Efferent projections of Nps-expressing neurons in the parabrachial region

Abstract

In the brain, connectivity determines function. Neurons in the parabrachial nucleus (PB) relay diverse information to widespread brain regions, but the connections and functions of PB neurons that express Nps (neuropeptide S) remain mysterious. Here, we use Cre-dependent anterograde tracing and whole-brain analysis to map their output connections. While many other PB neurons project ascending axons through the central tegmental tract, NPS axons reach the forebrain via distinct periventricular and ventral pathways. Along the periventricular pathway, NPS axons target the tectal longitudinal column and periaqueductal gray then continue rostrally to target the paraventricular nucleus of the thalamus. Along the ventral pathway, NPS axons blanket much of the hypothalamus but avoid the ventromedial and mammillary nuclei. They also project prominently to the ventral bed nucleus of the stria terminalis, A13 cell group, and magnocellular subparafasciular nucleus. In the hindbrain, NPS axons have fewer descending projections, targeting primarily the superior salivatory nucleus, nucleus of the lateral lemniscus, and periolivary region. Combined with what is known about NPS and its receptor, the output pattern of Nps-expressing neurons in the PB region predicts a role in threat response and circadian behavior.

Introduction

The parabrachial nucleus (PB) is a region of the brainstem that links sensory information to a wide variety of brain regions (Cechetto et al., 1985; Herbert et al., 1990; Saper & Loewy, 1980). Glutamatergic neurons in this region belong to two mutually exclusive macropopulations defined by the transcription factors Atoh1 and Lmx1b (Karthik et al., 2022). These two macropopulations have distinct projection pathways: Lmx1b neurons project axons to the amygdala and cerebral cortex, primarily via the central tegmental tract, while Atoh1 neurons project axons to the hypothalamus and other targets, primarily via a ventral pathway (Karthik et al., 2022). Subpopulations of neurons within these two macropopulations have distinct connections (Huang et al., 2020; Huang et al., 2021; Pauli et al., 2022) and functions, which include thermoregulation, pain, itch, sodium appetite, and conditioned taste aversion (Carter et al., 2015; Gasparini et al., 2019; Geerling et al., 2016; Mu et al., 2017; Nakamura & Morrison, 2008, 2010; Shin et al., 2011).

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). 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).

Little is known about the neurons that produce NPS, but the roles of this peptide and its receptor (NPSR1) have been explored in several genetic and pharmacologic studies. Injecting NPS into the rodent brain increased locomotion, arousal, and body temperature, as well as reducing food intake and producing analgesia (Ensho et al., 2017; Leonard et al., 2008; Li et al., 2009; Peng, Han, et al., 2010; Peng, Zhang, et al., 2010; Rizzi et al., 2008; Smith et al., 2006; Xu et al., 2004). The NPS receptor (NPSR1) is required for its arousal-promoting effect (Duangdao et al., 2009; Fendt et al., 2011; Ruzza et al., 2012; Zhu et al., 2010), and human genetic studies have linked NPSR1 gain-of-function variants to sleep deprivation, anxiety, asthma, and panic disorders (Domschke et al., 2011; Donner et al., 2010; Gottlieb et al., 2007; Laitinen et al., 2004; Xing et al., 2019).

The connections of NPS neurons in the PB region remain unclear, and mapping their axonal projections would help predict possible functions. Previous studies immunolabeled NPS and identified immunoreactive fibers in the septum, hypothalamus, and midline thalamus (Adori et al., 2016; Clark et al., 2011). However, immunolabeling intensity varied with arousal state (Adori et al., 2016), and it remains unclear which population of NPS neurons projects immunolabeled fibers to each target.

In light of these uncertainties, and given the diverse projection patterns of PB neurons, a comprehensive map of axonal projections from NPS neurons is needed. This information is necessary to elucidate the role of NPS neurons. The goal of this study is to analyze the projection pattern of NPS neurons in the PB region as a framework for future studies exploring their function. To accomplish this goal, we combined Cre-conditional axonal tracing with presynaptic labeling (Carter et al., 2013; Opland et al., 2013) and semi-automated bouton plotting (Grady et al., 2022). Based on their genetic identity as Atoh1-derived neurons, we hypothesized that NPS neurons project axons primarily to the hypothalamus, via a ventral pathway and not the central tegmental tract (Huang et al., 2022). Our results support this hypothesis and reveal that NPS neurons also project axons via a robust periventircular pathway and target a novel pattern of output sites.

Materials and Methods

Mice.

All mice were group-housed in a temperature- and humidity-controlled room on a 12/12-hour light/dark cycle with ad libitum access to water and standard rodent chow. Overall, we used n=7 Nps-2A-Cre, n=4 Nps-2A-Cre;R26-LSL-L10GFP, and n=4 C57BL6/J mice ranging in age from 8 to 10 weeks (20–40 g body weight, male and female). Detailed information about each knockin strain is provided in Table 1. All mice were hemizygous and maintained on a C57BL6/J background. All experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at the University of Iowa.

Table 1.

Cre driver and reporter mice used in this study.

Strain	Reference	Source information	Key gene
Nps-2A-Cre	Huang, D., Zhang, R., Gasparini, S., McDonough, M. C., Paradee, W. J., & Geerling, J. C. (2022). Neuropeptide S (NPS) neurons: Parabrachial identity and novel distributions. J Comp Neurol, 530(18), 3157–3178. https://doi.org/10.1002/cne.25400	Available from originating investigators	2A-Cre inserted immediately after terminal exon in endogenous neuropeptide S gene
R26-LSL-L10GFP reporter	Krashes, Michael J., et al. “An excitatory paraventricular nucleus to AgRP neuron circuit that drives hunger.” Nature 507.7491 (2014): 238.	Available from originating investigators http://www.informatics.jax.org/allele/MGI:5559562	Floxed transcription STOP cassette followed by EGFP/Rpl10 fusion reporter gene under control of the CAG promoter targeted to the Gt(ROSA)26Sor locus

Stereotaxic injections.

Mice were anesthetized with isoflurane (0.5–2.0%) and placed in a stereotactic frame (Kopf 1900 or 940). We made a midline incision and retracted the skin to expose the skull. Through a pulled glass micropipette (20–30 μm inner diameter), we injected 50–100 nL of AAV8-hEf1a-DIO-synatophysin-mCherry (AAV8-DIO-Syp-mCherry, x 2.5 10¹³ pfu/mL; purchased from Dr. Rachel Neve at the Massachusetts Institute of Technology McGovern Institute for Brain Research Viral Vector Core). In Nps-2A-Cre mice used for formal analysis in the coronal plane, we used separate coordinates to target the rostral or caudal cluster of NPS neurons unilaterally in each case. Rostral coordinates were: 1.90 mm right of midline, 4.80 mm caudal to bregma, and 3.90 mm deep to bregma. Caudal coordinates were: 1.05 mm right of midline, 5.35 mm caudal to bregma, and 4.00 mm deep to bregma. In Nps-2A-Cre;R26-LSL-L10GFP mice used for histology in the sagittal plane, we targeted the rostral PB bilaterally, using the following coordinates: left and right 1.80 mm lateral to midline, 4.80 mm caudal to bregma, and 3.90 mm deep to bregma. Each injection was made over a 5-minute period, using picoliter air puffs through a solenoid valve (Clippard EV 24V DC) pulsed by a Grass stimulator. The pipette was left in place for an additional 3 minutes, then withdrawn slowly before closing the skin with Vetbond (3M). Carprofen (5 mg/kg) was provided prior to incision and again 24 hours later for postoperative analgesia. AAV-injected mice were allowed to survive for 3–5 weeks after surgery to allow for production of Cre-conditional proteins.

Perfusion and tissue sections.

Mice were anesthetized with ketamine (150 mg/kg) and xylazine (15 mg/kg, dissolved with ketamine in sterile 0.9% saline and injected i.p.). They were then perfused transcardially with phosphate-buffered saline (PBS, prepared from 10X stock; P7059, Sigma), followed by 10% formalin-PBS (SF100-20, Fischer Scientific). After perfusion, we removed each brain and fixed them overnight in 10% formalin-PBS. We sectioned each brain into 40 μm-thick coronal slices using a freezing microtome and collected tissue sections into separate, 1-in-3 series. Sections were stored in cryoprotectant solution at −30 °C until further processing.

Immunohistology.

For brightfield labeling (immunohistochemistry), we removed tissue sections from cryoprotectant and rinsed them in PBS. To quench endogenous peroxidase activity, we incubated all tissue sections for 30 minutes in 0.3% hydrogen peroxide (#H325-100, Fisher) in a PBS solution containing 0.25% Triton X-100 (BP151-500, Fisher). After 3 washes in PBS, we loaded sections into a primary antibody solution prepared in PBT-NDS-azide, which is a PBS solution containing 0.25% Triton X-100, 2% normal donkey serum (NDS, 017-000-121, Jackson ImmunoResearch), and 0.05% sodium azide as a preservative (14314, Alfa Aesar). This PBT-NDS-azide solution contained either rabbit anti-dsRed or rat anti-mCherry, and we incubated tissue sections overnight, at room temperature, on a tissue shaker. On the following morning, after 3 PBS washes, we incubated sections for 2 hours in a PBT-NDS-azide solution containing a 1:500 dilution of either donkey anti-rabbit (#711-065-152) or donkey anti-rat (#712-065-153; Jackson ImmunoResearch) secondary antibody. Sections were then washed 3 times and placed for 1 hour in biotin-avidin complex (Vectastain ABC kit PK-6100; Vector), washed 3 times in PBS, and incubated in nickel-diaminobenzidine (NiDAB) solution for 10 minutes. Our stock DAB solution was prepared by adding 100 tablets (#D-4418, Sigma, Saint Louis, MO) into 200 mL ddH2O, then filtering it. We used 1 mL of this DAB stock solution, with 300 μL of 8% nickel chloride (#N54-500, Fisher Chemical) per 6.5 mL PBS. After 10 minutes in NiDAB, we added hydrogen peroxide (0.8 μL of 30% H2O2 per 1 mL PBS-DAB) and swirled sections for 4–5 min until observing dark color change. After two rapid PBS washes, we wet-mounted one or more sections, checked them in a light microscope to ensure optimal staining, and in rare cases replaced sections for up to one additional minute for additional enzymatic staining. Finally, after washing an additional 3 times in PBS, we mounted tissue sections on glass slides (#2575-plus; Brain Research Laboratories). Slides were air-dried, then dehydrated in an ascending series of alcohols and xylenes, coverslipped immediately with Cytoseal 60 (#8310-16 Thermo Scientific), and stored at room temperature until microscope imaging.

For immunofluorescence labeling, we removed tissue sections from cryoprotectant, rinsed them in PBS and loaded them into a PBT-NDS-azide solution containing one or more primary antisera (Table 2). We incubated these sections overnight at room temperature on a tissue shaker. The following morning, the sections were washed 3 times in PBS, then incubated for 2 hours at room temperature in a PBT-NDS-azide solution containing species-specific donkey secondary antibodies. These secondary antibodies were conjugated to Cy3, Cy5, Alexa Fluor 488, or biotin (Jackson ImmunoResearch #s 711-065-152, 711-165-152, 711-175-152, 705-065-147, 705-545-147, 713-545-147, 706-545-148, 706-165-148, 706-065-148, 715-065-15, 712-165-153; each diluted 1:1,000 or 1:500). When biotin was used, sections were washed 3 times in PBS, then incubated for an additional 2 hours in streptavidin-Cy5 (#SA1011; Invitrogen) prepared in PBT-NDS-azide. These sections were then washed 3 times in PBS and mounted on glass slides (#2575-plus; Brain Research Laboratories), then coverslipped using Vectashield with DAPI (Vector Labs). Slides were stored in slide folders at 4 °C until microscope imaging.

Table 2.

Antisera.

Antigen	Immunogen Description	Source, host species, RRID	Concentration
CGRP	Synthetic CGRP peptide.	Abcam, goat polyclonal, cat# ab36001, lot #: GR3330475-13, RRID: AB_725807	1:1,000
FoxP2	E. coli-derived recombinant human FoxP2 isoform 1 Ala640-Glu715	R&D Systems, sheep polyclonal cat# AF5647; lot#: CCUB0109061, RRID: AB_2107133	1:3,000
mCherry	Full length mCherry protein	Invitrogen, rat monoclonal, cat# M11217, lot#: VA 293192, lot# W6337875, lot# VH 307508, RRID: AB_2536611	1:2,000
GRP	Synthetic (human) bombesin coupled to bovine thyroglobulin with glutaraldehyde.	Immunostar, rabbit polyclonal, cat# 20073, lot# 1420001, RRID: AB_572221	1:4,000
AgRP	Human AgRP 83-132 Amide	Phoenix Pharmaceuticals, rabbit polyclonal, cat#: H-003-53, lot#: 01825-2, RRID AB_2313908	1:5,000
TH	Native tyrosine hydroxylase from rat pheochromocytoma in sheep host	Millipore, sheep polyclonal, cat#: Ab1542, lot#: 34756625, RRID: AB_90755	1:1,000
DsRed	DsRed-express, a variant of Discosoma sp. red fluorescent protein	Takara Bio, rabbit polyclonal, cat#: 632496, lot# 1805060, RRID: AB_10013483	1:2,000
GFP	Full length green fluorescent protein from the jellyfish Aequorea victoria	Invitrogen, chicken polyclonal, cat# A10262, lot# 2591656, lot# 1972783, RRID: AB_2534023	1:3,000
TH	Denatured tyrosine hydroxylase from rat pheochromocytoma in rabbit host	Millipore, rabbit polyclonal, AB152, 3199177, RRID: AB_390204	1:10,000
NPS	Synthetic NPS peptide	Abcam, rabbit polyclonal, #ab18252, lot: GR198504-1, RRID: AB_776718	1:2000

Nissl counterstaining.

After whole-slide microscope imaging (described below), all slides were Nissl-counterstained and re-imaged. First, coverslips were removed by soaking slides in xylenes for up to a week. After rehydration, through 1-minute dips in a graded series of alcohols, we rinsed the slides in water and dipped them in a 0.125% thionin solution (Fisher Scientific) for 1 minute. Slides were then rinsed in distilled water until the solution cleared, and then dehydrated in a series of ethanol solutions: 50% EtOH, 70% EtOH, 400mL of 95% EtOH with 10 drops of glacial acetic acid, 95% EtOH, 100% and 100% EtOH. Finally, after the second of two xylene solutions, slides were coverslipped immediately with Cytoseal.

Microscope imaging and figures.

All slides were imaged using an Olympus VS120 slide-scanning microscope. For brightfield images of NiDAB and then Nissl-counterstained sections, we used a 20x UPLSAPO 20x air objective (numerical aperture 0.75) and the extended focal imaging (EFI) to collect and combine in-focus images from 11 focal planes through the tissue. To collect epifluorescence images, we first used a 10x (NA 0.40) objective, then collected additional EFI or multifocal image stacks at 20x in regions of interest. After reviewing whole-slide images in OlyVIA (Olympus), we collected additional EFI or multifocal image stacks at higher magnifications in some regions of interest.

To plot Syp-mCherry-labeled boutons for illustrations from case 5569, we used BoutonNet (Grady et al., 2022). This method uses full-resolution source images (346 nm/pixel OME/TIFF exports from VS-ASW) and combines two separate algorithms to label boutons: an intensity-based “proposal” algorithm, followed by a convolutional neural-network based “confirmation” algorithm. In separate layers in Illustrator, we aligned the PNG output file containing plotted boutons, the NiDAB source image, and a Nissl-counterstained image of the same section. We used the aligned Nissl cytoarchitecture to trace borders, white matter tracts, and ventricles in an additional illustration layer. We inspected each plot to verify accuracy and remove any automated bouton detection atop histological artifacts rather than actual NiDAB-labeled boutons. Finally, we exported the illustration layer and BoutonNet PNG together as a TIF for the final figure layout.

For Figures containing brightfield or fluorescence images, we first used Adobe Photoshop to crop bitmap images from VS-ASW or cellSens (Olympus), adjust brightness and contrast, and combine raw fluorescence data for multicolor combinations. We used Adobe Illustrator to add lettering, trace scale bars atop calibrated lines from OlyVIA (to produce a clean white or black line) and to make all illustrations.

Whole-brain analysis of Syp-mCherry labeling.

To compare the spatial distribution of neurons transduced in the PB region, we first plotted Syp-mCherry-expressing neurons across five rostrocaudal levels from each case with a brain sliced in the coronal plane (approximately −4.7 to −5.6 mm caudal to bregma). We overlaid these injection site plots in Adobe Illustrator and used images of the same sections after Nissl counterstaining to illustrate brainstem borders and major white matter tracts in each section.

Next, we immunolabeled, imaged, and reviewed every tissue section from a 1-in-3 series of sections through the full brain (olfactory bulbs to cervical spinal cord). We began by identifying all regions with Syp-mCherry-labeled boutons in NiDAB-labeled images (without Nissl counterstaining). We then compared each region side-by-side with Nissl-counterstained images of the same sections to identify cytoarchitectural loci. For each region, we scored the density of Syp-mCherry-labeled boutons using a semi-quantitative scale (0–4), and for brain regions with minimal or “trace” labeling, we assigned an intermediate designation (0.5), as in previous work (Huang et al., 2021). Three independent raters (RZ, DH, & JCG) scored every region from every brain and reviewed the results to reach consensus.

Nomenclature.

We use the term “boutons” to refer to punctate Syp-mCherry labeling. For mouse genes, we used Mouse Genome Informatics (MGI) nomenclature. For mouse proteins, we used common abbreviations from the published literature. For neuroanatomical structures and cell populations, where possible, we used and referred to nomenclature defined in peer-reviewed neuroanatomical literature. In some instances, we used or referred to nomenclature derived from rodent brain atlases (Dong, 2008; Paxinos & Franklin, 2013), with preference to the taxonomy used in the publicly available Allen Brain Mouse Atlas (Wang et al., 2020).

Results

Brain-wide distribution of NPS neurons.

We began by examining the brain-wide distribution of NPS neurons in parasagittal tissue sections from Nps-2A-Cre;R26-LSL-L10GFP mice (Figure 1 and Supplemental Video 1). The PB region contained the most prominent clusters of NPS neurons, and the sagittal plane of section highlighted the rostrocaudal ribbons of NPS neurons in the lateral habenular nucleus, as well as epitheliod cells running along the central canal of the spinal cord (Figure 1e), complementing our previous description in the coronal plane (Huang et al., 2022). Also mathcing our previous description, fewer NPS neurons were scattered in the hindbrain reticular formation, nucleus incertus region, anterior hypothalamus, and medial amygdala.

FIGURE 1.

Neurons with L10GFP expression in Nps Cre-reporter mice (Nps-2A-Cre;R26-lsl-L10GFP) in a sagittal (a), coronal (b), and horizontal (c) planes. Each dot represents approximately 1–5 neurons. Additional non-neuronal L10GFP-expressing cells were found along the central canal of the spinal cord (d,e). All scalebars are 100 μm.

NPS-immunoreactive fibers.

We immunolabeled NPS and observed fiber labeling in a pattern consistent with a previous report (Clark et al., 2011). The anterior paraventricular thalamic nucleus contained the densest labeling (Figure 2b). We also found immunoreactive fibers in the preoptic area (Figure 2a), sparse labeling in the dorsal midbrain (Figure 2c), moderately dense labeling in the subparaventricular zone (Figure 2d), and a dense horizontal band dorsal to the third ventricle (Figure 2d). Further caudally in the medial hypothalamus, we found a broad patch of labeling in the DMH and dorsal to it (Figure 2e), as well as in the periventricular region alongside the mammillary recess of the third ventricle (Figure 2f). All mice had the same overall pattern of fiber labeling, but the intensity of labeling varied between cases.

FIGURE 2.

NPS immunofluorescence labeling in the BST (a), PVT (b), PAG (c), PVH (d), caudal hypothalamus (e), and posterior periventricular hypothalamic nucleus (f) varied between cases. Scalebars in every panel are 200 μm.

Injection sites.

Because the origins of NPS-immunoreactive fibers in each region are unclear, we next used Cre-conditional anterograde tracing in the PB region, which contains the densest concentration of Nps-expressing neurons. We injected a vector that delivers a Cre-conditional presynaptic marker into the PB of Nps-2A-Cre mice (AAV8-hEf1a-DIO-synaptophysin-mCherry, n=7). We targeted the rostral cluster of PB NPS neurons in three cases (5495, 5568, 5569) and the caudal cluster in four cases. Two caudal injections failed to transduce any neurons, and we analyzed the remaining two cases (5429, 5432).

Rostral-to-caudal plots of each injection site are shown in Figure 3. Among the rostral cases, 5568 transduced the most neurons, 5569 transduced an intermediate amount, and 5495 transduced the fewest. Case 5429 mainly transduced neurons in the caudal cluster and transduced fewer neurons in the lateral PB, while case 5432 transduced neurons exclusively in the caudal NPS cluster. We sliced each brain in the coronal plane and used them for brain-wide analysis of Syp-mCherry NiDAB labeling and BoutonNet plots, plus immunofluorescence labeling in select target regions. Rostral-to-caudal images of the full injection site in each case are shown in Supplementary Figure 1.

FIGURE 3.

Injection site plots of Syp-mCherry transduced neurons in all Nps-2A-Cre cases. Rostral cases (5569, 5568, 5495) had labeling in the rostral PB with no labeling caudally. In caudal cases (5432, 5429), somatic labeling was primarily located caudally, near the locus coeruleus, and caudal case 5429 had some labeling in the lateral parabrachial nucleus.

As a supplement to our primary analysis in the coronal plane, we injected the same AAV vector into the rostral PB of additional Nps-2A-Cre;R26-LSL-L10GFP mice (n=4). We sliced these four brains in the sagittal plane and used their parasagittal sections for immunofluorescence labeling. An injection site from one of these cases is shown in Figure 4.

FIGURE 4.

Parasagittal injection site of Cre-conditional Syp-mCherry in case 6730. L10GFP expression reveals cells in the rostral lateral PB with a history of Nps expression (green, in a and c). Syp-mCherry expression (red in b) was exclusive to a subset of these neurons (yellow in c). NPS neurons are immediately dorsal and caudal to the noradrenergic A7 group of neurons, which are immunoreactive for tyrosine hydroxlyase (TH; magenta, in a and c). Scalebar in (b) is 200 μm and applies to (a). Scalebar in (c) is 200 μm.

Brain-wide distribution of Syp-mCherry labeled boutons.

We analyzed the brain-wide distribution of Syp-mCherry labeling in each case (Figure 5). Overall, cases with injections into the caudal cluster of NPS neurons, near the locus coeruleus, had lighter labeling than those with injections into the larger, rostral cluster of NPS neurons. In rostral cases, nearly every brain region with prominent labeling also contained at least trace labeling in caudal cases. In all cases, labeling ipsilateral to the injection site was stronger. To show the brain-wide distribution of Syp-mCherry-labeled boutons, we performed BoutonNet analysis on tissue sections from a representative case (Figure 6).

FIGURE 5.

Density of Syp-mCherry labeling in 150 brain regions in each case, grouped by rostral vs. caudal injection site. Light grey indicates absence of labeling and darker shades of blue represent increasing density of labeling. For all abbreviations, see List of Abbreviations.

FIGURE 6.

Brain-wide distribution of Syp-mCherry labeling in case 5569. These 26 illustrated sections (a–z) were chosen to best represent the output pattern of NPS neurons in the PB region. Transduced neurons in the injection site are represented by black dots in panels (s–u). All abbreviations are found in List of Abbreviations.

Cerebral cortex and olfactory bulb.

Except for a few, scattered boutons in some cases, we did not find any labeling in the cerebral cortex or olfactory bulb. The anterior olfactory nucleus contained trace labeling in several cases, and the tenia tecta contained trace labeling only in rostral cases.

Septum.

The lateral septum contained labeling in all cases. This labeling concentrated in its rostral and ventral subregions, with virtually no labeling dorsally (Figure 6c–d). In rostral cases, the medial septal nucleus also contained light labeling, but no other region of the septum contained labeling in other cases.

Basal ganglia.

In every case, we found light labeling in the caudal nucleus accumbens shell, where it merges with the bed nucleus of the stria terminalis (Figure 6d–e). Rostral to this, the nucleus accumbens shell contained no more than trace labeling (Figure 6c). Trace labeling in the striatum, globus pallidus, and subthalamic nucleus was an inconsistent finding in rostral cases.

Amygdala.

The amygdala received very little input. The central nucleus of the amygdala contained trace Syp-mCherry labeling in some cases, and the medial amygdalar nucleus had trace labeling in all cases (Figure 6l). Only case 5568 had trace labeling in the basomedial nucleus and cortical amygdalar area.

Basal forebrain.

Most basal forebrain nuclei received no input. Some cases had light labeling in the substantia innominata or trace labeling in the diagonal band (Figure 6c–h).

Bed nucleus of the stria terminalis.

All cases had dense labeling in the bed nucleus of the stria terminalis (BST). Labeling was densest ventral to the anterior commissure (Figure 6e–f), and lighter labeling covered a larger expanse of the anterolateral and anteromedial BST while largely avoiding the oval subnucleus and posterior subnuclei. We immunolabeled AgRP and CGRP to contextualize the anterograde terminal field in this interesting area. Syp-mCherry-labeled boutons overlaid the focal cluster of CGRP-labeled axons that identify the fusiform BST subnucleus, and this medium-density labeling extended medially through ventral BST subnuclei that receive dense AgRP-immunoreactive fibers. In contrast, the oval BST subnucleus, which contains denser CGRP labeling, had significantly less Syp-mCherry labeling (Figure 7).

FIGURE 7.

In the ventral BST, Syp-mCherry immunofluorescence (red, a) partly overlapped CGRP-immunoreactive fibers (green, b) and extensively overlapped AgRP-immunoreactive fibers (blue, c–e). AgRP, CGRP, and mCherry combined immunolabeling in panel (e). All sections are from case 5568. All scalebars are 200 μm. Scalebar in (d) also applies to (a–c).

Lamina terminalis.

Both circumventricular organs of the lamina terminalis, the subfornical organ (SFO, Figure 4g) and vascular organ of the lamina terminalis (OVLT), had light labeling in several cases. In-between them, the median preoptic nucleus contained moderate labeling in each case (Figure 7).

Hypothalamus.

In every case, the hypothalamus received more labeling overall than any other brain region (Figure 5g–o). However, this labeling was not uniform across the hypothalamus. Syp-mCherry labeling blanketed the medial hypothalamus, except for the mammillary, ventromedial, and suprachiasmatic nuclei, which contained virtually no labeling. Also, a band extending dorsolaterally from the medial preoptic nucleus through the posterior BST received relatively less labeling than surrounding parts of the anterior hypothalamus.

Near the third ventricle and dorsal to the suprachiasmatic nucleus, we found a dense patch of Syp-mCherry labeling in a focal subregion of the subparaventricular zone (SPZ), in-between TH-immunoreactive neurons and axons in the PVH and GRP-immunoreactive fibers projecting dorsally from the SCN (Figures 6i & 8). The paraventricular hypothalamic nucleus (PVH) itself contained only light labeling, but a thin, horizonal band immediately dorsal to the ventral third ventricle contained denser labeling (Figure 8).

FIGURE 8.

Dense Syp-mCherry immunofluorescence (red, a) lies immediately ventral to TH-immunoreactive neurons and fibers in the paraventricular hypothalamic nucleus (green) and immediately dorsal to GRP-immunoreactive fibers in the SPZ (b–c). Combined mCherry, TH, and GFP labeling is shown in panel (c; case 5569). This level of the anterior hypothalamus also contained dense Syp-mCherry labeling in a band dorsal to the third ventricle. Both scalebars are 200 μm. Scalebar in (b) also applies to (a).

The dorsomedial hypothalamic nucleus (DMH) contained the densest labeling in the hypothalamus, and the parasubthalamic nucleus (PSTN) also contained dense labeling. Labeling in the rest of the lateral hypothalamic area and posterior hypothalamus was less dense. The dense blanket of labeling that covered the DMH extended dorsally to encompass the A13 cluster of catecholaminergic-GABAergic neurons (Negishi et al., 2020) in the zona incerta at the dorsal margin of the hypothalamus (Figure 9).

Figure 9.

Syp-mCherry labeling (red, b–c) in a parasagittal section though the thalamus and hypothalamus (case 6730). L10GFP Cre-reporter for Nps (green; a, c) identifies NPS neurons in the anterior hypothalamic nucleus and lateral habenula, while TH immunolabeling identifies catecholaminergic neurons and fibers (magenta; a, c). Anterogradely labeled axons produced dense concentrations in the paraventricular thalamic nucleus (PVT), magnocellular subparafasicular nucleus (mSPF), A13 group, and several hypothalamic subregions, while most of the thalamus and much of the mPOA, VMH, and MB lacked labeling. For remaining abbreviations, see List of Abbreviations. Scalebars are 800 μm and the scalebar in (b) also applies to (a).

Thalamus.

The median thalamus contained some of the densest labeling in the brain. Every case had dense labeling in the paraventricular nucleus of the thalamus (PVT). Profuse labeling enveloped FoxP2-immunoreactive neurons in the anterior PVT and extended laterally to surround those in the paratenial nucleus (Figure 10). This dense terminal field extended caudally through the full PVT. The reunions, rhomboid, xiphoid, and lateral habenular nuclei contained modest labeling.

Figure 10.

In the thalamus, dense Syp-mCherry labeling (red, a) concentrated in the anterior PVT and arced laterally around the paratenial nucleus (case 5495). These thalamic nuclei contained strong nuclear immunoreactivity for the transcription factor FoxP2 (green, b–c). Scalebar in (b) applies to (a) and is 200 μm. Scalebar in (c) is 200 μm.

We also found a small, dense patch of labeling in the caudal, ventral thalamus. The density of labeling in this small, round spot was similar to the density of Syp-mCherry labeling in the PVT. Syp-mCherry labeling here avoided neighboring catecholamine neurons and instead targeted a small spot referred to as the magnocellular subparafasciular nucleus (mSPF; Figure 6m, Figure 9c). In contrast, the parafasicular thalamic nucleus had sparse labeling, and the remaining subparafasicular region had light labeling that extended laterally toward the geniculate nuclei and peripeduncular nucleus.

In the caudal, lateral thalamus, we found sparse labeling in the intergeniculate leaflet, ventral lateral geniculate nucleus, and a region referred to as the posterior limitans nucleus (Paxinos & Franklin, 2013; Paxinos & Watson, 2007). Labeling in these regions was sparse in rostral cases and absent in caudal cases. No cases contained labeling in the dorsal lateral geniculate nucleus. Immediately ventral to the medial geniculate nucleus, we found moderately dense labeling in the peripeduncular nucleus.

Midbrain.

In rostral cases, the tectum contained one of the densest targets in the brain. There, we found a thin, dense stripe of Syp-mCherry-labeled boutons near the midline of the deep layers of the superior and inferior colliculi. This terminal field, immediately dorsal to the PAG, was apparent in serial coronal sections (Figure 11a–b) and even more prominent in the sagittal plane (Figure 11c–d). This rostrocaudally elonged zone appears to be the “tectal longitudinal column” (TLC) described in rats (Saldana et al., 2007). While the TLC was one of the most prominent targets in the brain in rostral cases, we found virtually no labeling in this region in caudal cases. Labeled axons and boutons reached this target zone via a periventricular pathway, through the periaqueductal gray matter, which contained moderately dense labeling as well.

Figure 11.

(a–b) Dense labeling in the deep midline SC (case 5569; coronal section, NiDAB labeling for Syp-mCherry). (c) Sagittal view of median collicular labeling with L10GFP Cre-reporter for Nps (green) plus immunofluorescence labeling for TH (magenta; case 6731) and magnified area of interest in (d). Scalebars are (a) 500 μm, (b) 100 μm, (c) 1 mm, and (d) 100 μm.

Separate from this periventricular pathway, the midbrain received moderate labeling along a ventrolateral pathway. Most axons coursed alongside the medial lemniscus and dorsal to the substantia nigra without arborizing in the midbrain, but many produced a concentration of branches and boutons in the retrorubral field and extended rostrally into the peripeduncular and subparafasciular thalamic target zones described above.

Cerebellum.

No part of the cerebellar cortex or deep cerebellar nuclei contained labeling in any cases.

Hindbrain.

While NPS neurons supply a descending pathway, these projections did not produce wide-ranging axonal labeling in the hindbrain. A thin rim of moderately dense labeling encased the superior olivary nucleus, trapezoid bodies, and ventral nucleus of the lateral lemniscus. We also found light labeling in the ventrolateral pontine reticular nucleus and forming a rim around the facial motor nucleus. Moderately dense labeling also targeted the superior salivatory nucleus, just rostral and dorsolateral to the facial motor nucleus. The nucleus of the solitary tract had no more than trace labeling. A region of the caudal ventrolateral medulla that included the A1 group and caudal pressor area (CPA) contained sparse labeling.

Discussion

Glutamatergic neurons in the PB form two macropopulations, an Atoh1-derived macropopulation and an Lmx1-expressing macropopulation. PB neurons project to target regions via four pathways: a periventricular pathway, the central tegmental tract, a ventral pathway, and a descending pathway (Figure 12a). Neurons in the Lmx1 macropopulation project axons primarily via the central tegmental tract, while neurons in the Atoh1-derived macropopulation project axons primarily via the ventral pathway. NPS neurons are a subset of the Atoh1 macropopulation and project axons via this same ventral pathway, as well as a robust periventricular pathway (Figure 12b). We found that Nps-expressing PB neurons project heavily to the midline thalamus, medial hypothalamus, and midline tectum. In contrast, we found little to no labeling in the cerebral cortex, olfactory bulb, amygdala, and cerebellum. After discussing limitations of our study, we compare our results to previous work and forecast the function of these neurons.

Figure 12.

Projection pathways and targets of (a) PB glutamatergic neurons in comparison to (b) the Nps-expressing subset of glutamatergic neurons. Nps-expressing neurons project prominently via a periventricular pathway.

Limitations.

Syp-mCherry tracing with BoutonNet algorithmic processing has several advantages compared to previous anterograde tracing techniques (Grady et al., 2022). Syp-mCherry concentrates in the presynaptic bouton, while previous tracers uniformly distribute in the axoplasm. However, a small portion of the putative boutons labeled by our method likely represent non-synaptic portions of axons cut in cross-section because Syp-mCherry must travel through the axon shaft to reach presynaptic boutons. To mitigate this limitation, we also visually inspected Syp-mCherry labeling in every brain region and applied semi-quantitative density standards used in previous tracing studies (Huang et al., 2020; Huang et al., 2021). Despite these efforts to identify boutons from the morphology of labeling, determining whether individual boutons form structural synapses would require the use of electron microscopy (Wouterlood & Jorritsma-Byham, 1993), and determining whether they form functional synapses would require the use of electrophysiology (Petreanu et al., 2007). Neither of these approaches is practical at the scale of this study.

We genetically targeted NPS neurons using a knockin-Cre strain with a 2A “self-cleaving” peptide inserted after the last exon of the endogenous Nps gene (Huang et al., 2022). We used a Cre-conditional construct (DIO-Syp-mCherry), so only neurons with sufficient Cre expression between the time of injection and perfusion were transduced. We performed these experiments in adult mice, so the results reflect axonal projections of neurons with adult Nps expression, not just developmental expression, which may be more widespread.

Additionally, the Syp-mCherry promoter is different from the Nps gene promoter. Therefore, neurons with sufficient Cre expression to activate the DIO construct produce large amounts of Syp-mCherry, without reflecting varying levels of Nps expression in those neurons. This may be relevant for NPS neurons due to the known variations in NPS protein and Nps mRNA expression with physiological state. Thus, while our approach has high sensitivity for neurons that express Nps, the axonal labeling density in our results does not reflect Nps expression level in those neurons.

Although we compared the projection patterns of the rostral and caudal group of NPS neurons, we did not distinguish subpopulations of neurons within these groups. The rostral group of NPS neurons concentrates between the tip of the superior cerebellar peduncle and the medial edge of the lateral lemniscus, resembling the cytoarchitecturally defined “extreme lateral” subnucleus in the rat PB (Fulwiler & Saper, 1984). However, this group also includes neurons scattered caudally through the lateral PB, and additional neurons expressing an Nps Cre-reporter extend rostrally along the medial edge of the lateral lemniscus, dorsal to the population of TIP39/Pth2-expressing neurons in the medial paralemniscal nucleus (Dobolyi et al., 2003; Sun et al., 2022; Varga et al., 2008) and overlapping a location described as the “semilunar nucleus” in rats (Gómez-Martínez et al., 2023). Neurons concentrated in the main (“extreme lateral”) NPS cluster express high levels of Nps mRNA, while the other neurons identifiable by Cre-reporter expression contain very little, so these neurons may form separate subpopulations with separate connections and functions. Discriminating differences in projection patterns would require first identifying additional genetic markers that can distinguish NPS subpopulations. Within the caudal cluster, most NPS neurons near the locus coeruleus express Foxp2, but Barrington’s nucleus includes a small number of neurons with Nps Cre-reporter expression that lack FoxP2 (Huang et al., 2022). Differentiating the projection pattern of Barrington’s NPS neurons that lack FoxP2 from that of peri-LC NPS neurons that express Foxp2 may be possible with intersectional genetic targeting.

Comparison with previous neuroanatomical literature.

Generally, the pattern of Syp-mCherry labeling from NPS neurons in the PB region approximated the pattern of NPS fiber immunolabeling (Figure 2), as described in a previous study using the same NPS antiserum (Clark et al., 2011). This implies that neurons in the PB region supply most of the NPS peptide in the mouse brain. Neighboring, Cck-expressing neurons in the PB project heavily to the VMH (Garfield et al., 2014; Grady et al., 2020), and a previous study of NPS immunoreactivity reported dense fiber labeling in this hypothalamic nucleus (Clark et al., 2011). However, the VMH contained virtually no NPS-immunoreactive fibers and was largely devoid of Syp-mCherry axonal labeling in our study (Figure 2e).

Additionally, we identified output projections to regions of the brain that contain sparse immunolabeling, including the ventral BST, mSPF, and TLC. These target regions contained dense Syp-mCherry labeling and appear to be important targets for NPS neurons. A previous study in rats observed retrograde labeling in the “extreme lateral” PB subnucleus after a tracer injection spanning the rostral BST, nucleus accumbens, and lateral septum (Fulwiler & Saper, 1984). Another previous tracing study identified retrograde labeling in the PB region after tracer injections into the mSPF (Wang et al., 2006), but we are not aware of any previous retrograde tracing studies that described the dense connectivity from the PB region to TLC. This reigon has Npsr1 expression as part of a broader pattern of expression in the superior colliculi (Clark et al., 2011). To the best of our knowledge, the presence or absence Npsr1 expression in the mSPF has not been clarified in previous work.

NPS neurons are glutamatergic, Atoh1-derived, and Foxp2-expressing, but they do not express Pdyn. (Huang et al., 2022; Xu et al., 2007). The present results are largely consistent with previous tracing data on the efferent projections of Foxp2-expressing neurons in rats and mice, including a general lack of labeling in the olfactory bulb, cerebral cortex, amygdala (Huang et al., 2020; Shin et al., 2011). Our results contrast the projections of the PB Lmx1 macropopulation, including Calca-expressing neurons, which primarily target the amygdala and cerebral cortex and send much less output to the hypothalamus (Huang et al., 2021).

Most NPS projections identified here represent a subset of the efferent projections previously identified in our study of the larger population of glutamatergic Foxp2-expressing neurons in the PB region (Huang et al., 2020). However, NPS neurons densely targeted a small number of brain regions, including the TLC and NLL, that were less evident in our previous study. Interestingly, we found dense fiber labeling in the TLC in Atoh1-Cre mice with a tdTomato Cre-reporter (see Figure 20b of Karthik et al., 2022). The injection sites in our Foxp2 tracing study were centered caudal to the main concentration of NPS neurons, and the presence of labeling in the TLC in Atoh1-Cre tdTomato reporter mice supports the present finding that Atoh1-derived, Nps-expressing neurons in the rostral PB project to this region.

The median superior colliculus contains the tectal longitudinal column with ventral (TLCv) and dorsal (TLCd) components (Aparicio & Saldaña, 2014). Anterograde Syp-mCherry axonal labeling from Nps-expressing neurons in the PB appear to target TLCv with less dense labeling dorsally in the TLCd (Figure 11). Interestingly, the periolivary region, which receives input from the NPS neurons, appears to be reciprocally connected with the TLCv (Saldaña et al., 2009; Viñuela et al., 2011).

Functional implications.

The PB is a major integrator of interoceptive sensory inputs. The genetic identity and location of neurons in the PB is a useful way to delineate their different projections and functions. For example, Pdyn-expressing neurons in the dorsolateral PB relay warm thermosensory information (Geerling et al., 2016), Satb2-expressing neurons in the medial PB relay taste information (Fu et al., 2019; Jarvie et al., 2021), and Calca-expressing neurons in the external lateral subdivision induce taste aversion and inhibit appetite (Carter et al., 2015; Carter et al., 2013; Palmiter, 2018). In contrast to these known populations, Nps-expressing neurons are a neuroanatomically distinct group with a novel projection pattern. They target brain regions that regulate circadian rhythm and threat response.

The SCN is the master circadian regulator, and among brain regions with direct connectivity to the SCN, NPS axons project to the SPZ, PVT, DMH, and IGL. The SPZ is the main output target of the SCN and plays an important role in the circadian regulation of locomotor activity, body temperature, and sleep (Vujovic et al., 2015). Evidence exists for a relationship between NPS and sleep: a human NPSR1 gain-of-function mutation was associated with substantially reduced sleep, and mice carrying this mutation also slept less (Xing et al., 2019). Additionally, central injection of an NPSR1 antagonist reduced wakefulness in rats (Oishi et al., 2014). Another region of dense labeling in the midline thalamus was the mSPF. In our analysis of NPS neuron anterograde tracing, we were surprised to find dense labeling in the mSPF, whose neurons are medial and ventral to the fasciculus retroflexus and have not been comprehensively studied. This compact and unusual thalamic nucleus contains GABAergic neurons and sends a prominent output projection to the IGL (Vrang et al., 2003), which is implicated in circadian function due to its input from the retina and output to the SCN (Moore et al., 2000). In addition to the mSPF, the IGL also receives light axonal input from the NPS neurons. The anterior PVT and DMH also play important roles in circadian regulation, and each of these sites receives heavy input from NPS neurons. It is likely that NPS projections to these brain regions influence circadian patterns.

Adaptive responses to threats are fundamental to survival. NPS axons target several brain regions that are implicated in modulating threat responses: the DMH, ventral BST, PVT, and medial tectum. NPS axons blanked the medial hypothalamus while largely avoiding the VMH, mamillary bodies, and SCN, and they produced particularly dense labeling in the DMH. Cck-expressing neurons in the superior lateral PB project to the VMH and are activated by noxious, aversive input from the spinal cord (Hermanson et al., 1998), and neurons in the VMH cause aggressive attack behavior in male mice (Lin et al., 2011). However, the lack of Syp-mCherry labeling in the VMH and the mutual exclusivity of Cck and Nps expression suggest that NPS neurons do not regulate these behaviors. Dense labeling in the DMH spreads dorsally and laterally into the zona incerta and encompasses the A13 catecholaminergic cell group. Electrically stimulating this medial region of the hypothalamus triggers escape behaviors, while stimulating the lateral hypothalamus triggers exploratory locomotion (Lammers et al., 1988). The DMH contains Npsr1 expression, and central NPS injections increase locomotion, so it is tempting to speculate that NPS-DMH connectivity is responsible for escape responses to threats.

NPS axons avoided the dorsal BST, while broadly targeting a subregion of the ventral BST that receives AgRP input from the arcuate hypothalamic nucleus. The BST appears to play a role in sustained responses to threat (Davis et al., 2010; Gewirtz et al., 1998; Walker et al., 2003) and is implicated in general anxiety disorder in humans (Lebow & Chen, 2016; Yassa et al., 2012). Based on associations between human NPSR1 gain-of-function mutations with panic and anxiety disorders (Domschke et al., 2011; Donner et al., 2010; Okamura et al., 2007; Pape et al., 2010), NPS projections to the BST may play a role in regulating long-term responses to threats.

The PVT is the most prominent region with Syp-mCherry labeling, as well as NPS-immunoreactive fibers and Npsr1 mRNA expression (Clark et al., 2011). Notably, activating PB projections to the PVT is aversive (Zhu et al., 2022). Also, deleting either Nps or Npsr1 attenuates conditioned fear responses, and these responses can be rescued by injecting NPS into the PVT (Garau et al., 2022). NPS neuron connectivity to the PVT is probably important for relaying aversive information.

In the midbrain, we found a dense patch of labeling in the midline of the tectum. This labeling was most prominent in cases with rostral injections sites and virtually absent in caudal cases. This area of the brain was identified in rats as the TLC (Saldana et al., 2007). Neurons in this region may be important for encoding threat salience, as opposed neurons in the dorsal PAG that regulate escape behavior (Evans et al., 2018). We postulate that NPS axons targeting the medial tectum are part of a neural circuit that encodes for threat salience.

Descending projections to the hindbrain targeted the superior salivatory nucleus, while skirting around the edges of the VII. Via their peripheral output projections to the pterygopalatine ganglion, SSN neurons control the lacrimal gland, along with mucous secretion and dilation of cerebral blood vessels. Along with their output to other target regions implicated in aversive responses, this connection to the SSN may allow NPS neurons to drive autonomic responses to distress, such as mucous secretion from the nasal mucosa and tear secretion from the lacrimal glands.

Conclusion.

In pharmacological studies, NPS promotes arousal and increases locomotion. Receptor mutations are associated with reduced sleep time, as well as panic and anxiety disorders. In this study, we mapped and analyzed the output projections of NPS neurons in the PB region. Their target regions predict that NPS neurons influence circadian rhythms and compute responses to threat.

Supplementary Material

Supplement 1

Supplemental Figure 1. Full rostral-to-caudal Nissl-counterstained sections with Syp-mCherry NiDAB immunolabeling in all injection sites cut in the coronal plane.

Supplement 2

Supplemental Video 1. Rotations of a 3D model of NPS neuron locations in the mouse brain.

Abbreviations

A1

A1 catecholaminergic group

A8

A8 catecholaminergic group

ac

anterior commissure

AcbSh

accumbens nucleus, shell

ACA

anterior cingulate cortical area

AgRP

agouti-related peptide

AHA

anterior hypothalamic area

AI

agranular insular cortex

AmbC

ambiguus nucleus compact part

AON

anterior olfactory nucleus

AP

area postrema

APir

amygdalopiriform transition area

APN

anterior pretectal nucleus

Arc

arcuate nucleus

AVP

arginine vasopressin

BLA

basolateral amygdalar nucleus

BMA

basomedial amygdalar nucleus

BST

bed nucleus of the stria terminalis

BSTaL

BST anterolateral subnucleus

BSTam

BST anteromedial subnucleus

BSTfu

BST fusiform subnucleus

BSTov

BST oval subnucleus

BSTp

BST posterior subnucleus

CeA

central nucleus of the amygdala

CeAc

CeA capsular subdivision

CeAl

CeA lateral subdivision

CeAm

CeA medial subdivision

CGRP

calcitonin gene-related peptide

Cla

claustrum

CLi

central linear raphe

CM

central medial thalamic nucleus

CoA

cortical amygdalar nucleus

CPA

caudal pressor area

Cun

cuneiform nucleus

DCN

deep cerebellar nuclei

DCO

dorsal cochlear nucleus

Dl

dysgranular insular cortex

DMH

dorsomedial hypothalamic nucl.

DP

dorsal peduncular cortical area

DR

dorsal raphe nucleus

DTN

dorsal tegmental nucleus

Ent

entorhinal cortical area

Epd/Epv

endopiriform nucleus, dorsal/ventral subdivisions

Epy

ependymal cell layer of cerebral aqueduct

EW

Edinger-Westphal nucleus

FL

flocculus of the cerebellum

fr

fasciculus retroflexus

fx

fornix

GI

granular insular cortex

GiRN

gigantocellular reticular nucleus

GPe

globus pallidus external

GPi

globus pallidus internal

GRP

gastrin-related peptide

Hc

hippocampus

IA

intercalated amygdalar nucleus

IC

inferior colliculus

IFV

interfascicular trigeminal nucleus

IG

induseum griseum

IGL

intergeniculate leaflet

ILA

infralimbic cortical area

IMD

intermediodorsal thalamic nucl.

IO

inferior olivary complex

IPN

interpeduncular nucleus

IRN

intermediate reticular nucleus

L6 PFC

layer 6 prefrontal cortex

LAr

lateral amygdaloid nucleus

LDT

laterodorsal tegmental nucleus

LGd

lateral geniculate nucleus, dorsal subdivision

LGv

lateral geniculate nucleus, ventral subdivision

LHA

lateral hypothalamic area

LHb

lateral habenular nucleus

LM

lateral mammillary nucleus

LPOA

lateral preoptic area

LRN

lateral reticular nucleus

LSi

lateral septum, intermediate subdivision

LSr

lateral septum, rostral subdivision

LSv

lateral septum ventral subdivision

MA

magnocellular nucleus

MB

mammillary bodies

MD

mediodorsal thalamic nucleus

MeA

medial amgydalar nucleus

MGN

medial geniculate nucleus

MHb

medial habenular nucleus

MM

medial mammillary nucleus

MnPO

median preoptic nucleus

MOB

main olfactory bulb

MPN

medial preoptic nucleus

mPOA

medial preoptic area

MR

median raphe

MRF

midbrain reticular formation

MS

medial septum

mSPF

magnocellular subparafascicular nucleus

NBM

nucleus basalis of Meynert

NDB

nucleus of the diagonal band

NeoCtx

neocortex

NLL

nucleus of the lateral lemniscus

NPS

neuropeptide S

NTB

nucleus of trapezoid body

NTS

nucleus of the solitary tract

NTS-Lat

NTS lateral subdivision

NTS-Med

NTS medial subdivision

OVLT

organum vasculosum of lamina terminalis

PAG

Periaqueductal gray matter

PAG-L

PAG lateral subdivision

PAGdl

PAG dorsolateral subdivision

PAGdm

PAG dorsomedial subdivision

PAGvL

PAG ventrolateral subdivision

PaRN

parvicellular reticular nucleus

PB

parabrachial nucleus

PC

paracentral thalamic nucleus

PeriRh

perirhinal cortical area

PF

parafascicular thalamic nucleus

PG

pontine grey

PH

posterior hypothalamic nucleus

Pir

piriform cortical area

PL

prelimbic cortical area

PLi

posterior limitans thalamic nucleus

PMd/v

posterior mammillary nucleus, dorsal/ventral

Po

posterior thalamic complex

PP

peripeduncular nucleus

PPN

pedunculopontine tegmental nucleus

PreT

pretectal region

PRN

pontine reticular nucleus

PS

parastrial nucleus

PSTN

parasubthalamic nucleus

PSV

principal sensory trigeminal nucl.

PT

paratenial thalamic nucleus

PVH

paraventricular hypothalamic nucleus

PVT

paraventricular thalamic nucleus

RCA

retrochiasmatic area

Re

reuniens thalamic nucleus

Rh

rhomboid thalamic nucleus

RLi

rostral linear raphe

RMg/Ob/Pa

raphe magnus/obscurus/pallidus

RMTg

rostromedial tegmental nucleus

RN

red nucleus

RR

retrorubral area

SC

superior colliculus

SC-Lat

SC lateral aspect

SC-Sen

SC sensory-related

SCN

suprachiasmatic nucleus

SCO

subcommissural organ

SEZ

subependymal germinal zone

SF

septofimbral nucleus

SFO

subfornical organ

SHi

septohippocampal nucleus

SI

Substiantia inominata

SNc

Substiantia nigra pars compacta

SNr

Substiantia nigra pars reticularis

SOC

superior olivary complex

SON

supraoptic nucleus

SPFp

subparafasicular nucleus, parvicellular part

SpV

spinal trigeminal nucleus

SPZ

subparaventricular zone

SSN

superior salivatory nucleus

STN

subthalamic nucleus

Str (CP)

striatum (caudoputamen)

Str/GP bdr

striatum/pallidum border

SUM

supramammillary nucleus

TH

tyrosine hydroxylase

TLC

tectal longitudinal column

TM

tuberomammillary nucleus

TRN

tegmental reticular nucleus

TR

amygdalopiriform transition area

TT

tenia tecta

V

trigeminal motor nucleus

VII

facial motor nucleus

VLPO

ventrolateral preoptic nucleus

VM

ventromedial thalamic nucleus

VMH

ventromedial hypothalamic nucl.

VNC

vestibular nuclear complex

VPpc

parvicellular ventral posterior thalamic nucleus

VTA

ventral tegmental area

Xi

xiphoid thalamic nucleus

XII

hypoglossal motor nucleus

ZI

zona incer