory and K) expiratory times, L) significantly higher peak inspiratory flow, and M) significantly higher peak expiratory flow following CNO injection, in comparison to mCherry-injected control animals. There were no significant differences observed between groups in tidal volume. Data is expressed as a ratio (CNO or saline post-injection period normalized to same-day 2-hour baseline recording). [I: repeated-measures ANOVA, main effect of drug: F(1,19) = 4.729, p = 0.042, drug × virus interaction: F(1,19) = 2.340, p = 0.143; J: repeated-measures ANOVA, main effect of drug: F(1,19) = 11.038, p = 0.004, drug × virus interaction, F(1,19) = 27.090, p = 0.00005; K: repeated-measures ANOVA, main effect of drug: F(1,19) = 0.179, p = 0.677, drug × virus interaction: F(1,19) = 10.189, p = 0.005; L: repeated-measures ANOVA, main effect of drug: F(1,19) = 9.428, p = 0.006, drug × virus interaction: F(1,19) = 25.301, p = 0.00007; M: repeated-measures ANOVA, main effect of drug: F(1,19) = 0.354, p = 0.559, drug × virus interaction: F(1,19) = 9.430, p = 0.006; N: repeated-measures ANOVA, main effect of drug: F(1,19) = 1.553, p = 0.228, drug × virus interaction: F(1,19) = 1.199, p = 0.287]. All graphs are expressed as mean ± s.e.m. * represents p<0.05, ** p<0.01, ***<0.001.

See also Figure S5 and Figure S6.

Lateral parabrachial area-restricted NPS+ activation enhances respiratory rate

Because the LPBA is well-known to play a critical role in breathing rate^51–54, breathing is known to impact arousal^55,56, and the cellular mechanisms of LPBA-mediated respiration are not fully understood, we postulated that NPS may be a regulator of respiration in the LPBA. Thus, we employed whole body plethysmography recordings in Nps^Cre mice with hM3Dq targeted to the LPBA. CNO activation of NPS-expressing neurons selectively in the LPBA increased breathing rate, decreased inspiratory and expiratory time, and increased peak inspiratory and expiratory flow, relative to saline, in comparison to control mice injected with mCherry virus (Figure 6I–N). These results suggest that NPS-expressing neurons of the LPBA may play a role in respiratory regulation.

Peri-locus coeruleus-restricted NPS+ manipulation does not impact arousal or breathing

To determine if the sleep and respiratory alterations caused by LPBA NPS+ activation might be specific to this subregion, rather than a generalized feature of NPS+ neurons, Nps^Cre mice were infused bilaterally into the peri-LC with AAV carrying Cre-dependent hM3Dq. There were no changes observed in sleep/wake parameters upon peri-LC-restricted NPS+ CNO activation, in comparison to saline (Figure S6B–D, F–H). Moreover, there were no differences recorded in whole body plethysmography between peri-LC hM3Dq-infused Nps^Cre mice and peri-LC mCherry-infused Nps^Cre mice, nor were there any interactions between infusion groups and CNO/saline injection conditions (Figure S6K–P). Together, these findings indicate that sleep/wake and breathing are not significantly impacted by NPS-expressing neurons of the peri-LC. Rather, wake promotion, REM sleep reduction, and respiratory phenotypes derived from the NPS system are principally driven by NPS neurons of the LPBA cluster.

Discussion

In this study, we generated Nps^Cre and Npsr1^Cre knock-in mice and characterized the expression and impact of neurons expressing these genes in adult mice on sleep and breathing. We identified prominent clusters of neurons expressing the Cre-driver line in the LPBA, peri-LC, and DMT/PVT. Another Nps^-IRES-Cre mouse line was recently reported with very similar expression pattern to that observed in our study, except the DMT/PVT labeling observed in our study is noticeably absent in the investigation by Huang et al¹⁹. Interestingly, the DMT/PVT proved extremely difficult to manipulate using viral vectors in our Cre-dependent retrograde and anterograde tracing studies, which subsequently led us to double-labeling NPS+ and Cre+ mRNA in the DMT/PVT region of adult Nps^Cre mice. Indeed, in contrast to the LPBA, which expressed NPS+ RNA in adulthood (Figure S3M–O), the DMT/PVT had no cells positive for NPS or Cre mRNA (Figure S3P–Q). This data suggests that NPS expression in the DMT/PVT is transient, and NPS is not expressed in substantial quantities in the DMT/PVT in the adult mouse.

We characterized the afferents and efferents of NPS neuronal clusters (summarized in Figure 3E,F and Table S1, columns 4–8). Tracing of monosynaptic inputs revealed upstream projections from brain regions involved in arousal, REM sleep regulation, and anxiety. Similarly, synaptophysin-mRuby labeling of axon terminals revealed NPS+ outputs to several known wake- and REM sleep-regulating regions. Neurons of the Barrington’s nucleus, where NPS+ neurons of the peri-LC are located proximal to, project almost exclusively to the descending lumbosacral spinal cord⁵⁷. Previous studies, however, suggest that NPS either does not overlap^10,17, or overlaps minimally with known markers of the Barrington’s nucleus¹⁹. One limitation of our virus-based approaches in the peri-LC is the proximity of NPS+-expressing cells in the adjacent nucleus incertus (NI) and pontine central gray (PCG), which both contained some NPS+ starter cells in our YPet-2a-mGFP / synaptophysin-mRuby experiments (Figure 3D). Indeed, most of the forebrain regions receiving NPS+ synapses from the peri-LC/NI/PCG cluster are known projection targets of the NI⁵⁸. Thus, it cannot be discounted that at least some of the NPS+ outputs from the peri-LC cluster may arise from NPS+ neurons of the nucleus incertus (NI) or pontine central gray (PCG).

A few brain regions, including the PVT, lateral hypothalamus, and NTS received direct NPS+ inputs from both LPBA and peri-LC/NI/PCG clusters, potentially indicating a level of redundancy critical to NPS functionality. NPS+ outputs to the lateral hypothalamus may be of particular importance for the arousal-promoting effects of NPS. Hypocretin (also called orexin) neurons of the lateral hypothalamus are strongly wake-inducing and REM-suppressing^59,60, and NPS interactions with the hypocretin system have been shown to play a role in addiction and alcohol reinstatement^61–63. Additionally, NPS+ axon terminals and NPSR1 were observed in the dopaminergic VTA / extended VTA (primarily from the peri-LC/NI/PCG cluster) as well as the dopaminergic cell group A8 of the retrorubral field (primarily from the LPBA cluster). Activation of the dopaminergic system is wake-inducing⁶⁴, and NPS projections to major dopamine centers may underlie NPS’s role in arousal. It is interesting to note that significant NPS outputs originating from both the LPBA and peri-LC/NI/PCG cluster were observed in the NTS and PVT. The NTS is involved in taste and feeding behavior⁶⁵, and recent papers have implicated the peri-LC, as well as a PVT→parabrachial→pre-LC circuit, in the regulation of feeding behavior^66,67. While beyond the scope of this study, it is possible that these circuits contribute to the food intake inhibition observed upon NPS peptide infusion^68,69.

Behaviorally, chemogenetic inhibition of NPS-expressing neurons reduced wakefulness and increased sleep time during the dark (active) phase. Chemogenetic activation of NPS cells did not elicit arousal transitions; however, chemogenetic activation of NPSR1-expressing neurons modestly increased wakefulness in the 3-hours after CNO injection in the light phase. The lack of arousal elicitation upon NPS+ neuronal activation may be due to insufficient peptide release following chemogenetic manipulation in the Nps^Cre∷Rosa26^LSL-hM3Dq transgenic line, or from counteracting effects of NPS release from some of the sparser NPS+-expressing regions (i.e., the amygdala, preoptic area, anterior hypothalamus, and/or habenula) that were not interrogated in our current study. Most strikingly, activation of NPS neurons globally, as well as activation of NPSR1-expressing neurons, resulted in dramatic REM sleep suppression. This effect appears to be mostly REM-specific, as few alterations in NREM sleep time or parameters were observed upon hM3Dq activation of NPS+, NPSR1+, or LPBA-restricted NPS+ neuron populations (Figures 1 and 6; Figures S2 and S5). Fiber photometry recordings during endogenous sleep/wake behavior in the various NPS clusters may lend insight into the NPS populations differentially modulating aspects of NPS-related sleep regulation. For example, NPS+ Ca2+ activity increases in the LPBA over the ~15s immediately preceding transitions into wakefulness. Conversely, NPS Ca2+ activity steadily and consistently decreases in the LPBA and DMT/PVT clusters during REM sleep but is unaltered in the peri-LC during transitions into REM (Figure 5). It is possible that the brainstem-derived NPS+ clusters may fire in unison preceding transitions to wakefulness; however, from our chemogenetic studies, only the LPBA cluster promotes arousal, while the peri-LC cluster simply tracks with vigilance state. We cannot rule out the possibility that the activity of other cell populations in the LPBA, peri-LC, and/or DMT/PVT may similarly track with vigilance state. In fact, it would not be entirely surprising given the redundancy of sleep/wake circuitry⁷⁰. Nevertheless, our behavioral data cumulatively describes an important role for the Neuropeptide S system in sleep/wake regulation.

Given the prominence of NPS expression in both human and mouse LPBA^17–20,71, the monosynaptic inputs into the LPBA from known sleep- and anxiety-regulating centers (Figure 2), and the known role of the LPBA in breathing regulation^40,41,72,73, we specifically characterized the role of NPS in the LPBA on sleep/wake and respiratory parameters. Fascinatingly, chemogenetic activation of the LPBA recapitulated the arousal and REM-suppressing phenotypes observed by global NPS or NPSR1 activation. Further, we observed strong and consistent alterations in respiratory parameters associated with enhanced breathing rate following LPBA-restricted NPS activation. While the LPBA has been known to impact breathing inspiration, few specific cellular subtypes responsible for LPBA-mediated breathing have been described. To date, μ-opioid-expressing neurons of the LPBA have been shown to play a critical role in opioid-induced respiratory depression, and their activation/inactivation bidirectionally alters respiration rate⁷³. Moreover, calcitonin gene-related peptide (CGRP) expressing neurons of the LPB have been shown to be important for awakening from hypercapnia^41,72, and high frequency optogenetic activation of LPB^CGRP neurons decreases respiratory rate⁷². Conversely, optogenetic activation of LPB neurons expressing tachykinin1 (Tac1), but not CGRP, increases breathing rate and shortens the respiratory cycle. Interestingly, the respiratory alterations observed from both LPB^CGRP and LPB^Tac1 activation are blocked by anesthesia, suggesting a state-dependent role for LPB subpopulations on respiratory regulation⁵¹.

How do NPS+ neurons of the LPBA modulate respiration? It is tempting to search for links between LPBA NPS-expressing neurons and the pre-Bötzinger complex (preBötC) of the ventrolateral medulla, which is responsible for generating the respiratory rhythm^74,75. While our monosynaptic afferent and efferent tracing did not demonstrate direct linkage between NPS-expressing neurons of the LPBA and the preBötC, it is interesting to note that both the preBötC-adjacent lateral paragigantocellular nucleus and spinal trigeminal nucleus are target regions of LPBA-, but not peri-LC-derived NPS-expressing neurons (Table S1, column 7–8). The NTS, an important inspiration generator of the dorsal respiratory group^76,77, receives inputs from NPS+ cells of both the LPBA and peri-LC clusters. It is also possible that potential connections between NPS-expressing neurons of the LPBA and the preBötC may not be monosynaptic. Interestingly, the preBötC receives input from the central amygdala and BNST⁷⁸, projects to the inferior colliculus (IC)⁷⁹, and has bidirectional connectivity with the LPBA and PAG^78,79— all regions represented in the top ten of inputs to NPS+ neurons of the LPBA. Likewise, the preBötC projects to the parafacial zone, lateral preoptic area, and IC while bidirectionally connecting with the NTS, LH, and PAG^78,79—all regions that also receive projections from NPS-expressing neurons of the LPBA cluster. The specific circuit(s) underlying NPS’s LPBA-derived role in respiration will be the subject of future investigations. For LPB^CGRP neurons, connections with the BNST and rostral central amygdala were the only ones of those studied to significantly alter breathing rate⁷². Given the connections between the BNST, central amygdala, and LPBA^NPS neurons, it is tempting to speculate that NPS and CGRP may represent an overlapping neuronal population; however, NPS-expressing and CGRP-expressing neurons in the LPBA are mutually exclusive^19,71.

This study highlights a sufficiency for LPBA NPS-expressing neurons on breathing and sleep/wake regulation, particularly in comparison to NPS+ neurons of the peri-LC and nearby NI/PCG, which do not significantly affect sleep/wake behavior or respiration. Future studies will assess the functional contributions of specific outputs/circuits of these NPS clusters, aiming to disentangle and distinguish the interrelated phenomena of arousal, respiration, and anxiety. One interesting avenue of investigation is the effects of central nervous system (CNS) NPS activation on the peripheral nervous system. In humans, while NPS expression is restricted to the brain, NPSR1 is ubiquitously expressed outside of the CNS, including in epithelial cells and smooth muscles of the bronchus⁸⁰. Mutations in NPSR1³² and NPS²³ are significantly associated with asthma, which is an inflammatory disease of the lung airways. The expression of NPSR1 is increased in alveolar macrophages in inflamed lung tissue⁸¹ and in eosinophils of severe asthmatics⁸². It remains difficult to speculate on the relationship between hyper- or hypo-functional NPSR1 and asthma, as genetic associations between NPSR1 and asthma to date have identified haplotypes instead of SNPs⁸³. LPBA NPS+ activation increases inspiratory and expiratory flow, which are decreased during asthma, but also increases breathing rate and decreases expiratory time, which may lead to intrinsic positive end-expiratory pressure and hyperinflation⁸⁴. As of now, no CNS-mediated mechanisms of asthma pathogenesis have been established⁸⁵. From our dataset, the presence of NPS+ fibers surrounding the cerebral aqueduct in the far caudal brainstem suggest outputs to the periphery (Figure S3K). The impact of specific manipulation of these peripheral NPS+ efferents on NPSR1 activation in the bronchial epithelium may unveil a mechanistic link between the NPS system and asthma pathogenesis. Beyond this, interrogation of the NPS-expressing clusters described in this paper may also lead to insight into other diverse roles of the NPS system, which encompass a myriad of phenotypes including anxiolysis^10,44,86, hyperlocomotion^10,44,87, memory-promotion^88–90, fear extinction⁹¹, food intake inhibition^68,69, and drug seeking/reward^61,92–94. The possibility must also be considered that NPS-expressing neurons may co-express other neuropeptides, as seen for neuropeptide-expressing cortical neurons, which dedicate a significant portion of their transcriptional machinery to peptide production⁹⁵. Thus, future studies will also need to address the possible contributions of other neuropeptide/neurotransmitter systems co-expressing with NPS on physiology and behavior.

STAR Methods

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Luis de Lecea (llecea@stanford.edu)

Materials availability

Mouse lines Neuropeptide S-IRES-Cre (Nps^Cre) and NPSR1-IRES-Cre (Npsr1^Cre) generated in the study are available and will be shared upon request.
This study did not generate new unique reagents.
This study did not generate new unique plasmids.

Data and code availability

All data reported in this paper will be shared by the lead contact upon request.
All original code has been deposited at figshare and is publicly available as of the date of publication. DOIs are listed in the key resources table.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
chicken anti-Tyrosine Hydroxylase	Aves Labs	TYH; RRID:AB_10013440
rabbit anti-GFP	NovisBio	NB600-308; RRID:AB_10005904
chicken anti-GFP	Abcam	ab13970; RRID:AB_300798
goat anti-chicken Alexa 488	Invitrogen	A-11039; RRID:AB_142924
donkey anti-rabbit Alexa 488	Invitrogen	A-21206; RRID:AB_2535792
Bacterial and Virus Strains
AAV-DJ-EF1α-DIO-GCaMP7s	Addgene	Addgene AAV9; 104491-AAV9
SAD ΔG EnvA-GFP	Salk Institute Gene Targeting and Therapeutics Core	Addgene Plasmid #32635
AAVDJ-CAG-DIO-RG	Stanford Gene Vector and Virus Core	GVVC-AAV-59-DJ
AAVDJ-EF1α-DIO-TVA-mCherry	Stanford Gene Vector and Virus Core	GVVC-AAV-67-DJ
AAVDJ-EF1α-DIO-hM3Dq-mCherry	Stanford Gene Vector and Virus Core	GVVC-AAV-130
AAVDJ-EF1α-DIO-mCherry	Stanford Gene Vector and Virus Core	N/A
AAV-DJ-hSyn-DIO-Synaptophysin-mRuby	Stanford Gene Vector and Virus Core	GVVC-AAV-100
AAV-DJ-EF1α-DIO-YPet-2a-mGFP	Stanford Gene Vector and Virus Core	N/A
Chemicals, Peptides, and Recombinant Proteins
Clozapine N-oxide	ENZO Life Sciences	BML-NS105
Critical Commercial Assays
RNAscope^® Fluorescent Multiplex Kit User Manual	Advanced Cell Diagnostics	Document 320513 (part 1) and 320293 (part 2)
Experimental Models: Organisms/Strains
Mouse: Neuropeptide S-IRES-Cre	This paper	N/A
Mouse: Neuropeptide S Receptor-IRES-Cre	This paper	N/A
Mouse: B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J	The Jackson Laboratory	RRID:IMSR_JAX:007914
Mouse: B6.129-Gt(ROSA)26Sortm1(CAG-CHRM4*,-mCitrine)Ute/J	The Jackson Laboratory	RRID:IMSR_JAX:026219
Mouse: B6N;129-Tg(CAG-CHRM3*,-mCitrine)1Ute/J	The Jackson Laboratory	RRID:IMSR_JAX:026220
Oligonucleotides
RNAscope^™ Probe-Mouse-Nps	Advanced Cell Diagnostics	accession number NM_001163611.1, target region 5-846
RNAscope^™ Probe-Mouse-Npsr1	Advanced Cell Diagnostics	accession number NM_175678.3, target region 2022-3048
RNAscope^™ Probe-Cre	Advanced Cell Diagnostics	accession number KC845567.1, target region 1058-2032
Software and Algorithms
HALO software	Indica Labs	https://indicalab.com/halo/; RRID:SCR_018350
MATLAB R2022B	MathWorks	https://www.mathworks.com; RRID: SCR_001622
FIJI	ImageJ	https://imagej.net/; RRID:SCR_003070
GraphPad 9.3	Prism	www.graphpad.com; RRID:SCR_002798
SPSS for Windows V25.0	IBM	https://www.ibm.com/spss; RRID:SCR_016479
IOX2	emka Technologies	https://www.emkatech.com/product/iox2software/; RRID:SCR_022973
LabChart	ADInstruments	https://www.adinstruments.com/products/labchart/versions-and-licenses; RRID:SCR_023643
Photometry_EEG_Alignment MATLAB script	This paper; Figshare	https://figshare.com/s/307ed2bd819a44b8aa1d; DOI: 10.6084/m9.figshare.22299703

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EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Animals

Mice were housed in plexiglass chambers at constant temperature (22 ± 1 °C) and humidity (40–60 %) and kept on a standard 12h:12h light/dark cycle (lights on at 7:00 am and lights off at 7:00 pm). Food and water were available ad libitum. All mice were adults between 8 – 20 weeks old at the start of experiments. Subjectively, no differences in NPS expression, NPS+ inputs/outputs, or NPSR1 expression were observed between male and female mice. Statistically, with one exception, there were no main effects of sex, nor sex × drug (saline or CNO) interactions, nor sex × time bin interactions for any sleep/wake, Calcium activity, or respiratory behavior analyzed. For Wake to NREM transitions in the LPBA (Figure 5F), there was a significant sex × time bin interaction on Ca²⁺ activity for one time bin, encompassing the last 15 seconds of wake preceding the transition to NREM. Due to the overall lack of sex differences observed in our study, and in the NPS system reported to date, male and female mice were pooled randomly for behavioral testing and collapsed together for statistical analysis. All mice were group housed in groups of 3 – 5 same-sex littermates until adulthood (2 – 5 months old). For EEG/EMG and fiber photometry experiments, mice were singly-housed and then plugged in/tethered in preparation for experimentation. All mice were allowed to acclimate to the EEG/EMG tethers and/or fiber-optic patch cords, as well as the singly-housed homecage for at least 5 days prior to the commencement of experimentation. For all other experiments, mice remained group housed. All experiments were performed in accordance with the guidelines described in the US National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by Stanford University Administrative Panel on Laboratory Animal Care.

Generation of NPS-IRES-Cre (Nps^Cre) knock-in mice was outsourced to Cyagen Biosciences (Santa Clara, CA) using CRISPR-cas9 technology. The internal ribosomal entry sequence (IRES) linked to Cre recombinase was inserted immediately downstream of the TGA stop codon in exon 3 on the mouse NPS gene on chromosome 7. Cas9, gRNA, and the targeting vector was co-injected into fertilized eggs. Genomic fragments comprised of homology arms and the conditional knockout region were amplified from BAC clone via high fidelity Taq DNA polymerase. The targeting vector, recombination sites, and selection markers are shown in Figure S1A–C. Four pups (1 male, 3 females) were identified by PCR screening and used as founders of the Nps^Cre mouse line. Heterozygous male and female Cre-expressing mice were continuously backcrossed to C57BL/6J wild-type mice from the Jackson Laboratory for several generations (JAX-West; Sacramento, CA).

Npsr1^Cre knock-in mice were generated at the University of Washington. A cassette encoding IRES-mnCre:GFP was inserted just 3’ of the termination codon in the last coding exon of the Npsr1 gene. The 5′ arm (11 kb with PacI and SalI sites at 5’ and 3’ ends, respectively) and 3′ arm (~3.5 kb with XhoI and NotI sites at 5’ and 3’ ends, respectively) of the Npsr1 gene were amplified from a C57BL/6 BAC clone by PCR using Q5 Polymerase (New England Biolabs) and cloned into polylinkers of a targeting vector that contained IRES-mnCre:GFP, a frt-flanked Sv40Neo gene for positive selection, and HSV thymidine kinase and Pgk-diphtheria toxin A chain genes for negative selection. The IRES-mnCre:GFP cassette has an internal ribosome entry sequence (IRES), a Myc-tag, and nuclear localization signals at the N-terminus of Cre recombinase, which is fused to green fluorescent protein followed by a SV40 polyadenylation sequence. The construct was electroporated into G4 ES cells (C57Bl/6 × 129 Sv hybrid) and correct targeting was determined by Southern blot of DNA digested with BamH1 using a ³²P-labeled probe outside of the targeting construct. One of 36 clones analyzed was correctly targeted; it was injected into blastocysts and resulted in good chimeras that transmitted the targeted allele through the germline. The progeny were bred with Rosa26^FLPo mice to remove the frt-flanked SV-Neo gene. Mice were then continuously backcrossed to C57Bl/6 mice for several generations.

We used the following additional previously-generated Cre-dependent mouse lines: B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (Rosa26^LSL-tdTomato Ai14; JAX #007914), B6N;129-Tg(CAG-CHRM3*,-mCitrine)1Ute/J (Rosa26^LSL-hM3Dq; JAX #026220), B6.129-Gt(ROSA)26Sortm1(CAG-CHRM4*,-mCitrine)Ute/J (Rosa26^LSL-hM43Di; JAX #026219). Transgenic hM3Dq and hM3Di mouse lines also express mCitrine yellow fluorescent protein upon Cre-recombination and removal of upstream lox-stop-lox (LSL) cassette.

METHOD DETAILS

Surgery

Mice underwent stereotaxic surgery for viral infusion, fiber-optic implantation, and/or EEG/EMG implantation as previously described⁹⁶. Mice were anesthetized with ketamine and xylazine (100 and 10 mg/kg, respectively, intraperitoneal; i.p.) and placed on a stereotaxic frame (David Kopf Instruments, Tujunga, CA). Adeno-associated or Rabies virus constructs were infused through a stainless steel 28-gauge internal microinjector (Plastics One, Inc., Roanoke, VA) connected to a 10-μl Hamilton syringe. 300–400 nL of virus was infused and after injection, the cannula was held in place for 10 minutes, followed by a slow removal, pausing for 1 minute at every 0.8 mm interval. For fiber photometry experiments, AAV9-EF1α-DIO-gCaMP7s was mixed 1:1 with silk fibroin⁹⁷, dripped upon the 400-μm fiber-optic tip (0.48 NA; Doric Lenses) 30 μL at a time, and allowed to dry for 1–2 minutes between applications. We used the following stereotactic coordinates: DMT (−1.3 A/P, ±0.3 M/L, −3.4 D/V for virus), LPBA (−5.0, ±1.75 M/L, −4.3 DV for virus), peri-LC (−5.4 A/P, ±0.68 M/L, −4.4 D/V for virus). For EEG/EMG experiments, insulated mini-wire was stripped ~3 mm on each end. One end was soldered to a mini-screw (US Micro Screw) and the other end was soldered to a gold pin aligned in an electrode socket. Each electrode socket contains four channels with two mini-screw channels for EEG recording and two stripped mini-wires for EMG recording. The resistance of all the channels was evaluated with a digital multimeter (Fluke), and those lower than 1.5 ohms were utilized as ideal conductance. The two mini-screws were placed in the skull above the frontal [anteroposterior (AP), 1 mm; mediolateral (ML), ±1 mm] and temporal (AP, −2 mm; ML, ±1.5 mm) cortices for sampling EEG signals, and the two exposed mini-wires were placed in the neck muscles for EMG signal acquisition. Electrode sockets were secured with Metabond and dental acrylic on the skull for recording freely moving mice. After surgery, mice were administered buprenorphine-SR (0.5 mg/kg; 30 min before and 24 to 48 hours after surgery, subcutaneously) for pain relief.

Virus Preparation

Adeno-associated viruses (AAVs) carrying Cre-inducible (double inverse orientation; DIO) transgenes were purchased from the Gene Vector and Virus Core at Stanford University. Glycoprotein-deleted rabies virus for retrograde tracing (SAD ΔG EnvA-GFP, “RVdG-GFP”; 1.51 × 10⁸ or 9.59 × 10⁹) was purchased from Salk Institute Gene Transfer Targeting and Therapeutics Core. We used the following AAVs: AAV-DJ-EF1α-DIO-GCaMP7s (3 × 10¹³, 300 nL per hemisphere, Addgene, 104491), AAVDJ-CAG-DIO-RG (2.47 × 10¹², 200 nL per hemisphere, Stanford Vector Core), AAVDJ-EF1α-DIO-TVA-mCherry (4.81 × 10¹², 200 nL per hemisphere, Stanford Vector Core), AAVDJ-EF1α-DIO-hM3Dq-mCherry (4.83 × 10¹³, 300 nL per hemisphere, Stanford Vector Core), AAVDJ-EF1α-DIO-mCherry (7.30 × 10¹², 300 nL per hemisphere, Stanford Vector Core), AAV-DJ-hSyn-DIO-Synaptophysin-mRuby (6.56 × 10¹², 200 nL per hemisphere, Stanford Vector Core), AAV-DJ-EF1α-DIO-YPet-2a-mGFP (1.77 × 10¹², 200 nL per hemisphere, Stanford Vector Core).

Monosynaptic Modified-Rabies Viral Tracing

Modified-rabies viral tracing was performed as previously described^34,35,98. Briefly, 3 weeks following unilateral viral infusion of AAVDJ-DIO-TVA-mCherry and AAVDJ-DIO-RG into either the LPBA, peri-LC, or the DMT (1:1 viral mixture, 400 nL total volume), 300 nL of the RVdG-GFP modified rabies was injected into the same site. Mice were anesthetized and perfused, and brains were dissected and processed as described below. Starter cells were identified as neurons co-expressing TVA-mCherry and RVdG-GFP in the injection site. Input cells were defined as all RVdG-GFP-labeled cells that were not starter cells. For all brains with identified starter cells, we imaged coronal sections sliced at 30 μm and the entire brain was mounted from the rostral olfactory bulbs to the caudal brainstem. We counted the number of input cells and categorized them into brain subregions based on cytoarchitectural DAPI-assisted analysis, according to major delineations made by the Allen Institute and Paxinos & Watson mouse brain atlases. After generating the total sum of all input cells from each brain, we converted the number of input cells from each region into the percentage of total input cells. Graphs in Figure 2 are comprised of an average of 2114 GFP+ cells per animal for the LPBA (n=2, 1 male and 1 female), 1303 GFP+ cells per animal for the peri-LC (n=2, 1 male and 1 female), and a total of 246 GFP+ cells for the habenula/PVT/DMT (n=4, 3 males and 1 female), expressed as a percentage.

HALO quantification of NPSR1 expression

Images were obtained on an Olympus Fluoview 3000 confocal microscope (Evident Scientific, Waltham, MA) and analyzed with HALO software (V. 3.2, Indica Labs, Albuquerque, NM). Each image was opened in the HALO software and boundaries for brain regions were set. DAPI-positive cells were then registered and used as markers for individual cells. The area of Npsr1 expression was analyzed via an area quantification module (HALO, Indica Labs) and expressed as a percentage (the area of positive tissue / total area of the tissue of interest), as previously described^99,100. An observer blind to experimental conditions then set thresholds for each channel, determining the minimum intensity of fluorescence necessary for a probe to be included and counted. These thresholds were validated by manual spot-check throughout the image to ensure fluorescence was appropriately detected. For each marker identified, the input parameter and HALO software allows for the semi-quantitative specification of thresholds that define negativity and three levels of positivity (weak, moderate, and strong). Weak intensity threshold– pixels with intensity values below this threshold are considered negative for the stain. Pixels with intensities greater than or equal to this threshold, but also less than the moderate intensity threshold, are considered weakly positive. Moderate intensity threshold– pixels with intensity values greater than or equal to the moderate intensity threshold, but also less than the strong intensity threshold, are considered moderately positive. Strong intensity threshold – pixels with intensity values greater than or equal to the strong intensity threshold are considered strongly positive. For our purposes, tissue marked positive for fluorescence detection were conservatively determined by the percentage having fluorescence intensity equal to or greater than the minimum strong intensity threshold (Figure 4). HALO software reported the area analyzed (μm²), fluorescence area (μm²; weak, moderate, strong), fluorescence % of tissue (%; weak, moderate, strong). One-two separate slices from each brain region were used for each animal (n=3, 1 male and 2 females), and the fluorescence % was aggregated and plotted (Figure 4I).

RNAscope In Situ Hybridization and HALO Analysis

Following rapid decapitation of Nps^Cre mice, brains were rapidly frozen in 100 mL −50°C isopentane and stored at −80°C. Coronal sections corresponding to the site of interest were cut at 16 μM at −20°C and thaw-mounted onto SuperFrost Plus slides (Fisher Scientific, Waltham, MA). Slides were stored at −80°C until further processing. Fluorescent in situ hybridization was performed according to the RNAscope 2.0 Fluorescent Multiple Kit User Manual for Fresh Frozen Tissue (Advanced Cell Diagnostics, Newark, CA), as described by Wang et al.¹⁰¹. Briefly, sections were fixed in 4% PFA, dehydrated, and treated with pretreatment 3 protease solution. Sections were then incubated for target probes for mouse NPS (ACD, accession number NM_001163611.1, target region 5–846), NPSR1 (accession number NM_175678.3, target region 2022–3048), and/or Cre (accession number KC845567.1, target region 1058–2032). All targets consisted of 20 ZZ oligonucleotides and were obtained from Advanced Cell Diagnostics (ACD, Newark, CA). Following probe hybridization, sections underwent a series of probe signal amplification steps followed by incubation of fluorescently labeled probes designed to target the specific channel associated with the probes¹⁰¹. Slides were counterstained with DAPI, and coverslips were mounted with Vectashield HardSet mounting medium (Vector Laboratories, Newark, CA). Images were obtained on an Olympus Fluoview 3000 confocal microscope (Evident Scientific, Waltham, MA) and analyzed with HALO software (V. 3.2, Indica Labs, Albuquerque, NM). To analyze the images, each image was opened in the HALO software and boundaries were set for the area to be analyzed. DAPI positive cells were then registered and used as markers for individual cells. The maximum area around each cell for probes to be detected was then set, approximately 3 microns. An observer blind to the origin of the brain tissue and identity of the probes then set thresholds for each channel, determining the minimum intensity of fluorescence necessary for a probe to be included and counted. These thresholds were validated via manual spot check to ensure that cells and probes were being appropriately counted. A positive cell consisted of an area within the radius of a DAPI nuclear staining that measured at least 3 positive pixels for receptor probes, or 10 total positive pixels for neuropeptide probes. The HALO software reported the total counts of cells and levels of overlap, which are reported in the data, as previously described¹⁰⁰. Two - three separate slices from the PVT and LPBA (Nps^Cre, n=1) were used and the total cell count is presented in the data (Figure S3M–Q).

Synaptophysin-mRuby Anterograde Viral Tracing

AAV-DJ-EF1α-DIO-YPet-2a-mGFP and AAV-DJ-hSyn-DIO-Synaptophysin-mRuby were mixed (1:1 viral mixture, 400nL total volume) and injected into the LPBA, peri-LC/NI/PCG, and DMT/PVT of Nps^Cre mice. 3 weeks later, mice were anesthetized and perfused, and brains were dissected and processed as described below. Co-labeling (yellow) of YPet-2a-mGFP and synaptophysin-mRuby at the injection sites indicated successful co-expression of the viral mix. For brains with successful co-labeling, we imaged coronal sections sliced at 30 μm. Every 4^th brain section was mounted from the rostral olfactory bulbs to the caudal brainstem, and regions with NPS terminal outputs were defined by expression of red mRuby puncta. NPS efferent targets were classified into brain subregions based on cytoarchitectural DAPI-assisted analysis, according to major delineations made by the Allen Institute and Paxinos & Watson mouse brain atlases.

Histology, Immunostaining, Microscopy, and Image Analysis

Mice were anesthetized with ketamine and xylazine (100 and 10 mg/kg, respectively, i.p.) and transcardially perfused with 8 ml 1x phosphate buffered saline (PBS), followed by 8 ml paraformaldehyde (PFA; 4%, in PBS). Brains were rapidly extracted, postfixed overnight (12 – 18 hr) in 4% PFA at 4°C, and cryoprotected for at least 48 – 96 h at 4°C in sucrose solution (30% sucrose in PBS containing 0.1% NaN3) until sunk. Brains were sliced in 30 μm coronal sections at −21°C on a cryostat (Leica Microsystems), collected consecutively in 24-well plates containing PBS with 0.1% NaN3, covered in light-protective material, and stored at 4°C until imaging and/or immunohistochemical processing. We used the following antibodies: chicken anti-Tyrosine Hydroxylase (1:1000, Aves Labs, TYH), rabbit anti-GFP (1:250, NovisBio, NB600–308), chicken anti-GFP (1:250, Abcam, ab13970), goat anti-chicken Alexa 488 (1:500, Invitrogen, A-11039), donkey anti-rabbit Alexa 488 (1:500, Invitrogen, A-21206). Sections were washed in PBS for 10 min and then incubated for 2 hr in a blocking solution of PBS with 0.3% Triton X-100 (PBST) containing 4% bovine serum albumin (BSA). Next, we incubated slices in primary antibodies overnight for 12 – 18 hr, in 4% BSA/PBST block solution. After three 5-min PBS washes, sections were again incubated in blocking solution for 1 hr, and then incubated for an additional 2 hr in blocking solution containing secondary antibodies. After three final 5-min PBS washes, sections were mounted onto gelatin-coated glass slides (FD Neurotechnologies, Inc.; PO101), and coverslipped with Fluoroshield containing DAPI Mounting Media (Sigma; F6057). Images were collected on a Zeiss LSM 710 confocal microscope (Hebron, KY) using ZEN software, and minimally processed using ImageJ (NIH) to enhance brightness and contrast for optimal representation.

EEG/EMG recordings

Mice were allowed to recover for at least 1 week following completion of polysomnography surgery. Mice were singly-housed and connected to a flexible cable at least 5 days prior to EEG/EMG recording. EEG/EMG signals were amplified through a multi-channel amplifier (Grass Instruments) and collected by VitalRecorder (Kissei Comtec Co.) at a 256 Hz sampling rate and filtered at 0.5 – 30 Hz and 1 – 100 Hz, respectively, for offline sleep scoring. EEG/EMG data was analyzed in 4 s epochs as wake, non-rapid eye movement (NREM) sleep, or rapid eye movement (REM) sleep by a trained experimenter blind to experimental conditions. Wake was classified by increases in higher frequency waves (>10 Hz), decreases in amplitude of the EEG, and high activity in the EMG. NREM was classified by increases in delta power (0.5 – 4 Hz) and amplitude of the EEG, along with low amplitude activity in the EMG. REM was determined by high EEG activity in the theta range (4 – 8 Hz) and very low activity in the EMG. Wake and NREM bouts were identified as 30 s (8 epochs) or greater of consecutive wake or NREM, respectively, while bouts of REM were identified as 20 s (5 epochs) or greater of consecutive REM, as described previously¹⁰². Bouts of NREM and REM were considered broken once interrupted by 8 epochs of any other state, while bouts of wake were terminated by a single epoch of any other state. For EEG spectral analysis, fast Fourier transform (FFT; Hanning window; 0.5 – 20 Hz, 0.25 Hz resolution) was performed across the total 3-hour trace (Figure S2D–E,I–J,N–O and Figure S5D–E) or the 10-minute representative recording periods on artifact-free epochs (Figure 5C,J,Q). For the 10-minute representative traces (Figure 5), spectrograms were generated using the standard MATLAB spectrogram function.

General Procedures

Following recovery from fiber optic implantation and GCaMP7s viral injection (7 – 10 days), mice were singly-housed, habituated to EEG/EMG and/or fiber-optic patch cables, and allowed to move freely with the tether in their homecages for an additional 5 – 7 days before data collection. For plethysmography behavioral studies, mice were habituated to experimenter handling in the days and weeks prior to testing. For all pharmacological and chemogenetic studies, mice were fully habituated to intraperitoneal (i.p.) saline control injections for at least 5 days, at the same circadian time, prior to testing.

Fiber Photometry

Fiber photometry recordings were performed similarly to previously described⁹⁶. Separate Nps^Cre adult mice were implanted with a fiber-optic cannula and AAV-DJ-EF1α-DIO-GCaMP7s was infused into either the LPBA, peri-LC, or DMT/PVT (n = 6 per region). Calcium recordings were performed on one of two photometers. Briefly, we sinusoidally modulated blue light from a 470 nm excitation LED (M470F3, Thorlabs, NJ, USA) at 256 Hz using a custom MATLAB program (MathWorks, Natick, MA, USA) and a multifunction data acquisition device (NI USB-6259, National Instruments, Austin, TX, USA). Blue light was transmitted through a GFP excitation filter (MF469–35, Thorlabs), reflected off a dichroic mirror (MD498, Thorlabs), and coupled using a fiber collimation package (F240FC-A, Thorlabs) into a low-fluorescence patch cord (2 m, 0.48 NA, 400 μm core; Doric Lenses, Quebec, Canada) connected to the fiber-optic implant via a zirconia sleeve (Doric Lenses). GCaMP7s fluorescence was collected through the patch cord, passed through a GFP emission filter (MF525–39, Thorlabs), and focused onto a photodetector (Model 2151, Newport, Irvine, CA, USA) using a lens (LA1540-A, Thorlabs). The signal was sent to a lock-in amplifier (30 ms time constant, Model SR830, Stanford Research Systems, Sunnyvale, CA, USA) that was synchronized to 256 Hz. Signals from the amplifiers were collected at 1 KHz using a custom MATLAB program and a multifunction data acquisition device (National Instruments). Some animals were collected using a Neurophotometrics FP3002 system (Neurophotometrics, San Diego, CA, USA). For these, photometry signals were sampled at 32Hz and smoothed with a moving average window read (156 ms).

Fiber photometry analysis

Fiber photometry analysis was performed in MATLAB R2022b using custom scripts. Data collected at 256 Hz was downsampled to 32 Hz and all data was analyzed together. Raw GCaMP and the simultaneously collected isosbestic 415nm UV signals were filtered using the MATLAB built-in finite impulse response (FIR) filter function, fir1, with a Hamming window bandpass of 0.1 to 2 Hz. The filtered GCaMP and isosbestic UV data were then fitted to a polynomial fit and GCaMP signal was normalized to the UV to isolate Ca²⁺-dependent activity. Due to variable GCaMP expression in individual animals, the photometry signal was converted to a Z-score for each animal using the formula: z = (x-μ)/σ, where z is the Z-score, x is the raw score, μ is the within-recording population mean, and σ is the within-recording population standard deviation. Photometry signal expressed in Z-scores were temporally aligned to scored EEG/EMG for sleep-state specific analysis.

Transient detection was performed similarly to previously described^37,38. Briefly, a 4 Hz low pass filter was applied to the dataset and the derivative of the squared differences between the original signal and low pass filtered signal was calculated. Transients were detected and included if both the original Z-scored data and the derivative computed were >1.5 standard deviations above the Z-score mean. Transient detection was done on rolling 30-minute time bins to account for photobleaching. Transients were aligned to scored EEG/EMG data and mean transient amplitude, within-bout instantaneous transient frequency, and transient rate per second were calculated for wake, NREM, and REM sleep in each brain region (Figure S4).

Chemogenetic excitation and inhibition

Designer receptor ligand clozapine-N-oxide (CNO) (Enzo Life Sciences, Farmingdale, NY) was dissolved in 100% sterile saline at a concentration of 1.0 mg/kg and administered intraperitoneally (i.p.). This dose was chosen based upon prior experiments showing efficacy and negligible off-target effects on sleep and wake³⁸. For all chemogenetic experiments, mice received saline injections i.p. for at least 5 days prior to experimentation. CNO injections for EEG/EMG experiments were performed at ZT2 (light cycle) and ZT14 (dark cycle). All mice received both saline vehicle and 1.0mg/kg CNO injections at both circadian time points, and comparisons were made within-subject. Experiments were counterbalanced: some mice received saline injections first, while others received CNO injections first, with at least 5 days of washout allowed prior to experimental recordings, and daily saline injections between experimental days.

Plethysmography

Individual mice were placed in a 450 mL whole body plethysmography chamber (VivoFlow, Scireq, Montreal, QC, Canada) at room temperature (22°C) and supplied with room air, as previously described¹⁰³. Mice were allowed to acclimate to the plethysmography recording room for 3 days, and the plethysmography recording chambers for 4 hours on the day prior to plethysmography data collection. Each plethysmography chamber was calibrated before each recording session. Ventilation pump flow was provided at 0.6 L/min – 0.8 L/min and scaled input ranges between ±1000 mL/s and ±1875 mL/s following a calibration injection of 10mL air were deemed suitable for experimentation. We recorded all groups of mice during the light cycle between ZT2 and ZT11, with experimental and control mice randomly intermixed. Respiratory parameters were determined using EMKA iox2 software. Breathing data was acquired by iox software (emka Technologies, Sterling, VA, USA) and analyzed by LabChart software (AD Instruments, Sydney, Australia). Mice were analyzed for a 2-hour baseline recording period where their breathing was allowed to stabilize. Importantly, only “quiet wake” periods were utilized for analysis, encompassing the range of breathing events occurring during sleep or quiet/relaxed arousal, while omitting more “active” behaviors including grooming and running, which significantly alter respiration rate independent of experimental manipulation. This threshold was determined individually for each animal from baseline breathing analyses (however, the threshold for all animals was set at breathing rates ≤200 – 210 breaths per minute). Following the 2 hr baseline, all mice received either saline or 1.0 mg/kg CNO injections i.p., on separate days. Respiratory parameters were recorded for an additional 2 hours following saline or CNO injection, with all quiet wake periods > 30min after injection (time of CNO onset) included for analysis. Data is expressed and analyzed as a within-day ratio (CNO or saline post-injection period normalized to same-day 2-hour baseline recording), to eliminate possible temporal differences arising from equipment or environmental differences between saline and CNO recording days. One peri-LC mCherry-infused Nps^Cre outlier mouse was excluded from plethysmography analyses due to outputs >2.5 standard deviations outside of the group mean for several respiratory outputs.

QUANTIFICATION AND STATISTICAL ANALYSIS

All data are presented as mean ± standard error of the mean (s.e.m.) and statistical details for all experiments are presented in the main and supplemental figure legends. All statistical analyses were performed using SPSS for Windows (V. 25.0) or MATLAB (V. R2022b), and p-values < α = 0.05 were deemed statistically significant. Our sample sizes are comparable to those reported in previous publications using chemogenetic tools, sleep behavioral recording, fiber photometry, and plethysmography^37,96,103, and no statistical methods were used to pre-determine sample sizes. Student’s paired T-tests were used to compare total sleep time and sleep state bout counts, duration, and transitions. For time-binned sleep state analysis (half hour intervals over 3h recording) and FFT spectral analysis, mixed design repeated-measures ANOVAs were used with experimental condition (saline or CNO) as the between-subjects factor and time (or in the case of FFT analysis, frequency bin) as the within-subjects factor. For plethysmography respiratory factors, mixed design repeated-measures ANOVAs were used with experimental condition (AAV-hM3Dq-mCherry or AAV-mCherry) as the between-subjects factor and day (saline or CNO) as the within-subjects factor. One-way ANOVA was performed for analyses of GCaMP transients across wake, NREM, and REM sleep states. In instances where the assumption of sphericity was violated, Greenhouse-Geisser corrected F values are used. We ran a linear mixed effects model to compare GCaMP Z-scores in 15 s bins surrounding state transitions (30 s before, and 60 s after state transition), accounting for the possible lack of independence between time bins. Time bin was set as a fixed effect and mouse/subject was set as a random effect. The linear fixed effect model was analyzed using the fitlme function in MATLAB under the formula: Z-score ~ Time_bin + (1 | Mouse_ID). Mann-Whitney U-tests were performed on normalized FFT data binned into low delta, high delta, theta, alpha, and beta frequency bins. Post hoc multiple comparisons were performed using Bonferroni’s adjustment for multiple comparisons for repeated-measures ANOVAs and linear mixed effect models, and Tukey’s post hoc test was used for one-way ANOVAs. We plotted data using Prism 9.3 (GraphPad Software).

Supplementary Material

NIHMS1950328-supplement-1.pdf^{(2.5MB, pdf)}

Table S1. Summary of the expression, inputs, and projections of the Neuropeptide S system. Related to Figure 2, 3, and 4.

NPS+ Reporter (Column 3): + indicates sparse NPS+ cell bodies, +++ indicates large clusters of NPS+ cells, “fibers” indicates the presence of NPS+ fibers, but not cell bodies. LPBA Inputs (Column 4): + indicates 1% - 3% of total inputs to NPS+ cells of the LPBA, ++ indicates 3% - 10% of total inputs to NPS+ cells of the LPBA, +++ indicates >10% of total inputs to NPS+ cells of the LPBA. Peri-LC Inputs (Column 5): + indicates 1% - 3% of total inputs to NPS+ cells of the peri-LC, ++ indicates 3% - 5% of total inputs to NPS+ cells of the peri-LC, +++ indicates >5% of total inputs to NPS+ cells of the peri-LC. DMT/habenula Inputs (Column 6): + indicates 1% - 3% of total inputs to NPS+ cells of the DMT/habenula, ++ indicates 3% - 10% of total inputs to NPS+ cells of the DMT/habenula, +++ indicates >10% of total inputs to NPS+ cells of the DMT/habenula. LPBA Outputs (Column 7): + indicates sparse / few synaptophysin-mRuby puncta detected, +++ indicates substantial / more synaptophysin-mRuby puncta detected. Peri-LC Outputs (Column 8): + indicates sparse / few synaptophysin-mRuby puncta detected, +++ indicates substantial / more synaptophysin-mRuby puncta detected. NPSR1+ Reporter (Column 9): + indicates 1% - 20% of total tissue area expresses NPSR1, ++ indicates 20% - 50% of total tissue area expresses NPSR1, +++ indicates >50% of total tissue area expresses NPSR1. Organized from rostral to caudal.

NIHMS1950328-supplement-2.xlsx^{(13.4KB, xlsx)}

Highlights.

Mapping of the NPS system reveals connections with regions critical for behavior
The NPS system augments arousal, suppresses REM sleep, and regulates respiration
In vivo Calcium recordings reveal that NPS+ activity tracks with vigilance state
Selective activation of LPBA NPS+ neurons recapitulates NPS system phenotypes

Acknowledgements

We acknowledge the helpful support of all members of the de Lecea lab. This work was funded by National Institutes of Health grants R01 MH116470 (L.d.L.), R01 MH128140 (L.d.L.), F32 HL154792 (C.C.A.), T32 HL 1109 (C.C.A.), R01 MH112355 (M.R.B), F32DA055480 (K.S.G), R56 MH114313 (S.D.C), and HHMI (R.D.P.). We also recognize support from Dr. Andrew Olson and the Stanford Neuroscience Microscopy Service, NIH NS069375.

Footnotes

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Declaration of interests: The authors declare no competing interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS1950328-supplement-1.pdf^{(2.5MB, pdf)}

Table S1. Summary of the expression, inputs, and projections of the Neuropeptide S system. Related to Figure 2, 3, and 4.

NIHMS1950328-supplement-2.xlsx^{(13.4KB, xlsx)}

Data Availability Statement

All data reported in this paper will be shared by the lead contact upon request.
All original code has been deposited at figshare and is publicly available as of the date of publication. DOIs are listed in the key resources table.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
chicken anti-Tyrosine Hydroxylase	Aves Labs	TYH; RRID:AB_10013440
rabbit anti-GFP	NovisBio	NB600-308; RRID:AB_10005904
chicken anti-GFP	Abcam	ab13970; RRID:AB_300798
goat anti-chicken Alexa 488	Invitrogen	A-11039; RRID:AB_142924
donkey anti-rabbit Alexa 488	Invitrogen	A-21206; RRID:AB_2535792
Bacterial and Virus Strains
AAV-DJ-EF1α-DIO-GCaMP7s	Addgene	Addgene AAV9; 104491-AAV9
SAD ΔG EnvA-GFP	Salk Institute Gene Targeting and Therapeutics Core	Addgene Plasmid #32635
AAVDJ-CAG-DIO-RG	Stanford Gene Vector and Virus Core	GVVC-AAV-59-DJ
AAVDJ-EF1α-DIO-TVA-mCherry	Stanford Gene Vector and Virus Core	GVVC-AAV-67-DJ
AAVDJ-EF1α-DIO-hM3Dq-mCherry	Stanford Gene Vector and Virus Core	GVVC-AAV-130
AAVDJ-EF1α-DIO-mCherry	Stanford Gene Vector and Virus Core	N/A
AAV-DJ-hSyn-DIO-Synaptophysin-mRuby	Stanford Gene Vector and Virus Core	GVVC-AAV-100
AAV-DJ-EF1α-DIO-YPet-2a-mGFP	Stanford Gene Vector and Virus Core	N/A
Chemicals, Peptides, and Recombinant Proteins
Clozapine N-oxide	ENZO Life Sciences	BML-NS105
Critical Commercial Assays
RNAscope^® Fluorescent Multiplex Kit User Manual	Advanced Cell Diagnostics	Document 320513 (part 1) and 320293 (part 2)
Experimental Models: Organisms/Strains
Mouse: Neuropeptide S-IRES-Cre	This paper	N/A
Mouse: Neuropeptide S Receptor-IRES-Cre	This paper	N/A
Mouse: B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J	The Jackson Laboratory	RRID:IMSR_JAX:007914
Mouse: B6.129-Gt(ROSA)26Sortm1(CAG-CHRM4*,-mCitrine)Ute/J	The Jackson Laboratory	RRID:IMSR_JAX:026219
Mouse: B6N;129-Tg(CAG-CHRM3*,-mCitrine)1Ute/J	The Jackson Laboratory	RRID:IMSR_JAX:026220
Oligonucleotides
RNAscope^™ Probe-Mouse-Nps	Advanced Cell Diagnostics	accession number NM_001163611.1, target region 5-846
RNAscope^™ Probe-Mouse-Npsr1	Advanced Cell Diagnostics	accession number NM_175678.3, target region 2022-3048
RNAscope^™ Probe-Cre	Advanced Cell Diagnostics	accession number KC845567.1, target region 1058-2032
Software and Algorithms
HALO software	Indica Labs	https://indicalab.com/halo/; RRID:SCR_018350
MATLAB R2022B	MathWorks	https://www.mathworks.com; RRID: SCR_001622
FIJI	ImageJ	https://imagej.net/; RRID:SCR_003070
GraphPad 9.3	Prism	www.graphpad.com; RRID:SCR_002798
SPSS for Windows V25.0	IBM	https://www.ibm.com/spss; RRID:SCR_016479
IOX2	emka Technologies	https://www.emkatech.com/product/iox2software/; RRID:SCR_022973
LabChart	ADInstruments	https://www.adinstruments.com/products/labchart/versions-and-licenses; RRID:SCR_023643
Photometry_EEG_Alignment MATLAB script	This paper; Figshare	https://figshare.com/s/307ed2bd819a44b8aa1d; DOI: 10.6084/m9.figshare.22299703

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PERMALINK

A cluster of neuropeptide S neurons regulates breathing and arousal

Christopher Caleb Angelakos

Kasey S Girven

Yin Liu

Oscar C Gonzalez

Keith R Murphy

Kim J Jennings

William J Giardino

Larry S Zweifel

Azra Suko

Richard D Palmiter

Stewart D Clark

Mark A Krasnow

Michael R Bruchas

Luis de Lecea

Summary

eTOC Blurb

Introduction

Results

Chemogenetic manipulation of NPS system reveals a role in arousal and REM sleep suppression

Figure 1. Chemogenetic manipulation of NPS system reveals a role in arousal and REM sleep suppression.

NpsCre is expressed in distinct neuronal clusters with broad afferent and efferent projections

Figure 2. NpsCre is expressed in distinct neuronal clusters with broad afferent inputs.

Figure 3. NPS+ outputs and summary of NPS+ tracing studies.

Npsr1Cre is expressed in areas critical for sensory processing, behavior, and arousal

Figure 4. Npsr1Cre is expressed in areas critical for sensory processing, behavior, and arousal.

NPS-expressing cells increase activity preceding transitions to wake and suppress activity during REM sleep

Figure 5. NPS-expressing cells increase activity preceding transitions to wake and suppress activity during REM sleep.

Lateral parabrachial area-restricted NPS+ activation augments wake and suppresses REM sleep

Figure 6. Lateral parabrachial area-restricted NPS+ neuronal activation augments wake, suppresses REM sleep, and enhances respiratory rate.

Lateral parabrachial area-restricted NPS+ activation enhances respiratory rate

Peri-locus coeruleus-restricted NPS+ manipulation does not impact arousal or breathing

Discussion

STAR Methods

RESOURCE AVAILABILITY

Lead contact

Materials availability

Data and code availability

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Animals

METHOD DETAILS

Surgery

Virus Preparation

Monosynaptic Modified-Rabies Viral Tracing

HALO quantification of NPSR1 expression

RNAscope In Situ Hybridization and HALO Analysis

Synaptophysin-mRuby Anterograde Viral Tracing

Histology, Immunostaining, Microscopy, and Image Analysis

EEG/EMG recordings

General Procedures

Fiber Photometry

Fiber photometry analysis

Chemogenetic excitation and inhibition

Plethysmography

QUANTIFICATION AND STATISTICAL ANALYSIS

Supplementary Material

Highlights.

Acknowledgements

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Nps^Cre is expressed in distinct neuronal clusters with broad afferent and efferent projections

Figure 2. Nps^Cre is expressed in distinct neuronal clusters with broad afferent inputs.

Npsr1^Cre is expressed in areas critical for sensory processing, behavior, and arousal

Figure 4. Npsr1^Cre is expressed in areas critical for sensory processing, behavior, and arousal.