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

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.


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][3][4][5][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 (NP Calb ) 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 NP Calb 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 NP Calb . In addition, we screened the AGEA (Anatomic Gene expression Atlas) [12] at the ABA, focusing on genes expressed in the NP Calb /DPGi area.

Animals
For analyzing the expression of several proteins potentially coexpressed in the NP Calb , 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.

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 NP Calb , at the following Bregma coordinates: rostro-caudal: −6.36 mm, mediolateral: −0.2 mm, and dorso-ventral: −4.35 mm. Two weeks after the stereotactic injections, the animals were anesthetized The antigen, host species, manufacturer, catalog number, and working dilution are given for all primary antibodies used in this study.    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 NP Calb . First, we downloaded threedimensional 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.

Data mining for genes expressed in the Nucleus papilio in the mouse brain
The NP Calb was previously defined as a symmetric cluster of Calbexpressing 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].
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 NP Calb 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 NP Calb . Figures 2 and 3 illustrate examples of genes that manifest such restricted expression patterns, with a focus on the area corresponding to the NP Calb . The genes fall into several main categories ( Figure 4; Tables 2-4):

Identifying neurotransmitters and neuropeptides expressed in the Nucleus papilio
We have started previously to investigate the neurotransmitter status of NP Calb 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 NP Calb neurons were not glycinergic ( Figure 5A-C′). Similarly, double immunostaining for Calb and Chat revealed the absence of Chat expression in NP Calb neurons ( Figure 5D-F′).
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 NP Calb ( 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).

Cart-expressing neurons within the DPGi are glutamatergic
With the aim of analyzing the neurotransmitter status of the Cart-expressing neurons in the NP Calb /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.

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][48][49], and particularly the NP Calb 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. 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.

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 NP Calb . Several of these genes encode proteins involved in the synthesis/transport of neurotransmitters, namely, Slc17a7 and Slc17a6 (encoding VGlut1-and VGlut2glutamate 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 NP Calb were of glutamatergic nature [9]. Analyzing the list of potential genes expressed in the NP Calb , 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/Ca 2+ -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 NP Calb . 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 NP Calb 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 NP Calb 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 NP Calb in regulating some aspects of REM sleep.