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. 2020 Apr 28;43(11):zsaa085. doi: 10.1093/sleep/zsaa085

Gene expression analysis in the mouse brainstem identifies Cart and Nesfatin as neuropeptides coexpressed in the Calbindin-positive neurons of the Nucleus papilio

Franck Girard 1,✉, Michelle von Siebenthal 1, Fred P Davis 2,✉, Marco R Celio 1
PMCID: PMC7658639  PMID: 32343818

Abstract

Study Objectives: The brainstem contains several neuronal populations, heterogeneous in terms of neurotransmitter/neuropeptide content, which are important for controlling various aspects of the rapid eye movement (REM) phase of sleep. Among these populations are the Calbindin (Calb)-immunoreactive NPCalb neurons, located in the Nucleus papilio, within the dorsal paragigantocellular nucleus (DPGi), and recently shown to control eye movement during the REM phase of sleep. Methods: We performed in-depth data mining of the in situ hybridization data collected at the Allen Brain Atlas, in order to identify potentially interesting genes expressed in this brainstem nucleus. Our attention focused on genes encoding neuropeptides, including Cart (Cocaine and Amphetamine Regulated Transcripts) and Nesfatin 1. Results: While nesfatin 1 appeared ubiquitously expressed in this Calb-positive neuronal population, Cart was coexpressed in only a subset of these glutamatergic NPCalb neurons. Furthermore, an REM sleep deprivation and rebound assay performed with mice revealed that the Cart-positive neuronal population within the DPGi was activated during REM sleep (as measured by c-fos immunoreactivity), suggesting a role of this neuropeptide in regulating some aspects of REM sleep. Conclusions: The assembled information could afford functional clues to investigators, conducive to further experimental pursuits.

Keywords: Nucleus papilio, NPCalb, REM sleep, DPGi, calbindin, Cart, nesfatin

Statement of Significance.

Several physiological and behavioral features are characteristics of the rapid eye movement (REM) phase of sleep, also called paradoxical sleep. These include muscle atonia, desynchronized EEG activity, vivid dreaming, and rapid eye movements. A small cluster of Calbindin-immunoreactive neurons (namely, the Nucleus papilio) has been recently identified in the brainstem and shown to be both necessary and sufficient for triggering eye movement during REM sleep. In the present study, we performed data mining of the in situ hybridization data collected at the Allen Brain Atlas, in order to identify genes expressed in these neurons. Our data show that the neuropeptide Cart (Cocaine and Amphetamine Regulated Transcript) is expressed in some of these Calbindin-immunoreactive neurons and that these Cart-neurons are activated during REM sleep.

Introduction

Several physiological and behavioral features are characteristics of the REM phase of sleep, also called paradoxical sleep. These include rapid eye movements, vivid dreaming, desynchronized electroencephalogram activity, and atonia of the postural muscles [1]. Among several brainstem structures that have been shown to be involved during REM sleep [2–6], the dorsal paragigantocellular nucleus (DPGi) appears as a very crucial area in sleep regulation, as it contains neurons of different nature, which apparently play particular roles in regulating some aspects of REM sleep. Indeed, GABAergic neurons of the DPGi were proposed to inhibit the noradrenergic wake-promoting neurons of the Locus ceruleus, the dorsal raphé nucleus, and the ventrolateral periaqueductal gray, thereby favoring the initiation of REM sleep [4, 7, 8]. Within the DPGi we recently identified the Nucleus papilio (NPCalb) as a bilateral, symmetric cluster of glutamatergic neurons expressing the calcium-binding protein Calbindin-D28k (Calb) [9]. Calb immunoreactivity in this nucleus is conserved in rodents (mouse and rat), monkey, and human. In mouse, it densely projects to the three contralateral eye-muscle nuclei (abducens, trochlear, and oculomotor), but also to several brain areas contributing to REM sleep control including the MCH-neurons of the lateral hypothalamus, the subcoeruleus nucleus (SubC), the pontine reticular formation (PnC), and the gigantocellular reticular nucleus (Gi). Noteworthy, activating or inactivating these neurons by means of optogenetics demonstrated both the necessity and sufficiency of the NPCalb for triggering eye movement during REM sleep [9].

The automated ALLENMINER search [10] of in situ hybridization (ISH) images in the Allen Brain Atlas (ABA) can be implemented to identify genes that are expressed in rather small agglomerations of cells, such as the PV1/Parvafox nucleus (parvalbumin-FoxB1 immunoreactive nucleus) in the lateral hypothalamus [11]. Using this protocol, potentially interesting information respecting very small neuronal populations can be elicited. By this means, ISH on adjacent sections has hitherto revealed most of the genes that were tested to be coexpressed with the mRNA for Pvalb [11]. We were therefore sanguine that a similar search would facilitate molecular and potentially functional characterization of the Calb-expressing neurons of the NPCalb. In addition, we screened the AGEA (Anatomic Gene expression Atlas) [12] at the ABA, focusing on genes expressed in the NPCalb/DPGi area.

Methods

Animals

For analyzing the expression of several proteins potentially coexpressed in the NPCalb, 15 C57BL/6J mice (from our animal facility) of both sexes, aged 10–14 weeks, as well as 3 Wistar rats (Janvier, Lyon, France), were used. For the analysis of the neurotransmitter status of the Cart-expressing neurons, two mice of each genotype were used (Slc17a6::Cre and Slc32a1::Cre encoding, respectively, VGlut2 glutamate and VGat GABA transporters, both obtained from the Jackson Laboratory; Slc6a5-GFP::Cre mice encoding the Glyt2 glycine transporter, obtained from Dr Zeilhofer, Pharmacology, Zurich). Fifteen C57BL/6J female mice were included in the REM sleep deprivation and rebound assay.

All animals were housed in our animal facilities and in accordance with the relevant Swiss laws. The Veterinary Commission for Animal Research of the Canton of Fribourg (Switzerland) approved this study.

Animals were anesthetized with pentobarbital (100 mg/kg of body weight) and then perfused via the left ventricle, first with chilled (4°C) physiological (0.9%) saline and then with chilled (4°C) 4% paraformaldehyde. The brains were excised and post-fixed overnight at 4°C in 4% paraformaldehyde and subsequently immersed in 0.1 M Tris buffer (pH 7.3) containing 20% sucrose in preparation for cryo-sectioning.

Immunohistochemistry

The various brain specimens were cryo-sectioned into 30, or 40, µm coronal sections and collected directly in 0.1 M Tris buffer containing 0.02% sodium azide, within which they were maintained until the time of analysis. The sections were immunostained according to standard protocols. Free-floating sections were incubated for 1–3 days at 4°C with primary antibody mixture diluted in TBS containing 0.1% Triton X-100 and 10% calf serum. The primary antibodies used are described in Table 1. Depending on the experiment, secondary antibodies included Cy3- or Cy2-conjugated anti-rabbit/mouse, Alexa488-conjugated anti-rabbit/mouse, Cy3- or Cy2-conjugated Streptavidin (Jackson Immunoresearch, Suffolk, UK), biotinylated anti-rabbit/mouse (Vector Laboratories, Servion, Switzerland), all used at the dilution recommended by the suppliers.

Table 1.

Description of Primary Antibodies Used for Immunohistochemistry

Antibody to Host species Antigen Manufacturer Catalog number Dilution used
Calbindin D-28K Mouse Whole chicken protein from gut Swant, Marly, Swizerland CB300 1–2,000
Calbindin D-28K Rabbit Recombinant rat calbindin D-28K Swant, Marly, Swizerland CB38 1–2,000
Cart Rabbit Rat Cart aa 55–102 Phoenix Pharmaceuticals,Karlsruhe, Germany H-003-62 1–2,000
Nesfatin Rabbit Rat Nesfatin aa 1–82 Phoenix Pharmaceuticals, Karlsruhe, Germany H-003-22 1–1,000
c-fos Mouse Recombinant human c-fos aa 1–380 Abcam, Cambridge, UK ab208942 1–2,000
ChAT Rabbit Pig ChAT aa 150–250 Abcam, Cambridge, UK ab178850 1–1,000
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The antigen, host species, manufacturer, catalog number, and working dilution are given for all primary antibodies used in this study.

Stereotactic injections in mouse brains

The experiment was conducted essentially as already described [9, 13]. Briefly, AAV2/1.CAG.Flex.Tomato.WPRE.bGH viral construct (Vector Core, University of Pennsylvania, USA) was stereotactically injected in the brain of either Slc17a6::Cre or Slc32a1::Cre mice. Injections were performed in the NPCalb, at the following Bregma coordinates: rostro-caudal: −6.36 mm, medio-lateral: −0.2 mm, and dorso-ventral: −4.35 mm. Two weeks after the stereotactic injections, the animals were anesthetized and perfused with 4% paraformaldehyde. The brains were excised and cryo-sectioned, and the specimens were analyzed immunohistochemically for Cart and Tomato expression. In these conditions, a specific and accurate Tomato expression is obtained in Cre-expressing neurons, as previously shown in Calb1::Cre mice [9].

REM sleep deprivation and rebound assay

Mice were deprived of REM sleep by implementing a modified version of the flower-pot technique, which spared the animals of major stress [14, 15]. Three groups were established: in the first group (“REM sleep deprivation and rebound” = REMS-D + R), the animals (n = 5) were maintained together for 72 h on six small stone platforms (7 × 4 cm for rats, 3 × 3 for mice), placed in a water tank. The surface of the platform was 1 cm above the water level. During this 72 h period, the animals had free access to food and water. Owing to the loss of the muscular tone that characterizes the onset of REM sleep, the animals fell into the water and were thereby deprived of REM sleep. After 72 h, the animals were transferred to a conventional cage in a quiet room and were permitted for 3 h to undergo REM sleep (=rebound). In the second group (“REM sleep deprivation” = REMS-D), the animals (n = 5) were sacrificed immediately after the termination of the 72 h REM sleep deprivation period, without recovery. In the third group (“control” = C), the animals (n = 5) were maintained in their cages under standard conditions for 72 h prior to sacrifice. The animals were anesthetized and perfused with fixative as described above under the “Animals” section. The brains were then excised and cryo-sectioned. The sections were immunostained for Cart as well as for c-fos, a surrogate marker of neuronal activity [16] The number of double-stained c-fos/Cart was determined on alternating 30 µm coronal sections, on the following brain areas: dorsal motor nucleus of vagus (10N)/nucleus of the solitary tract (Sol), Nucleus prepositus (Pr), DPGi, gigantocellular reticular nucleus (Gi), medial longitudinal fasciculus (mlf), and lateral paragigantocellular nucleus (LPGi). Only cells with strong Cart immunoreactivity were taken into account. The percentage of Cart+Fos+ relative to the total Cart+ cells was statistically compared between the three different conditions using a one-tailed Student’s t-test, for each of the anatomical areas investigated. Data from n = 4 animals in each of the conditions were used.

Image analysis

The specimens were evaluated either in a Leica epifluorescence microscope, a Nikon Eclipse Ni fluorescence microscope, a Leica TCS SP5 confocal laser microscope, or a Hamamatsu Nanozoomer scanner. Postprocessing of the images and contrast adjustments therein were performed using the Adobe Photoshop and Nanozoomer slide-processing software.

Informatics

A search of the adult mouse ABA (https://portal.brain-map.org/) was undertaken to identify genes that might be coexpressed with Calb1 in the targeted NPCalb. First, we downloaded three-dimensional expression data measured by ISH from coronal sections of the adult mouse brain using ALLENMINER (v2.0) [10]. Since the Calb1 gene expression in the target nucleus was restricted to a small region, we only used data from the coronal ISH series (4,216 datasets available for 3,968 genes), which are more densely sampled than the sagittal ones. Next, we defined a region of interest (ROI), which bilaterally encompassed the Calb1-expressing cells and the local neighborhood as a cube with boundaries in ABA coordinate space of 11–12 mm rostro-caudal, 5.4–6.4 mm dorso-ventral, and 5–6.4 mm medio-lateral. Within this ROI, the patterns of gene expression were then ranked relative to that for Calb1 (ABA image series 71717640, https://mouse.brain-map.org/experiment/show/71717640 and 79556672, https://mouse.brain-map.org/experiment/show/79556672), as measured by the Pearson’s correlation of the expression energy reported in corresponding voxels in the region (ALLENMINER run mode—sim search). An additional search was performed using the AGEA facility available at the ABA, allowing users to screen for genes expressed in a selected ROI [12]. The area investigated, focused on the DPGi, corresponded to AGEA coordinates: 11.154/5.422/5.887.

Results

Data mining for genes expressed in the Nucleus papilio in the mouse brain

The NPCalb was previously defined as a symmetric cluster of Calb-expressing neurons, lodged in the DPGi, which in the mouse brain spans a distance of ~0.6 mm, from Bregma levels −5.8 to −6.4 mm [9]. The ABA ISH data available for the Calb1 gene show that Calb1 mRNA expression fully recapitulates the protein expression (Figure 1) [9, 17].

Figure 1.

Figure 1.

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Expression of Calb1 mRNA (A and B) and protein (C) in the NPCalb. ISH images (A and B) were taken from the Allen Brain Atlas (Image credit: Allen Institute; http://mouse.brain-map.org/experiment/show/79556672). (B) It is a higher magnification of the image shown in (A), focusing only on one hemisphere. (C) It is a confocal image of a mouse brain coronal section immunostained for Calb protein, showing immunoreactivity in neuronal cell bodies within the DPGi and in neuritis in surrounding areas (Pr and Gi). DPGi, dorsal paragigantocellular nucleus; Gi, gigantocellular reticular nucleus; MVeMC, medial vestibular nucleus, magnocellular; MVePC, medial vestibular nucleus, parvicellular; NP, Nucleus papilio; Pr, prepositus nucleus; V4, fourth ventricle.

For the ALLENMINER search, we drew only on data that were derived from coronally sectioned ISH series (4,216 datasets available for 3,968 genes—see the “Methods” section). Consequently, they relate to only a fraction of the murine genome. Additional data mining using AGEA yielded 107 pages each comprising 20 genes, with ISH data also corresponding to coronal sections. The genes that were selected by both automated searches were further screened by visual inspection of the imaged ISH sections that traversed the NPCalb area. After this second round of screening, we eliminated genes for which the signals were so low as to raise doubts respecting their specificity and those with ubiquitous expression in the medulla oblongata.

The screens identified 141 genes, which are restrictedly expressed in discrete regions of the medulla oblongata, including the area comprising the NPCalb. Figures 2 and 3 illustrate examples of genes that manifest such restricted expression patterns, with a focus on the area corresponding to the NPCalb. The genes fall into several main categories (Figure 4; Tables 2–4):

Figure 2.

Figure 2.

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Genes showing expression in the area of the NPCalb—Part 1. The expression profile of the following genes is shown: (A) Calb1, (B) Cacna1g, (C) Cartpt, (D) Cck, (E) Cnr1, (F) Crh, (G) Crhr1, (H) Ecel1, (I) Fxyd7, (J) Grik1, (K) Grm8, (L) Htr2c, (M) Kcng3, (N) Kcnip3, and (O) Ly6h. For the complete name of the genes shown, as well as the function of the encoded proteins, see Tables 1–3. Red, respectively white, dashed lines delimit the NPCalb area. Black, respectively white, lines delimit the fourth ventricle. Data for the Calb1 gene are given in panel (A) as reference. For each gene are shown both the ISH image (left; obtained with a digoxygenin-based method) and the corresponding colored image (right; ranging from blue to red, respectively from low to high expression) (Image credit: Allen Institute; Calb1: http://mouse.brain-map.org/experiment/show/79556672; Cacna1g: http://mouse.brain-map.org/experiment/show/71587822; Cartpt: http://mouse.brain-map.org/experiment/show/72077479; Cck: http://mouse.brain-map.org/experiment/show/200; Cnr1: http://mouse.brain-map.org/experiment/show/79591675; Crh: http://mouse.brain-map.org/experiment/show/292; Crhr1: http://mouse.brain-map.org/experiment/show/297; Ecel1: http://mouse.brain-map.org/experiment/show/70231305; Fxyd7: http://mouse.brain-map.org/experiment/show/73592536; Grik1: http://mouse.brain-map.org/experiment/show/75749751; Grm8: http://mouse.brain-map.org/experiment/show/73771227; Htr2c: http://mouse.brain-map.org/experiment/show/73636098; Kcng3: http://mouse.brain-map.org/experiment/show/71717451; Kcnip3: http://mouse.brain-map.org/experiment/show/71587887; Ly6h: http://mouse.brain-map.org/experiment/show/71924388).

Figure 3.

Figure 3.

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Genes showing expression in the area of the NPCalb—Part 2. The expression profile of the following genes is shown: (A) Necab2, (B) Nell2, (C) Nnat, (D) Nos1, (E) Nptx1, (F) Ntng1, (G) Nucb2, (H) Nxph1, (I) Pnoc, (J) Ptpro, (K) Rgs10, (L) Scn3b, (M) Sez6, (N) Slit1, and (O) Sncg. See the legend of Figure 2 for details (Image credit: Allen Institute; Necab2: http://mouse.brain-map.org/experiment/show/73788010; Nell2: http://mouse.brain-map.org/experiment/show/72103854; Nnat: http://mouse.brain-map.org/experiment/show/77887874; Nos1: http://mouse.brain-map.org/experiment/show/75147762; Nptx1: http://mouse.brain-map.org/experiment/show/73520998; Ntng1: http://mouse.brain-map.org/experiment/show/71924185; Nucb2: http://mouse.brain-map.org/experiment/show/75774683; Nxph1: http://mouse.brain-map.org/experiment/show/75084479; Pnoc: http://mouse.brain-map.org/experiment/show/75038402; Ptpro: http://mouse.brain-map.org/experiment/show/72340109; Rgs10: http://mouse.brain-map.org/experiment/show/74511849; Scn3b: http://mouse.brain-map.org/experiment/show/71064082; Sez6: http://mouse.brain-map.org/experiment/show/71063725; Slit1: http://mouse.brain-map.org/experiment/show/73788105; Sncg: http://mouse.brain-map.org/experiment/show/72081426).

Figure 4.

Figure 4.

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Proportions of the several categories of genes expressed in the NPCalb area. See also Tables 1–3 for details on gene name and function.

Table 2.

Genes Expressed in the DPGi/NPCalb Region—Part 1

Gene Complete name Molecular activity Biological process
Adarb1 Adenosine deaminase, RNA-specific, B1 Enzyme Nucleic acid processing
Adcyap1 Adenylate cyclase activating polypeptide 1 (=PACAP) Neuropeptide Neurotransmission/synapse functioning (neuropeptide) [18, 19]
Adk Adenosine kinase Enzyme Metabolism (adenine) [20]
Ajap1 Adherens junction associated protein 1 (=Shrew1) Cell adhesion/ECM/axon guidance
Apba1 Amyloid beta (A4) precursor protein binding, family A, member 1 (=X11/Mint1) Adaptator protein Neurotransmission/synapse functioning (neurotransmitter release)
Arl10 ADP-ribosylation factor-like 10 GTPase activity Multiple
Asic2 Acid-sensing (proton-gated) ion channel 2 Sodium channel Ion channel
Baiap3 BAI1-associated protein 3 Neurotransmission/synapse functioning (SNARE-dependent exocytosis)
Btbd11 BTB (POZ) domain containing 11
Cacna1g Calcium channel, voltage-dependent, T type, alpha 1G subunit (=Cav3.1) Calcium channel Ion channel [21–23]
Cacna1h Calcium channel, voltage-dependent, T type, alpha 1H subunit (=Cav3.2) Calcium channel Ion channel [21–23]
Cacna2d1 Calcium channel, voltage-dependent, alpha2/delta subunit 1 (=a2d1) Calcium channel Ion channel
Cacna2d3 Calcium channel, voltage-dependent, alpha2/delta subunit 3 (=a2d3) Calcium channel Ion channel
Cacng5 Calcium channel, voltage-dependent, gamma subunit 5 Calcium channel Ion channel
Cadps2 Ca2+-dependent activator protein for secretion 2 Neurotransmission/synapse functioning (dendritic spine maintenance)
Calb1 Calbindin 1 (=Calbindin D28k) EF-hand Ca binding; calcium sensor/buffer Calcium homeostasis [9]
Calb2 Calbindin 2 (=Calretinin) EF-hand Ca binding; calcium sensor/buffer Calcium homeostasis
CamkV CaM kinase-like vesicle-associated Enzyme Neurotransmission/synapse functioning
Cartpt CART prepropeptide Neuropeptide Neurotransmission/synapse functioning (neuropeptide) [24–26]
Cck Cholecystokinin Neuropeptide Neurotransmission/synapse functioning (neuropeptide) [27, 28]
Cdh8 Cadherin 8 Protein binding Cell adhesion/ECM/axon guidance
Cdh13 Cadherin 13 (=Tcad) Protein binding Cell adhesion/ECM/axon guidance
Cd24a CD24a antigen Protein binding Cell adhesion/ECM/axon guidance
Chrm2 Cholinergic receptor, muscarinic 2, cardiac (=AChR-M2) GPCR Neurotransmission/synapse functioning (cholinergic receptor) [29–31]
Chrm3 Cholinergic receptor, muscarinic 3, cardiac (=AChR-M3) GPCR Neurotransmission/synapse functioning (cholinergic receptor) [29–31]
Cnr1 Cannabinoid receptor 1 (brain) (=CB1) GPCR Neurotransmission/synapse functioning (cannabinoid receptor) [32, 33]
Cntnap2 Contactin associated protein-like 2 (=Caspr2) Protein binding Cell adhesion/ECM/axon guidance [34]
Coch Cochlin Protein binding Immunity
Col6a1 Collagen, type VI, alpha 1 ECM structural component Cell adhesion/ECM/axon guidance
Col27a1 Procollagen, type XXVII, alpha 1 ECM structural component Cell adhesion/ECM/axon guidance
Cpne6 Copine VI Ca binding; Ca sensor Neurotransmission/synapse functioning
Crh Corticotropin releasing hormone Neuropeptide Neurotransmission/synapse functioning (neuropeptide) [35]
Crhr1 Corticotropin releasing hormone receptor 1 GPCR Neurotransmission/synapse functioning (neuropeptide receptor)
Crtac1 Cartilage acidic protein 1 (=Lotus) Ca binding; protein binding Cell adhesion/ECM/axon guidance
Ctxn1 Cortexin 1
Cux2 Cut-like homeobox 2 Transcription factor Nucleic acid processing
Cyp26b1 Cytochrome P450, family 26, subfamily b, polypeptide 1 Enzyme Metabolism
Deptor DEP domain containing MTOR-interacting protein (=Depdc6) Cell signaling
Dkk3 Dickkopf WNT signaling pathway inhibitor 3 Secreted ligand Cell signaling
Dpp10 Dipeptidylpeptidase 10 Enzyme Proteolysis
Ecel1 Endothelin converting enzyme-like 1 Enzyme Multiple
Esyt1 Extended synaptotagmin-like protein 1 Ca/lipid/protein binding Intracellular lipid dynamics
Fbxw7 F-box and WD-40 domain protein 7 Protein binding Cell signaling
Foxa1 Forkhead box A1 Transcription factor Nucleic acid processing
Foxp1 Forkhead box P1 Transcription factor Nucleic acid processing
Fxyd6 FXYD domain-containing ion transport regulator 6 Na-K ATPase regulator Ion channel regulation
Fxyd7 FXYD domain-containing ion transport regulator 7 Na-K ATPase regulator Ion channel regulation
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A list of the genes that are restrictedly expressed in discrete regions of the murine medulla oblongata, including the region embracing the Nucleus papilio, as revealed by ALLENMINER and AGEA searches of the ISH images in the ABA. The abbreviated as well as the full name of each gene are given, together with their known or putative molecular activity and functions (in ontologic terms). In gray: genes that have been experimentally implicated in the regulation of the sleep/wake cycle, with references indicated.

Table 3.

Genes Expressed in the DPGi/NPCalb Region—Part 2 (see the footnote of Table 2)

Gene Complete name Molecular activity Biological process
Gabra1 Gamma-aminobutyric acid (GABA) A receptor, subunit alpha 1 Transmitter gated ion channel activity Neurotransmission/synapse functioning (GABA receptor)
Gad1 Glutamic acid decarboxylase 1 Enzyme Neurotransmission/synapse functioning (GABA synthesis)
Gad2 Glutamic acid decarboxylase 2 Enzyme Neurotransmission/synapse functioning (GABA synthesis)
Glra1 Glycine receptor, alpha 1 subunit Transmitter gated ion channel activity Neurotransmission/synapse functioning (glycine receptor)
Glra4 Glycine receptor, alpha 4 subunit Transmitter gated ion channel activity Neurotransmission/synapse functioning (glycine receptor)
Gpr125 G protein-coupled receptor 125 (=Adgra3) GPCR Cell signaling
Gpr137 G protein-coupled receptor 137 GPCR Cell signaling
Grid1 Glutamate receptor, ionotropic, delta 1 Transmitter gated ion channel activity Neurotransmission/synapse functioning (glutamate receptor)
Grik1 Glutamate receptor, ionotropic, kainate 1 Transmitter gated ion channel activity Neurotransmission/synapse functioning (kainate/glutamate receptor)
Grin3a Glutamate receptor ionotropic, NMDA3A Transmitter gated ion channel activity Neurotransmission/synapse functioning (NMDA/glutamate receptor)
Grm8 Glutamate receptor, metabotropic 8 GPCR Neurotransmission/synapse functioning (glutamate receptor)
Grsf1 G-rich RNA sequence binding factor 1 RNA binding Nucleic acid processing
Gsta4 Glutathione S-transferase, alpha 4 Enzyme Metabolism (glutathione metabolism)
Hap1 Huntingtin-associated protein 1 Protein binding Axonal transport
Hcn1 Hyperpolarization-activated, cyclic nucleotide-gated K+ 1 Potassium channel Ion channel
Htr2c 5-Hydroxytryptamine (serotonin) receptor 2C GPCR Neurotransmission/synapse functioning (serotonin receptor) [36]
Igsf21 Immunoglobulin superfamily, member 21 Protein binding Cell adhesion/ECM/axon guidance
Itm2c Integral membrane protein 2C
Kcna1 Potassium voltage-gated channel, shaker-related subfamily, member 1 (=Kv1.1) Potassium channel Ion channel
Kcnab1 Potassium voltage-gated channel, shaker-related subfamily, beta member 1 (=Kv1.3) Potassium channel Ion channel
Kcnc2 Potassium voltage gated channel, Shaw-related subfamily, member 2 (=Kv3.2) Potassium channel Ion channel [37]
Kcnc3 Potassium voltage gated channel, Shaw-related subfamily, member 3 (=Kv3.3) Potassium channel Ion channel [38]
Kcng3 Potassium voltage-gated channel, subfamily G, member 3 (=Kv6.3) Potassium channel Ion channel
Kcng4 Potassium voltage-gated channel, subfamily G, member 4 (=Kv6.4) Potassium channel Ion channel
Kcnj3 Potassium inwardly rectifying channel, subfamily J, member 3 Potassium channel Ion channel
Kcnip1 Kv channel-interacting protein 1 (=KCHIP1) K channel regulator (Ca binding) Ion channel regulation
Kcnip4 Kv channel interacting protein 4 (=KCHIP4) K channel regulator (Ca binding) Ion channel regulation
Lrrn1 Leucine rich repeat protein 1, neuronal
Ly6h Lymphocyte antigen 6 complex, locus H Prototoxin Neurotransmission/synapse functioning (modulation of AchR activity)
Megf11 Multiple EGF-like-domains 11 Cell adhesion/ECM/axon guidance
Mesdc2 Mesoderm development candidate 2 Chaperone LDL Cell signaling
Myo5b Myosin VB Multiple Cytoskeleton dynamics
Ndst4 N-deacetylase/N-sulfotransferase (heparin glucosaminyl) 4 Enzyme Metabolism (glycosaminoglycan)
Necab2 N-terminal EF-hand calcium binding protein 2 EF-hand Ca binding
Necab3 N-terminal EF-hand calcium binding protein 3 EF-hand Ca binding
Nell2 NEL-like 2 (neural EGF like 2) Ca/protein/ECM binding Cell adhesion/ECM/axon guidance
Nnat Neuronatin Multiple
Nos1 Nitric oxide synthase 1, neuronal Enzyme Neurotransmission/synapse functioning (NO synthesis) [39–41]
Nos1ap Nitric oxide synthase 1 (neuronal) adaptor protein (=Capon) Protein binding Neurotransmission/synapse functioning (NO synthesis)
Npnt Nephronectin Ca/protein/ECM binding Cell adhesion/ECM/axon guidance
Nptx1 Neuronal pentraxin 1 (=NP1) Neurotransmission/synapse functioning
Nrg1 Neuregulin 1 Protein binding Cell signaling
Nrn1 Neuritin 1 Neurotransmission/synapse functioning
Ntng1 Netrin G1 Protein binding Cell adhesion/ECM/axon guidance
Nucb2 Nucleobindin 2 Ca2+/DNA binding/neuropeptide Neurotransmission/synapse functioning (neuropeptide) [42, 43]
Nxph1 Neurexophilin 1 Neurexin ligand Neurotransmission/synapse functioning (neuropeptide-like)
Nxph4 Neurexophilin 4 Neurexin ligand Neurotransmission/synapse functioning (neuropeptide-like)
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Table 4.

Genes Expressed in the DPGi/NPCalb Region—Part 3 (see the footnote of Table 2)

Gene Complete name Molecular activity Biological process
Pcp4 Purkinje cell protein 4 Ca/protein binding Multiple
Pcp4l1 Purkinje cell protein 4-like 1 Ca/protein binding Multiple
Penk Preproenkephalin Neuropeptide Neurotransmission/synapse functioning (neuropeptide)
Pnoc Prepronociceptin Neuropeptide Neurotransmission/synapse functioning (neuropeptide)
Psd Pleckstrin and Sec7 domain containing (=EFA6) GEF activity Axonal transport
Ptpro Protein tyrosine phosphatase, receptor type, O Receptor/enzyme Neurotransmission/synapse functioning (promotes synapse formation)
Pvalb Parvalbumin EF-hand Ca binding; calcium sensor/buffer Calcium homeostasis [44]
Rec8 REC8 meiotic recombination protein Chromatin binding Nucleic acid processing
Rgs4 Regulator of G-protein signaling 4 GTPase activator Cell signaling
Rgs10 Regulator of G-protein signaling 10 GTPase activator Cell signaling
Scn3b Sodium channel, voltage-gated, type III, beta Sodium channel Ion channel
Scn4b Sodium channel, type IV, beta Sodium channel Ion channel
Scrt1 Scratch family zinc finger 1 Transcription repressor Nucleic acid processing
Sdk2 Sidekick homolog 2 (chicken) Cell adhesion/ECM/axon guidance
Slc6a7 Solute carrier family 6 (neurotransmitter transporter, l-proline), member 7 Proline transporter Multiple
Slc8a1 Solute carrier family 8 (sodium/calcium exchanger), member 1 (=Ncx1) Ca/Na antiporter Calcium homeostasis
Slc17a6 Solute carrier family 6 (sodium-dependent inorganic phosphate cotransporter), member 6 Vesicular neurotransmitter transporter Neurotransmission/synapse functioning (=VGlut2, glutamate transporter)
Slc17a7 Solute carrier family 6 (sodium-dependent inorganic phosphate cotransporter), member 7 Vesicular neurotransmitter transporter Neurotransmission/synapse functioning (=VGlut1, glutamate transporter)
Slc32a1 Solute carrier family 32 (GABA vesicular transporter), member 1 Vesicular neurotransmitter transporter Neurotransmission/synapse functioning (=VGAT, GABA transporter)
Slc36a1 Solute carrier family 36 (proton/amino acid symporter), member 1 Transmembrane transporter Multiple
Sema3a Semaphorin 3A Protein/ECM binding Cell adhesion/ECM/axon guidance
Sema6a Semaphorin 6a Protein/ECM binding Cell adhesion/ECM/axon guidance
Sez6 Seizure related gene 6 (=BSRP-C) Neurotransmission/synapse functioning (shaping dendritic arborization)
Sez6l Seizure related 6 homolog like Neurotransmission/synapse functioning (shaping dendritic arborization)
Sh3bgrl2 SH3 domain binding glutamic acid-rich protein like 2
Slit1 Slit guidance ligand 1 Ca/protein/ECM binding Cell adhesion/ECM/axon guidance
Slit2 Slit guidance ligand 2 Ca/protein/ECM binding Cell adhesion/ECM/axon guidance
Snca Synuclein, alpha Protein binding Multiple (involved in synucleinopathies including PD; RBD) [45, 46]
Sncg Synuclein, gamma Protein binding Multiple
Sphkap SPHK1 interactor, AKAP domain containing (=SKIP) A kinase anchoring protein
Spp1 Secreted phosphoprotein 1 (=OPN) Cytokine/ECM binding Cell adhesion/ECM/axon guidance
Steap2 Six transmembrane epithelial antigen of prostate 2 Enzyme Multiple
Sv2b Synaptic vesicle glycoprotein 2 b Transmembrane transporter Neurotransmission/synapse functioning (vesicular transport/exocytosis)
Sv2c Synaptic vesicle glycoprotein 2c Transmembrane transporter Neurotransmission/synapse functioning (vesicular transport/exocytosis)
Syt4 Synaptotagmin IV Ca/protein/lipid binding Neurotransmission/synapse functioning (vesicular transport/exocytosis)
S100a10 S100 calcium-binding protein A10 (=calgizzarin) EF-hand Ca binding/protein binding Multiple
S100b S100 protein, beta polypeptide, neural EF-hand Ca binding/protein binding Multiple (marker in sleep disturbance syndromes and PD)
Tesc Tescalcin EF-hand Ca binding/protein binding Multiple
Tmem65 Transmembrane protein 65
Tpbg Trophoblast glycoprotein
Usp11 Ubiquitin-specific peptidase 11 Enzyme Proteolysis
Vat1l Vesicle amine transport protein 1 homolog-like
Whrn Whirlin Cytoskeleton dynamics
Zfp385b Zinc finger protein 385B Transcription factor Nucleic acid processing
Zfhx4 Zinc finger homeodomain 4 Transcription factor Nucleic acid processing
Zfp365 Zinc finger protein 365 Transcription factor Nucleic acid processing
Zkscan16 Zinc finger with KRAB and SCAN domains 16 Transcription factor Nucleic acid processing
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  • “Neurotransmission”: Neuropeptides: Adcyap1; Cartpt, Cck, Crh, Nucb2, Nxph1, Nxph4, Penk, Pnoc. Neurotransmitter/neuropeptide receptor: Chrm2, Chrm3, Cnr1, Crhr1, Gabra1, Glra1, Glra4, Grid1, Grik1, Grin3a, Grm8, Htr2c. Neurotransmitter synthesis/transport/release/exocytosis: Apba1, Baiap3, Gad1, Gad2, Nos1, Nos1ap; Slc17a6, Slc17a7, Slc32a1, Sv2b, Sv2c, Syt4.

  • “Synapse functioning”: Cadps2, Nrn1, Ptpro, Sez6, Sez6l, Sv2b, Sv2c.

  • “Ion channel”: For calcium: Cacna1g, Cacna1h, Cacna2d1, Cacna2d3, Cacng5. For potassium: Hcn1, Kcna1, Kcnb1, Kcnc2, Kcnc3, Kcng3, Kcng4, Kcnj3. For sodium: Asic2, Scn3b, Scn4b. Ion channel regulation: Fxyd6, Fxyd7, Kcnip1, Kcnip4.

  • “Cell adhesion/Extracellular matrix/Axon guidance”: Ajap, Cdh8, Cdh13, Cd24a, Cntnap2, Col6a1, Col27a1, Crta1, Igsf21, Megf11, Nell2, Npnt, Ntng1, Sdk2, Sema3a, Sema6a, Slit1, Slit2, Spp1.

  • “Nucleic acid processing”: Adarb1, Cux2, Foxa1, Foxp1, Grsf1, Rec8, Scrt1, Zfp365, Zfp385b, Zfhx4, Zkscan16.

Some other less represented categories included metabolism, cell signaling, and calcium homeostasis.

Identifying neurotransmitters and neuropeptides expressed in the Nucleus papilio

We have started previously to investigate the neurotransmitter status of NPCalb neurons, by showing that a significant proportion (33.8%) of Calb-positive neurons were glutamatergic and that none were GABAergic [9]. Analyzing the ABA ISH data on sagittal sections revealed that the Slc6a5 gene, encoding the Glyt2 glycine vesicular transporter, and the Chat gene, encoding choline acetyltransferase, were also expressed in the region of the medulla oblongata. Immunostaining for Calb on coronal sections from GlyT2-GFP mouse brain revealed that NPCalb neurons were not glycinergic (Figure 5A–C′). Similarly, double immunostaining for Calb and Chat revealed the absence of Chat expression in NPCalb neurons (Figure 5D–F′).

Figure 5.

Figure 5.

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NPCalb neurons are neither glycinergic nor cholinergic. (A–C′) Coronal sections from a GlyT2-GFP mouse brain, stained for Calb (red) and GFP (green). The dashed square in (C) marks the area shown at higher magnification in panels (A′–C′). (D–F′) Coronal sections from a C57Bl6 mouse brain, stained for Calb (red) and ChAT (green). The dashed square in (F) marks the area shown at higher magnification in panels (D′–F′). Bars represent 100 µm.

Our search identified several genes encoding neuropeptides that are likely to be coexpressed with Calb1, namely, Cartpt (encoding Cart, cocaine-and-amphetamine-regulated transcript), Cck (encoding cholecystokinin), Crh (encoding corticotrophin-releasing hormone), Nucb2 (encoding nesfatin 1), Penk (encoding preproenkephalin), and Pnoc (encoding prepronociceptin) (see Figures 2 and 3 for the ABA ISH data). For the immunohistochemical revelation of coexpression, coronal sections through murine brains were double-stained for Calb and two of these neuropeptides. Nesfatin immunoreactivity was present in all Calb-immunoreactive neurons of the NPCalb (Figure 6A–C and A′–C′), whereas that for Cart (Figure 6D–F and D′–F′) was apparent in some, but not all. In addition, some cells lying close to the Calb-immunoreactive neurons were positive for each of the tested peptides, but not for Calb. Noteworthy, similar observations were made using sections from rat brains (not shown). We estimated that 27.2% ± 5.6% of the DPGi Calb+ cells were Cart+ (counting performed on each second sections in n = 8 mice) and that 65.9% ± 13.7% of the DPGi Cart+ cells were Calb+ (counting performed in n = 3 mice).

Figure 6.

Figure 6.

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Cart and Nesfatin neuropeptide expression in NPCalb neurons. Coronal sections from a C57Bl6 mouse brain, stained for Calb and either Nesfatin (A–C) or Cart (D–F) in the NPCalb. All Calb-positive NPCalb-neurons coexpress Nesfatin, while coexpression with Cart is limited to few neurons (marked by arrows in D′–F′). Panels (A′–C′) and (D′–F′) are higher magnifications. All images were obtained with confocal microscope. Bars represent 100 µm.

Cart-expressing neurons within the DPGi are glutamatergic

With the aim of analyzing the neurotransmitter status of the Cart-expressing neurons in the NPCalb/DPGI area, glutamatergic, respectively GABAergic, neurons within the medulla oblongata were labeled by means of fluorescent adenovirus tracer injection in Slc17a6::Cre mouse brains (encoding the VGlut2 glutamate transporter) and Slc32a1::Cre (encoding the VGAT GABA transporter). In these conditions, glutamatergic, respectively GABAergic, cell bodies could be easily identified by their strong fluorescence. Co-staining with anti-Cart antibody revealed that all Cart-positive cells within the DPGi were VGlut2-positive and that none was VGat-positive, highlighting their glutamatergic nature (Figure 7). In addition, Cart-positive cells located in either the adjacent Nucleus prepositus or the gigantocellular reticular nucleus were also mostly glutamatergic, while only very few Cart-positive cells within the Nucleus prepositus appeared as GABAergic.

Figure 7.

Figure 7.

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Cart-expressing neurons within the DPGi are glutamatergic. Immunostaining with Cart antibody reveals the glutamatergic nature of all Cart-expressing neurons in the DPGI. Shown are representative coronal sections through the medulla oblongata of a brain from (A–C′) Slc7a6::Cre (VGlut2-Cre) and (D–F′) Slc32a1::Cre (VGAT-Cre) mice, injected with Cre-dependent AAV-Tomato. In (C), respectively (F), a dashed square indicates the area presented at higher magnification in (A′–C′), respectively (D′–F′). White arrows in (A) and (D) point to Cart+ cells. Within the DPGi and the Gi, all Cart+ neurons are glutamatergic (yellow arrows in B), while none are GABAergic (white arrows in E). On the contrary, in the prepositus nucleus both glutamatergic and GABAergic Cart+ neurons are visible (yellow arrows pointing to Pr neurons in B and E). Bars represent 100 µm.

Cart-expressing neurons within the DPGi are activated during REM sleep

In an experimental paradigm consisting of REM sleep rebound following a 72 h REM sleep deprivation, we could demonstrate that neurons of the DPGi [47–49], and particularly the NPCalb neurons [9], were activated during REM sleep, as demonstrated by neuronal c-fos immunoreactivity. A similar test performed on mice revealed that Cart-positive neurons localized within the DPGi of the medulla oblongata were activated during REM sleep too. Only in the DPGi significant differences between the three different groups analyzed could be observed (REM sleep deprivation group; REM sleep deprivation + rebound group; Control group) (Figure 8). Indeed, in the REM sleep rebound group, we quantified 41.6% ± 6.9% of DPGi Cart-positive neurons displaying c-fos immunoreactivity and 31% ± 6.5% of DPGi c-fos+ cells being Cart+. Analyzing the other areas (including the Gi, the LPGi, the mlf, the 10N/Sol), no significant difference between the three groups was observed (Figure 8). This experiment suggests that Cart-expressing neurons in the DPGi are activated during the REM phase of sleep.

Figure 8.

Figure 8.

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Analysis of c-fos immunoreactivity in Cart-expressing neurons in the medulla oblongata during REM sleep. (A–C) Representative coronal section from the brain of an animal of the group “REM sleep deprivation and rebound,” stained for Cart (red) and c-fos (green). The two arrows in panel C point to Cart+c-fos+ neurons. (D–F) Representative brain coronal sections from animals of each three groups used for the REM sleep deprivation and rebound assay (REMSD + R, REM sleep deprivation and rebound; REMSD, REM sleep deprivation; C, Control), immunostained for c-fos. (G) Percentage of c-fos immunoreactive cells within the Cart+ population, in different brain areas. Counting was performed on alternating coronal sections from three animals of each three groups (REMSD + R; REMSD; C). Statistical significance at p < 0.05 between the different groups was achieved only in the DPGi; group REMS-D+R versus group C: t = 10.78389 and p = 0.000019; group REMS-D+R versus group REMS-D: t = 10.78389 and p = 0.000019. DPGi, dorsal paragigantocellular nucleus; Gi, gigantocellular reticular nucleus; LPGi, lateral paragigantocellular nucleus; mlf, medial longitudinal fasciculus; MVe, medial vestibular nucleus; Pr, prepositus nucleus; 4V, fourth ventricle; 10N, dorsal motor nucleus of vagus/Sol: nucleus of the solitary tract.

Discussion

The Nucleus papilio is a recently described brainstem structure, characterized by Calb immunoreactivity, and by its involvement in triggering eye movement during the REM sleep period [9]. Through an extensive data mining of the ABA, we propose here a list of genes that are likely to be expressed in the NPCalb. Several of these genes encode proteins involved in the synthesis/transport of neurotransmitters, namely, Slc17a7 and Slc17a6 (encoding VGlut1- and VGlut2-glutamate transporter, respectively), Gad1/2 (encoding glutamic acid decarboxylase, which is responsible for the production of GABA), and Slc32a1 (encoding a GABA transporter). By injecting Cre-dependent AAV-Tomato virus in Slc17a6::Cre and Slc17a7::Cre (both specific for glutamatergic neurons) and Slc32a1::Cre mice (specific for GABAergic neurons), we were able to show the absence of Calb immunoreactivity in the GABAergic neurons of the DPGi, whereas a significant proportion of the Calb-positive neurons composing the NPCalb were of glutamatergic nature [9]. Analyzing the list of potential genes expressed in the NPCalb, several glutamate receptors and a single GABA receptor were found (Tables 2–4).

Of special interest for us were the genes encoding neuropeptides. Nesfatin 1 is derived from a precursor, nucleobindin 2, which via posttranslational cleavage, yields either the neuropeptide nesfatin 1 or the DNA/Ca2+-binding proteins nesfatin 2 and 3. Nesfatin 1 has been identified as a satiety molecule in the hypothalamus [50]. Albeit so, its widespread extra-hypothalamic expression indicates that it might exert endocrine and autonomic effects on energy expenditure [51]. Cart peptides have been implicated in the regulation not only of food intake and body weight, but also of a variety of physiological processes, including drug reward/reinforcement and stress [52, 53], findings that are consistent with the complex pattern of Cart immunoreactivity in the rat brain [54]. Cart has been shown to coexist with Calb-D28k in granule cells of the dentate gyrus [55] and to be coexpressed with nesfatin in several hypothalamic and non-hypothalamic areas of the rat brain [51, 56, 57], including MCH-neurons. Data presented in the present study indicate that both peptides are coexpressed also in some Calb-immunoreactive neurons of the NPCalb. Several studies that have been recently conducted afford evidence for a role of nesfatin 1 in the regulation of REM sleep. Disruption of nesfatin signaling in the tuberal hypothalamic neurons, by the intra-cerebroventricular administration of either an antiserum against the neuropeptide or Nucb2 antisense, has been shown to suppress REM sleep [24]. Similarly, deprivation of REM sleep led to a downregulation of nesfatin 1 expression, which was reverted during REM rebound [25]. And finally, the activity of these hypothalamic nesfatin-positive neurons (which are also MCH positive), as monitored by c-fos immunostaining, was correlated with REM sleep [24, 25]. Clear evidence in favor of an involvement of Cart in the regulation of the sleep/wake regulation has not been forthcoming [26]. Indeed, although an intra-cerebroventricular injection of the Cart55-102-peptide promotes the wake phase in rats [18], both Cart-positive and Cart-negative MCH-immunoreactive neurons in the hypothalamus are activated during REM sleep [19, 20]. Our finding that a significant proportion of Cart-expressing neurons in the DPGi were c-fos-positive following REM sleep rebound is thus particularly interesting and suggests a possible involvement of Cart in regulating some aspects of REM sleep.

The Calb-expressing neurons forming the NPCalb appear to form a heterogeneous population, involved partly in controlling eye movement during REM sleep [9], with a substantial number being excitatory glutamatergic neurons, and with the neuropeptides Cart and nociceptin being expressed only in a subset of these neurons while nesfatin being present in all of them (this study). In addition, the surrounding DPGi also contains another pool of inhibitory GABAergic neurons involved in the initiation of REM sleep, as well as glycinergic and cholinergic neurons [4, 7, 8]. Deciphering the specific neuronal connections made by these particular neuronal populations will be challenging toward a better understanding of the functions of this nucleus in regulating various aspects of REM sleep. Indeed, apart from their connections to the three eye motor nuclei, we observed strong efferent connections of the NPCalb neurons to several of the brain areas involved in the initiation and the maintenance of REM sleep (including the subcoeruleus nucleus and the pontine reticular nuclei) [9], suggesting additional roles for the NPCalb in regulating some aspects of REM sleep.

Acknowledgments

We thank Simone Eichenberger, Drs Viktoria Szabolcsi, and Diana Waldmeyer-Roccaro for their technical assistance. We thank Dr Zeilhofer for the kind gift of GlyT2-GFP mice.

Funding

This work was funded by the Canton of Fribourg (Switzerland) and the Swiss National Foundation (31003A-144036 and 320030-179565).

Disclosure Statement

Financial disclosure: none.

Nonfinancial disclosure: none.

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