Journal of Chemical Neuroanatomy 61–62 (2014) 72–82

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Different distributions of preproMCH and hypocretin/orexin in the forebrain of the pig (Sus scrofa domesticus) S. Chometton a, G. Franchi a, C. Houdayer a, A. Mariot a, F. Poncet a, D. Fellmann a, Y. Tillet b, P.Y. Risold a,* a b

EA3922, UFR Sciences Me´dicales et Pharmaceutiques, SFR-FED 4234, Universite´ de Franche-Comte´, Besanc¸on 25000, France UMR 7247 INRA/CNRS/Universite´ Franc¸ois Rabelais de Tours/IFCE, SFR-FED 4226, Centre INRA Val de Loire, Nouzilly, Tours 37380, France

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 May 2014 Received in revised form 1 August 2014 Accepted 1 August 2014 Available online 11 August 2014

Neurons producing melanin-concentrating hormone (MCH) or hypocretin/orexin (Hcrt) have been implicated in the sleep/wake cycle and feeding behavior. Sleep and feeding habits vary greatly among mammalian species, depending in part of the prey/predatory status of animals. However, the distribution of both peptides has been described in only a limited number of species. In this work, we describe the distribution of MCH neurons in the brain of the domestic pig. Using in situ hybridization and immunohistochemistry, their cell bodies are shown to be located in the posterior lateral hypothalamic area (LHA), as expected. They form a dense cluster ventro-lateral to the fornix while only scattered cells are present dorsal to this tract. By comparison, Hcrt cell bodies are located mainly dorsal to the fornix. Therefore, the two populations of neurons display complementary distributions in the posterior LHA. MCH projections are, as indicated by MCH-positive axons, very abundant in all cortical fields ventral to the rhinal sulcus, as well as in the lateral, basolateral and basomedial amygdala. In contrast, most of the isocortex is sparsely innervated. To conclude, the distribution of MCH cell bodies and projections shows some very specific features in the pig brain, that are clearly different of that described in the rat, mouse or human. In contrast, the Hcrt pattern seems more similar to that in these species, i.e. more conserved. These results suggest that the LHA anatomic organization shows some very significant interspecies differences, which may be related to the different behavioral repertoires of animals with regard to feeding and sleep/wake cycles. ß 2014 Elsevier B.V. All rights reserved.

Keywords: Comparative anatomy Lateral hypothalamic area Amygdala Pig brain

Abbreviations: ac, anterior commissure; ACB, nucleus accumbens; AD, anterodorsal nucleus thalamus; AHN, anterior hypothalamic nucleus; AMY, amygdala; arb, arbor vitae; ARH, arcuate hypothalamic nucleus; AV, anteroventral nucleus thalamus; BL, basolateral complex amygdala; BLA, basolateral amygdalar nucleus; BM, basomedial complex amygdala; BMA, basomedial amygdalar nucleus; CA1-3, field CA1-3, Ammon’s horn; cc, corpus callosum; Cd, caudate nucleus; CER, cerebellum; CG, central gray; Cing, cingulate region; CLA, claustrum; CoA, cortical amygdalar nucleus; CPu, caudoputamen; cpd, cerebral peduncle; DG, dentate gyrus; DH, dorsal horn, spinal cord; DMH, dorsomedial hypothalamic nucleus; ENT, entorhinal area; fi, fimbria; fx, columns of the fornix; GP, globus pallidus; GRN, gigantocellular reticular nucleus; HYP, hypothalamus; IA, intercalated nucleus amygdala; ic, internal capsule; IF, interfascicular nucleus raphe´; IG, induseum griseum; INC, interstitial nucleus of Cajal; IO, inferior olivary complex; LA, lateral amygdalar nucleus; LD, lateral dorsal nucleus thalamus; LHAd, lateral hypothalamic area, dorsal region; LHAv, lateral hypothalamic area, ventral region; lot, lateral olfactory tract, body; lv, lateral ventricle; MB, mammillary body; MD, mediodorsal nucleus thalamus; MDRN, medullary reticular nucleus; ME, median eminence; MEA, medial amygdalar nucleus; mfb, medial forebrain bundle; MG, medial geniculate complex; MRN, midbrain reticular nucleus; MSN, medial septal nucleus; mtt, mammillothalamic tract; ND, nucleus of Darkschewitsch; NDB, diagonal band nucleus; NRT, reticular nucleus thalamus; NSO, supraoptic nucleus, proper; NTS, nucleus of the solitary tract; opt, optic tract; OT, olfactory tubercle; PAG, periaqueductal gray; PERI, perirhinal area; PH, posterior hypothalamic nucleus; PIR, piriform area; PMd, dorsal premammillary nucleus; Pu, putamen nucleus; PVH, paraventricular hypothalamic nucleus; PVT, paraventricular thalamic nucleus; py, pyramidal tract; Re, nucleus reuniens; rf, rhinal fissure; Rh, rhomboid nucleus; RL, rostral linear nucleus raphe; RM, raphe magnus; RN, red nucleus; RSP, retrosplenial area; SC, superior colliculus; SN, substantia nigra; st, stria terminalis; STN, subthalamic nucleus; Sub d, subiculum, dorsal part; Sub v, subiculum, ventral part; SubPVH, subparaventricular zone hypothalamus; SUM, supramammillary nucleus; VAL, ventral anterior-lateral complex thalamus; VCO, ventral cochlear nucleus; VH, ventral horn spinal cord; VMH, ventromedial hypothalamic nucleus; ZI, zona incerta; 3v, third ventricle; 4v, fourth ventricle proper; V, motor nucleus of the trigeminal nerve; VIIn, facial nerve; XII, hypoglossal nucleus. * Corresponding author. Tel.: +33 363 082223; fax: +33 363 082280. E-mail address: [email protected] (P.Y. Risold). http://dx.doi.org/10.1016/j.jchemneu.2014.08.001 0891-0618/ß 2014 Elsevier B.V. All rights reserved.

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MCH in situ hybridization

Introduction Neurons producing melanin-concentrating hormone (MCH) form a conspicuous cell population in the mammalian lateral hypothalamic area (LHA) (Baker, 1991; Bittencourt et al., 1992; Griffond and Baker, 2002; Nahon et al., 1989). In most species, they are described as being located in the dorsal and perifornical regions alongside hypocretin/orexin (Hcrt) producing neurons (Hahn, 2010; Swanson et al., 2005). However, we recently observed that the relative distribution of these cells could show some important interspecies differences (Croizier et al., 2013; Croizier et al., 2010). MCH cell bodies are found dorsal to the fornix and in the dorsolateral LHA in rodents while they are mostly dorsal to the fornix in the cat and are closer to the third ventricle in the dorsal and posterior hypothalamus in human (Croizier et al., 2013; Krolewski et al., 2010; Torterolo et al., 2006). By comparison, MCH neurons formed a dense cluster in the ventrolateral LHA of the sheep (Chaillou et al., 2003; Tillet et al., 1996). Based mostly in experiments made in rodents, these neurons are involved in several behaviors including the control of REM sleep and feeding (Boutrel et al., 2010; Eggermann et al., 2003; Griffond and Baker, 2002; Hassani et al., 2009; Jego et al., 2013; Qu et al., 1996; Verret et al., 2003). The behavioral repertoires of vegetarian/prey or predatory animals is obviously extremely different with regard to feeding and sleeping habits, and it may be possible to relate interspecies differences in MCH distribution to these behavioral divergences. However, to date, MCH distribution has been analyzed in only a very limited number of large mammals. The domestic pig is a preferentially vegetarian, omnivorous animal. It evolved from wild pig species that show a short sleep duration, as very often reported in prey animals. The object of the present study was to investigate the distribution of MCH cell bodies in the domestic pig brain. This distribution was compared to that of Hcrt containing neurons that overlap with MCH-expressing cells in many species. The distribution of the pig MCH and Hcrt projections was also researched. Materials and methods Animals Animal care and all procedures were carried out in accordance with French law (authorization A 37801 of the French Ministry of Agriculture). Four young adult female large-white pigs, weighing 70–80 kg, were killed by a lethal intravenous injection of Nesdonal 1 (16 mg/kg). The hormonal status of these animals was unknown, but they had just reached puberty. Heads were perfused through the carotid arteries with two liters of 1% sodium nitrite solution in 0.1 M phosphate buffer (pH 7.4) and four liters of a cold fixative solution made of paraformaldehyde (PFA) dissolved in the same phosphate buffer (40 g/L). The brain was removed, dissected out and sliced into 1 cm thick blocs. Then tissue samples were post-fixed for 24 h in the same fixative. Tissue cryoprotection Blocs were cryoprotected in a 15% sucrose solution (Sigma) and 0.1% sodium azide in 0.1 M (pH 7.4) phosphate buffer at 4 8C. Then, they were quickly frozen by immersion in isopentane at 74 8C using the Snap-FrostTM system (Excilone, France). The brains were cut in four series of 30 mm (2 brains) or in three series of 60 mm (2 brains) thick coronal sections, collected in a cryoprotector solution (1:1:2 glycerol/ethylene glycol/phosphate buffered saline or PBS) and stored at 20 8C.

A BLAST (Basic Local Alignment Search Tool) was performed to compare pig MCH mRNA with rat MCH mRNA. The percentage of homologies was high (81%) and in situ experiments were performed using a MCH RNA probe that was previously obtained in our laboratory (Brischoux et al., 2001). Free-floating sections were post-fixed in 4% PFA during 20 min at room temperature. Then they were incubated 1 h at 4 8C in 6% hydrogen peroxide (Sigma) in PBS with 1% Tween (PBT). Tissues were rinsed briefly in PBT, incubated in glycine solution (2 mg/ml PBT, Sigma) 10 min and in PBS with 0.3% Triton X100 (PBS-T) 15 min at room temperature. After rinsing in PBT, sections were incubated in proteinase K solution (10 mg/ml PBT, Roche) 15 min at 37 8C then in 4% PFA/0.2% glutaraldehyde (Electron Microscopy Sciences) 20 min at room temperature. Tissues were rinsed briefly in PBT and incubated in TEA (Triethanolamine, VWR) and acetic anhydride (Sigma) solution (TEA 1.33 ml, acetic anhydride 250 ml, HCl 410 ml qsp 100 ml water) 5 min at room temperature. Then, sections were incubated in hybridization buffer (50% formamide, 45% 10 SSC, 5% Denhart 100, 0.1 mg/ml salmon sperm DNA) at 37 8C during 10 min and overnight at 56 8C in humid chambers with hybridization buffer containing 100 ng labeled MCH RNA probes. The antisense and control sense rat MCH probes were produced by using the RNA transcription kit (Roche) and were DIG-UTP-labeled. After rinsing with solution I (50% formamide, 49% 5 SSC pH 4.5, 1% SDS) and solution III (50% formamide, 50% 2 SSC pH 4.5) for three 30 min washes at 37 8C, sections were incubated in 0.1% blocking reagent (Roche) in PBT 90 min at room temperature. They were incubated in anti-DIG Fab fragments conjugated to alkaline phosphatase (1:1300, Roche) overnight at room temperature and revealed with enzyme substrate NBT (nitro-blue tetrazolium chloride, Roche) BCIP (5-bromo-4-chloro-30 indolyphosphate phosphate, Roche) 2 h at room temperature. Sections were rinsed, mounted on gelatin-coated slides and cover-slipped with 60:40 glycerol:PBS-T. Control hybridization, including hybridization with sense DIG-labeled riboprobes, was performed.

Immunohistochemistry MCH, NEI, Hcrt After rinsing in PBS-T, free-floating sections were incubated with the primary antibody in milk solution (PBS-T, 1% bovine serum albumin, 10% lactoproteins and 0.01% sodium azide), during 65 h at 4 8C. The primary antibodies used were antiMCH [salmon or rat MCH, our laboratory (Risold et al., 1992), made in rabbit, 1:1000], anti-NEI [the MCH precursor neuropeptide glutamic acid-isoleucineamide, our laboratory (Risold et al., 1992), made in rabbit, 1:2000] and antihypocretin (Santa Cruz Biotechnology, made in goat, 1:1000). The labeling was then revealed through the avidin-biotin complex system for the rabbit antibodies and through the peroxidase-anti-peroxidase system for the goat antibody. Sections were incubated for 24 h at 4 8C in a solution of biotinylated goat antirabbit IgG (Vector Laboratories) at a dilution of 1:1000 in PBS-T, or for 8 h at room temperature in a solution of donkey anti-goat IgG (Jackson Immunoresearch) at a dilution of 1:1000 in PBS-T. Then, sections were placed in the mixed avidin-biotin horseradish peroxidase (HRP) complex solution (ABC Elite Kit, Vector Laboratories) for 1 h at room temperature, or in the goat peroxidase anti-peroxidase (PAP) solution (DAKO) at a dilution of 1:1000 for 16 h at 4 8C. The peroxidase complex was visualized by a 6 min exposure to a chromogen solution containing 0.04% 3,3’diaminobenzidine tetrahydrochloride (DAB, Sigma) with 0.006% hydrogen peroxide (Sigma) in PBS. The reaction was stopped by extensive washing in PBS. Sections were mounted on gelatin-coated slides, and then dehydrated and coverslipped with Canada balsam (Roth). An adjacent series was always stained in a solution of 1% toluidine blue (Roth) in water to serve as a reference series for cytoarchitectonic purposes. Some sections were revealed by immunofluorescence. After the incubation with the primary antibody, the tissue was incubated with Alexa Fluor 488-labeled goat anti-rabbit IgG antibody (Invitrogen, 1:1000) or with Cyanine-3 donkey anti-goat IgG antibody (Jackson Immunoresearch, 1:1500) diluted in PBS-T for 2 h at room temperature. Finally, the sections were washed with PBS-T three times 5 min, were mounted on gelatin-coated slides and coverslipped with 60:40 glycerol:PBS-T. Antibody characterization Please see Table 1 for a list of all antibodies used. The specificities of the sMCH, rMCH and NEI antisera were tested by blotting against the peptides salmon and rat MCH, NEI, NGE, GRF37, and alpha-MSH (Risold et al., 1992). The labeling provided by the two MCH AS on the pig brain sections

Table 1 Table of primary antibodies used. Antigen

Immunogen

Manufacturer

Dilution used

MCH MCH NEI Hypocretin I/Orexin A

Synthetic Salmon MCH; full 17 amino acid sequence Synthetic rat MCH; full 19 aa sequence Synthetic rat NEI; full sequence Human orexin A, 19 aa of the c-terminal fragment

Risold et al. (1992) Risold et al. (1992) Risold et al. (1992) Santa Cruz Biotechnology, goat polyclonal, # GO 813

1/1000 1/1000 1/2000 1/1000

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were eliminated by incubation with the synthetic peptide rMCH (1nmol/ml of AS). We also compared the labeling provided by all three MCH and NEI AS and found that they provided very similar patterns of staining on the pig brain sections. Finally, we performed double labeling experiments detecting the prepro-MCH mRNA by in situ hybridization and MCH or NEI peptides by indirect immunofluorescence and observed the co-expression of in situ and immunohistochemical signals in the same cell bodies in the posterior LHA. This double labeling experiment consisted in performing a regular in situ hybridization as described above, immediately followed by an indirect immunofluorescence (same conditions as described above) using AlexaFluor 488 secondary (dilution 1/1000, Invitrogen). Furthermore, incubation of synthetic human orexin A with the AS (1nmol of peptide/ml of AS) completely abolished the signal on the sections of the pig brain. The data sheet of the antibody identifies a crossreactivity with the pig orexin A. Finally, perikarya labeled by this antibody using standard indirect immunofluorescence corresponded to the expected pattern.

Line drawings were produced from pictures of toluidine blue stained sections at lowest magnification using a stereomicroscope SZX9 (Olympus, France) and the DP50 numeric camera. These pictures served as templates to draw sections and their cytoarchitectonic features using Adobe Illustrator CS2. Two atlases were used to determine nuclear borders (Felix et al., 1999; Saikali et al., 2010). Unfortunately, deep brain structures are often only partially illustrated or identified in these atlases, and when needed (for example in the thalamus and amygdala) we adapted the rat brain nomenclature (Swanson, 2004) on cytoarchitectonic structures identified in our material. Then, the immunohistochemical labeling was mapped from the adjacent sections stained with the MCH or NEI AS. Therefore, the data are illustrated from one brain, but all four brains were analyzed.

Results MCH neuron morphology

Microscopy and line drawings production Sections prepared for in situ hybridization or immunohistochemistry were analyzed on an Olympus fluorescence microscope BX51 configuration. Images were obtained through a DP50 numeric camera (Olympus, France), using the analySIS software (Olympus, France).

The distribution of MCH perikarya was analyzed using in situ hybridization and immunohistochemistry with sMCH, rMCH and NEI AS. An abundant population of cells was labeled in the posterior LHA using either one of these methods/AS (Figs. 1A–F and 2). The

Fig. 1. Distribution of MCH and Hcrt in the pig lateral hypothalamus. (A)–(E): Photomicrographs to illustrate the distribution of MCH perikarya in coronal sections passing through the posterior LHA. Neurons are detected using a sMCH AS (A, B, D) or in situ hybridization (C). Note that cell bodies are abundant ventro-lateral to the fornix, but scattered in the dorsal hypothalamus. At high magnifications, long dendritic processes extend across the path of the mfb (B). A dual labeling using in situ hybridization and immunofluorescence shows that cells that express the MCH mRNA also express the peptide (E). (F) Distribution of ppMCH mRNA on a coronal section passing through the posterior hypothalamus. Note the similarity of the distribution with immunohistochemical signal. (G) Distribution of Hcrt immunoreactive cell bodies in the caudal dorsal hypothalamus. Note their expected distribution mostly dorsal to the fornix. Scale bar: A = 350 mm; B = 50 mm; C–E = 100 mm; F–G = 400 mm.

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Fig. 2. Series of line drawings of coronal sections of the posterior hypothalamus arranged from rostral (A) to caudal (H) to schematically illustrate the comparative distribution of MCH (stars) and Hcrt (dots) containing neurons. Rostral and caudal perikarya of both cell populations are observed at the same anterior and posterior hypothalamic levels, but their pattern of distribution is clearly different through the length of the LHA. For practicable reasons and conserve a reasonable size to the illustration, dots and stars do not represent one single neuron each. However, their disposition on the illustration was done as to reflect as much as possible the distribution of neurons as observed at low magnification under the microscope.

MCH labeling was abolished after incubation of the MCH AS with synthetic peptides. No in situ hybridization signal was obtained using the sense riboprobe (data not shown). Detected by immunohistochemistry with either the MCH or NEI AS, many MCH cell bodies displayed a fusiform or polygonal shape often with at least 3 large dendritic trunks extending perpendicular and obliquely to the path of the medial forebrain bundle (mfb) (Fig. 1B). Dendrites often showed their first large ramifications

25 to 30 mm away from the cell bodies, but the immunohistochemical reaction did not reveal their distal segments. Thin axons were frequently observed emerging from a short cone at the base of a large dendrite. Some very lateral cells seemed more multipolar in shape, while periventricular somata often displayed a round appearance. Most cell bodies were around 15 to 20 mm in diameter, with the exception of medial most neurons, which were smaller.

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Distribution of MCH cell bodies in the posterior hypothalamus In coronal sections of the hypothalamus, the most anterior neurons were observed in the caudal anterior hypothalamic area at levels of the paraventricular nucleus (Fig. 2A). Some of them were lateral and ventral to the fornix, but a few were also found within the borders of the anterior hypothalamic nucleus (Fig. 2B). At more caudal levels, the number of labeled cells increased greatly. The densest cell concentration was observed lateral and ventral to the fornix. The positive neurons were scattered dorsal to the fornix and in dorsal lateral regions close to the cerebral peduncle. In the ventral LHA, these perikarya tended to avoid the tuberal nuclei. One or two positive neurons were found within the borders of the ventromedial nucleus (VMH) or close to the arcuate nucleus. The dorsomedial nucleus (DMH) was devoid of MCH cell bodies (Fig. 2D), but a significant group was located in the dorsomedial capsule of the VMH, between the cytoarchitectonic borders of the VMH and DMH. The dorsal hypothalamus contained a few perikarya as well as the rostromedial zona incerta (ZI). At premammillary levels, the number of labeled perikarya decreased dramatically, but a few were still observed around the fornix and in the posterior hypothalamic nucleus (Fig. 2G). A few more faintly

labeled neurons were also observed in the ventral ZI, just dorsal to the cerebral peduncle (Fig. 2F). Finally, the most caudal perikarya were observed in the rostral ventral tegmental area (VTA). Comparison with hypocretin/orexin containing neurons An abundant population of Hcrt cell bodies was labeled using immunohistochemistry on sections through the LHA of the pig (Figs. 1G and 2). These cell bodies were observed mainly dorsal to the fornix. They were numerous and spread over a large field of the dorsal LHA, extending from the ventricular surface to the cerebral peduncle. They were scattered ventral to the fornix. Very few were observed in the ventral rostromedial ZI. Compared to most MCH neurons, these perikarya were more dorsally located and both populations showed complementary patterns. However, overlaps were also noticed: the most rostral perikarya were observed at the same level of the anterior hypothalamus, lateral to the fornix and one or two just dorsal to the supraoptic nucleus. The distribution area of both cell populations also ended at similar posterior hypothalamic levels and with a similar distribution pattern in the posterior hypothalamic nucleus, and in the ventral tegmental area for a very few cells.

Fig. 3. Photomicrographs illustrating different patterns of cortical ppMCH innervations labeled with a NEI-AS and using the PAP method. (A), (B): Bright-field of a toluidin blue stained section (A) and dark-field photomicrograph of an adjacent section passing through the perirhinal cortex (B). Projections are very abundant in all layers. (C): The hippocampus, as illustrated in this dark-filed picture for the CA3 field, is moderately innervated. D: Isocortical fields are sparsely innervated (dark-field illumination). Scale bar = 100 mm for (A)–(D).

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Fig. 4. (A) to (F): Line drawings of coronal sections of the pig brain to illustrate the distribution of MCH axons labeled by the MCH or NEI-AS. They are arranged from rostral (A) to caudal (F). The medial septal nucleus contains a very abundant MCH innervation. Note also the very abundant innervation of the anterior piriform cortex as well as the amygdala. A, B, C, D in (D) correspond to microphotographs in Fig. 5.

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Fig. 4. (Continued ).

MCH projections (Figs. 3–5) MCH- and NEI-AS labeled axons with a similar pattern throughout the pig brain. However, the NEI-AS provided better dark-field photomicrographs. Therefore, dark-field illustrations are from immunohistochemistry obtained with the NEI-AS. For practical reasons, we will keep the term of MCH to describe all the projections instead of employing alternatively terms as ppMCH or MCH/NEI or NEI according to the circumstances and figures, but the used antiserum in illustrations are specified in the figure legend. MCH axons in varying densities were observed from the telencephalon to the medulla oblongata (Figs. 3–5). The telencephalon is a major target for MCH projections. MCH axons were abundant in several fields of the cerebral cortex. All cortical fields ventral to the rhinal sulcus contained a very abundant MCH innervation by well-labeled axons, including the ventral agranular insular, piriform, perirhinal and entorhinal cortical fields (Figs. 4A–E and 5B,B’). This innervation extended into the ventral subiculum from the entorhinal cortex. Dorsally in the cerebral cortex, MCH axons were also numerous in the medial prefrontal and ventral cingular fields. The taenia tecta and the indusium griseum contained a high number of MCH axons, providing an intense innervation. Many axons traveled through the cingulate bundle to reach the dorsal subiculum that was also densely innervated (Fig. 4E). The hippocampus proper contained only a moderate number of axons, mostly in the ammon’s horn while the dentate gyrus contained fewer axons (Fig. 4E). The claustrum, a subcortical sheet of gray matter, also received a clear input by labeled axons. Isocortical fields between cingular and peri-insular fields were little or very little innervated (Figs. 3 and 4A–E). The amygdala, including cortical amygdalar nuclei,

basomedial, basolateral and lateral complexes, was intensely innervated, but anterior, central and medial cell groups were less well targeted by MCH axons (Figs. 4C and D and 5A and D). These axons reached the amygdala through the ansa peduncularis and the stria terminalis, and provided moderate innervations of the substantia innominata. The bed nuclei of the stria terminalis also contained a moderate to light input. Most of the striatum contained very few MCH projections, with the notable exception of the olfactory tubercles that were moderately innervated. The nucleus accumbens as well as the very caudal caudate nucleus contained MCH axons and boutons as well. The lateral septal nucleus received a moderate input that concerned mostly rostral and lateral divisions (Fig. 4A and B). Pallidal structures were unevenly innervated. The globus pallidus itself contained very little immuno-labeled projections, but the medial septal nucleus displayed one of the densest MCH innervation in the central nervous system (CNS) (Fig. 4B). From this last structure, many axons continued rostrally, reaching rostral hippocampal rudiments (taenia tecta and indusium griseum) and cingular cortical fields by skirting around the genu of the corpus callosum. Few axons however could be followed from the medial septal nucleus into the fimbria. Projections reached the hippocampus mostly by following the cingulum bundle or expanding from the entorhinal fields. In the diencephalon, the thalamus was overall very little innervated. Only midline thalamic nuclei as the paraventricular nucleus contained some axons. Adjacent lateral habenular nucleus also contained few MCH projections (Fig. 4C and D). Therefore, the diencephalic MCH projections concerned mostly the hypothalamus. The lateral zone contained the densest concentration of MCH axons, many of them running rostrally through the medial forebrain bundle. Lateral preoptic and anterior LHA contained a

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Fig. 5. Photomicrographs illustrating the abundant MCH innervations (labeled by a NEI-AS) in the basolateral (A) and basomedial (D) nuclei of the amygdala, and the posterior piriform cortex (B). The putamen nucleus contained few labeled axons (C). B’ is a high magnification under bright-field illumination of axons in the piriform cortex (a different field from B) to illustrate the aspect of these axons. Scale bar: A, B, C, D = 100 mm; B’ = 50 mm.

moderate number of MCH axons. It is worthwhile to note that MCH projections were abundant in ventral parts of the LHA, but less abundant in dorsal regions. Medial and periventricular hypothalamic zones received a moderate to weak input, although the subparaventricular zone contained a dense cluster of labeled axons. Some very well-stained axons were noted in the external zone of the median eminence (Fig. 4C and D). In the posterior

hypothalamus, the posterior and the supramammillary nuclei contained moderate projections. A few were observed in both the medial and lateral mammillary nuclei. Many, however, were observed in the capsule of these nuclei. These projections provided an innervation of the ventral tuberomammillary nucleus. At the same level of sections, the subthalamic nucleus also contained a dense innervation. Fibers in this structure seemed of a thinner

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Fig. 6. Aspect of Hcrt distribution illustrating marked differences with MCH distribution illustrated in Fig. 4. A–D: Photomicrographs illustrating Hcrt axons labeled by immunohistochemistry in the piriform cortex (A), amygdala (B), paraventricular nucleus of the thalamus (C) and ventromedial hypothalamus (D). Note the poor innervation of both piriform cortex and amygdala, but abundant innervation of the PVT and ventro-medial hypothalamus. (E): Line drawing of a coronal section passing through the posterior hypothalamus and amygdala. The squares correspond to photomicrographs (A) to (D). Divergence with MCH projections are obvious when compared with Figs. 4C and 5. Scale bar: A, B, C, D = 100 mm.

caliber than in others, but many boutons were noted that were apparently in contact with unlabeled neuronal perikarya (Fig. 4E and F). Descending projections were abundant. These axons often seemed of a thinner caliber, resembling those in the subthalamic nucleus. The reticular formation contained moderate innervations through its rostrocaudal length. At the mesencephalic level, the tectum and the substantia nigra were innervated by MCH axons. The caudal grey, as well as the dorsal part of the paragigantocellular nucleus, contained the densest descending projections. Other structures as the parabrachial and nucleus of the solitary tract contained a moderate input. Even the cerebellar cortex contained

some axons. In the cervical spinal cord, the dorsal horn, especially its superficial lamina, was targeted. But a few axons were also noticed in the ventral horn. Comparison with Hcrt containing axons. The Hcrt AS labeled axons in the CNS, from the telencephalon to the spinal cord, as for the MCH or NEI AS. The distribution pattern of these projections was clearly reminiscent of that in rodent (Nambu et al., 1999). Medial septal nucleus, bed nucleus of the stria terminalis and parts of the substantia innominata were abundantly innervated. Axons were noted through the diencephalon, including the LHA but also many periventricular hypothalamic nuclei (paraventricular hypothalamic and arcuate nuclei) (Fig. 6D and E). In the thalamus, the

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paraventricular thalamic nucleus contained a dense innervation by axons exhibiting many boutons. Interestingly, this innervation included the ependymal epithelium (Fig. 6C and E). Descending projections were abundant in several nuclei as the dorsal raphe nucleus or the locus coeruleus, in that mimicking the descriptions of Hcrt axons in the rat or mouse. Many clear divergences were observed between the Hcrt and the MCH distribution patterns. The most spectacular divergence was in the temporal lobe in which we observed only sparse Hcrt projections through the amygdala (Fig. 6B and E), including nuclei abundantly innervated by MCH axons, and in the piriform cortex (Fig. 6A and E) which contained a weak Hcrt input while very abundantly innervated by MCH projections. The Hcrt thalamic input appeared also more significant than the MCH thalamic input, including in the paraventricular thalamic nucleus. Discussion The presence of MCH cell bodies in the caudal hypothalamus is a common feature observed in all vertebrates, and is confirmed in the pig brain by the present study. Furthermore, the same rostrocaudal hypothalamic levels contained Hcrt expressing neurons, also confirming that in other species. Pig MCH and Hcrt projections provided a very intense innervation of the basal forebrain and in particular of the medial septal complex, and were traced from the cerebral cortex to the spinal cord. These projection patterns being common to all mammals studied so far (Baker, 1991; Bittencourt et al., 1992; Griffond and Baker, 2002; Nahon et al., 1989). Conserved projections, as in the medial septal complex, suggest conserved functions for example in memory consolidation during REM sleep for MCH (Lima et al., 2013; Lu et al., 2013). However, besides these obvious common trends, the pig MCH system exhibited several very specific features in the distribution of both its perikarya and projections. The goal of this work was to point out the most remarkable of these specificities of the pig MCH system. In the pig hypothalamus, MCH cell bodies were found in large portions of the ventral lateral hypothalamus, ventrolateral to the fornix, being often closer to the pial (ventral) surface of the LHA than to the ependymal (ventricular) surface of the dorsal hypothalamus. This distribution pattern is strikingly different to that reported in most other mammals. In the rodent or in the cat, MCH perikarya sit in the dorsal perifornical LHA (Croizier et al., 2013; Croizier et al., 2010; Torterolo et al., 2006). The rodent patterns involve the zona incerta in which a large group of MCH neuron is observed. In human, they were described closer to the third ventricle in the posterior dorsal hypothalamus (Croizier et al., 2013; Krolewski et al., 2010). The pig MCH distribution was similar to that described in sheep (Tillet et al., 1996). In the pig, MCH perikarya are in the ventral LHA, but forming a dense group ventral to the fornix. Contrasting with the rodent pattern, few MCH cell bodies were found in the zona incerta and this structure cannot be regarded as a major site for MCH neurons in the pig. Hcrt containing neurons were mostly dorsal to the fornix in the pig brain. This distribution pattern resembles to that described in all species so far, including rat, mouse, cat and human (Croizier et al., 2010; Krolewski et al., 2010; Torterolo et al., 2006). Therefore, MCH expressing perikarya show divergent and species specific distribution patterns while Hcrt containing cells have a more conserved pattern of distribution. Differences in the distribution pattern of MCH neurons may reflect functional differences. The medial forebrain bundle (mfb) that runs longitudinally through the LHA is a complex but very organized tractus (Swanson et al., 1987; Veening et al., 1982).

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It plays a very important role in mediating responses from the LHA. Ascending or descending projections to or from the telencephalon take respectively more dorsal or ventral paths within this bundle. In particular, axons from several nuclei of the amygdala project through more ventral route within the mfb, especially olfactory projections. These projections reach the LHA mostly through the ansa peduncularis which form a shorter anatomical path than the stria terminalis between the telencephalic temporal lobe and the hypothalamus. Abundant MCH projections were also observed taking this direct hypothalamo-amygdala path. Therefore, it is tempting to correlate the ventral distribution of MCH perikarya in the pig lateral hypothalamus with the intense innervation of several nuclei of the amygdala, piriform and perirhinal cortical fields in this species. In the rodent, the temporal lobe appears far less innervated than in the pig. In mouse, few projections into the piriform cortex were described as compared to the rat (Croizier et al., 2010), MCH perikarya occupy mainly the dorsolateral sector of the LHA. Finally, Hcrt neurons that are dorsal in the LHA send only a modest projection into the amygdala or piriform cortex. Again this reinforces the hypothesis that a specific pattern of MCH perikarya distribution in the pig LHA is correlated with specific patterns of MCH projections, especially in the amygdala (Croizier et al., 2010). In human, MCH cell bodies are dorsal and periventricular in the posterior hypothalamus. These neurons sit medial to Hcrt containing neurons. Therefore, their projection patterns may show several clear cut differences in this species compared to that described in rat, mouse and obviously pig. Unfortunately, human MCH projections have not been fully investigated so far. The basomedial/basolateral amygdalar nuclei are integrated in networks with the medial prefrontal cortex, the hippocampus and the nucleus accumbens (Petrovich et al., 2005; Petrovich et al., 2002). They are involved in conditioned responses in relation with feeding behavior and in learning memory. They project through the mfb in the rodent LHA. With regard to the very abundant MCH projections in these structures in the pig, it is possible that pig MCH neurons are especially involved in mediating these responses in amygdalar sites, while it has been shown that in rodent, MCH influence feeding by interacting with its receptor MCHR1 in the shell of the nucleus accumbens (Georgescu et al., 2005; Pissios et al., 2008; Sears et al., 2010). Other differences between rat and pig projections were also noted, for example in the mammillary nuclei, as MCH axons are abundant in the medial mammillary nucleus in rat (Casatti et al., 2002), while only a modest input was noted in pig. Functional data plead for opposite/complementary roles for MCH and Hcrt in the rodent brain. Hcrt neurons are active during the waking period, while MCH neurons seem to be especially active during REM sleep (Hassani et al., 2009; Verret et al., 2003). The obvious divergent distribution patterns of the two cell populations in the pig may indicate that their roles are also clearly divergent in this species. We only provided a scant description of Hcrt projections in this work, but again the two peptidergic systems show different innervations of some key structures such as the thalamus (e.g. the paraventricular nucleus), the amygdala (very abundantly innervated by MCH axons) or within the hypothalamus with more abundant Hcrt projections in the periventricular hypothalamic zone. Any more morphofunctional consideration would be speculative in absence of experimental data. Nevertheless, the present analysis clearly points to fundamental divergences in the anatomy of the posterior LHA that is a structure known to coordinate the expression of behaviors such as REM sleep and feeding. Further studies of its comparative anatomy and physiology are of a huge importance in order to understand the functions of MCH neurons and of the LHA altogether in the human brain.

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