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, NPS) 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 already about NPS and its receptor, the output pattern of Nps-expressing neurons in the PB region predicts roles in threat response and circadian behavior.

Keywords: LPBN; PVA; TLC; parabrachial region; pre‐LC; subparaventricular.

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.

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.

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.

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 hydroxylase (TH; magenta, in a and c). Scalebar in (b) is 200 μm and applies to (a). Scalebar in (c) is 200 μm.

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.

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.

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.

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).

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).

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).

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.

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.

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, as well as a ventral pathway. These pathways converge in the anterior hypothalamus, and individual axons may reach diencephalic targets through either pathway.