Rhythmicity is a ubiquitous property of all living organisms (Turek and Van Reeth, 1996). Biological rhythms in mammals are driven by a circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus (Moore and Eichler, 1972; Stephan and Zucker, 1972; Inouye and Kawamura, 1979; Green and Gillette, 1982; Groos and Hendriks, 1982; Lehman et al., 1987). The SCN is a strong autonomous oscillator cycling with a period close to, but different from, 24 h and entrained to exactly 24 h by environmental cues, the Zeitgeber (Refinetti, 2006). This nucleus is a complex structure containing several neuronal populations that have specific afferents and efferents. In most species, especially in rodents, the SCN is divided into two subdivisions: the dorsomedial SCN or ‘shell’ and the ventrolateral SCN or ‘core’ (Abrahamson and Moore, 2001; Morin et al., 2006). The dorsomedial SCN contains neuronal perikarya immunopositive for arginine-vasopressin (AVP) (Ibata et al., 1999; Moore et al., 2002; Morin et al., 2006; Nascimento et al., 2010) while the ventrolateral SCN contains neurons expressing vasoactive intestinal polypeptide (VIP), gastrin releasing peptide and peptide histidine isoleucine (Stopa et al., 1984; Card et al., 1988; Mikkelsen et al., 1991; Swaab et al., 1994; Smale and Boverhof, 1999; Moore et al., 2002; Nascimento et al., 2010). A large number of other neuropeptides have been described in the SCN of mammals with some interspecies variations. It was demonstrated that somatostatin perikarya are generally located in the intermediate region of SCN (Card et al., 1988; Ibata et al., 1999) while neurophysin, neurotensin, thyrotropin-releasing hormone, enkephalins (Enk), and angiotensin II were described in different parts of the nucleus (Block et al., 1988; Tillet et al., 1989; Abrahamson and Moore, 2001; Thomas et al., 2004). Moreover, calcitonin gene-related peptide (Park et al., 1993), galanin (Skofitsch and Jacobowitz, 1985; Abrahamson and Moore, 2001), substance P (Morin et al., 1992; Mikkelsen and Larsen, 1993; Abrahamson and Moore, 2001; Piggins et al., 2001) and the calcium-binding protein calbindin (Silver et al., 1996; Ikeda and Allen, 2003; Menet et al., 2003) have also been reported in this nucleus in different species. Additionally, gamma-aminobutyric acid (GABA), an important neurotransmitter in the circadian system, is found in almost all neurons of the SCN (Okamura et al., 1989; Moore and Speh, 1993; Kalsbeek et al., 2000; Moore et al., 2002).

In addition to neuronal cell bodies, a typically dense innervation for various neuroactive agents exists in the SCN. Glutamate and pituitary adenylate cyclase activating peptide afferents originating from melanopsin retinal ganglion cells and forming the retinohypothalamic tract (RHT) are observed in the ventral region of the SCN containing VIP neurons (for review see Hannibal, 2002). Moreover, the ventrolateral SCN of rodents also contains serotonin (5-HT)-immunoreactive (ir) fibers that originate from the midbrain raphe nuclei. The core of the SCN also receives afferents originating from the intergeniculate leaflet (IGL) of the lateral geniculate complex containing neuropeptide Y (NPY), GABA and met-enkephalin (Met-Enk) (Moore and Eichler, 1972; Moore et al., 1978; Steinbush, 1981; van den Pol and Tsujimoto, 1985; Card and Moore, 1988, 1989; Tillet et al., 1989; Morin et al., 1992).

Functional relevance of the neurochemical composition of the master clock has not yet been understood fully. However, mechanisms and molecular processes underlying the synchronization of the SCN by environmental cues are well documented. It is known that in mammals, the light-dark cycle (LD) is the strongest Zeitgeber (Refinetti, 2006) imposing its period and phase to the circadian clock. The VIP neurons of the ventrolateral SCN, which are the target of the RHT (Tanaka et al., 1993), play an important role in the synchronization by light and transmit photic signaling to the pacemaker AVP neurons located dorsally in the dorsomedial SCN (Jacomy et al., 1999; Aton et al., 2005). In mammals, the molecular basis of clock function includes alternating activation and repression of gene expression by different proteins. These rhythmic self-regulating proteins are encoded by clock genes. The molecular machinery of the main clock is well known, and is based on two interdependent feedback loops (Reppert and Weaver, 2000, 2001; Shearman et al., 2000; Okamura et al., 2002; Takahashi et al., 2008; Mohawk et al., 2012).

In addition to photic entrainment by the LD cycle, a non-photic entrainment by other Zeitgebers and cues also exists. In particular, these include ambient temperature (Ta), food availability, pharmacological effect of melatonin and social cues. Experimental lesions have demonstrated that the geniculohypothalamic tract (Janik and Mrosovsky, 1994; Wickland and Turek, 1994; Challet et al., 1996; Miller et al., 1996; Schuhler et al., 1999; Goel et al., 2000) and serotoninergic afferents from the raphe nucleus (Cutrera et al., 1994; Miller et al., 1996; Challet et al., 1997) are involved in such non-photic entrainment. The relative importance of non-photic vs photic entrainment has not been widely studied.

Recently, we have demonstrated in the dromedary camel (one-humped camel, Camelus dromedarius) that a non-photic entrainment can be sufficiently strong to drive the circadian clock (El Allali et al., 2013). Indeed, in this species, environmental temperature cycles as well as the LD cycle synchronize (phase and period) melatonin and body temperature rhythms. The neural circuitry and neuropeptides involved in such entrainment are still unknown. This raises several questions about the structure and functioning of the SCN in this species. However, there are no data on the organization of the camel hypothalamus and only limited data are available on the brain of this species. For these reasons, the present study aimed at investigating the organization of the SCN in the dromedary camel and intended to fill the existing gap in our understanding of neuroanatomy. We carried out a cartography of the nucleus and studied its cytoarchitecture. The distribution of relevant neuropeptides involved in the circadian system was also investigated. These include AVP, VIP, Met-Enk, NPY, and oxytocin (OT). The enzymes tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) and the neurotransmitter serotonin (5-HT) were also studied.

This study was conducted by sampling the hypothalamus of 31 camels, slaughtered to provide meat for public consumption, at two sites: the Eddakhla slaughterhouse in the south of Morocco (latitude: 23^∘∘43′ N, 15°57′ W) and the Rabat slaughterhouse in the north of the country (latitude: 34°01′ N, 6°50′ W). The animals included 13 females and 18 males of local varieties (Piro et al., 2011) aged between 4 and 12 years (the lifespan of a camel is 20–30 years). They were kept outdoors under natural environmental (photoperiod and temperature) conditions with free access to water. Slaughter was carried out at different periods of the year and almost at the same time of the morning: 06h00. Brain samples were dissected out as animals were slaughtered. The study was performed in conformity with the Hassan II Agronomy and Veterinary Institute of Rabat and Moroccan Ministry of Agriculture recommendations, which are in accordance with international ethical standards (Touitou et al., 2006).

The heads were perfused immediately after slaughter. The perfusion was performed through the two external carotids using, first, a washing heparin (2.5 UI/ml) solution in saline (0.9% sodium chloride, Fluka^®), followed by tissue fixation (5–6 l), using either formaldehyde (Sigma–Aldrich^®; 10% in water) for cytoarchitectonic studies or 4% paraformaldehyde (Scharlau^®) in phosphate-buffered saline (PBS; 0.2M, pH 7.4) for immunohistochemistry. Thirty to forty minutes after perfusion, the brain was carefully removed and the hypothalamus dissected and post-fixed at 4°C for 3 days in the fixative solution.

This study is the first undertaken on camel SCN neuroanatomy including its cartography, cytoarchitecture and chemoarchitecture. The results show that the SCN of the camel displays peculiarities regarding its size, extent, morphology and cytoarchitecture. Based on histological and immunohistochemical criteria, we subdivided the camel SCN into rostral, main and caudal divisions (rSCN, mSCN and cSCN; respectively). The location of the rSCN, organized as a collection of neurons protruding from the preoptic area outside the main mass of the hypothalamus, is unusual. The SCN at these levels is located below the SoR, lining the dorsal surface of that portion of optic chiasm that is still detached at these levels from the ventral borders of the brain. Despite the fact that such a conformation is uncommon in most studied species, a similar organization has been described in sheep (Tillet et al., 1989; Tessonneaud et al., 1994), cow (Lignereux, 1986), and in humans (Swaab et al., 1985; Hofman et al., 1988; Hofman and Swaab, 1989). Similarities with these species were also demonstrated with regard to the rostrocaudal morphological variations of the SCN. Indeed, structures comparable to the median dome-shaped tissue (Mds), lateral suprachiasmatic swellings tissue (Lsw), and lateral expansions of the hypothalamus (Leh); have been observed in the rSCN of the above-mentioned species. Caudally, the mSCN exhibits the usual triangular shape and location at the base of the third ventricle, dorsally to the optic chiasm, as classically described in rodents and several other mammals (Spiegel and Zweig, 1917; Bleier et al., 1979; van den Pol, 1980; Cassone et al., 1988; Vrang et al., 1995).

Due to the existence of its two rostro-caudal subdivisions, the rSCN and cSCN, which are lacking in most species, the dromedary SCN seems to be the largest studied thus far. This can be attributed to the fact that the SCN starts far rostrally outside the main mass of the hypothalamus and elongates caudally to the optic chiasm. The SCN extends in the camel for 9.55 mm, while in sheep and in humans (species which exhibit a similar SCN morphology), the total rostrocaudal length is much shorter (2.8–3.1 mm and 1.47 mm, respectively; Swaab et al., 1985; Swaab and Hofman, 1990; Tessonneaud et al., 1994). The rostrocaudal extent of the SCN in other mammals depends on the species but ranges between 0.60 and 2.15 mm (0.60 mm in the Syrian hamster, 0.9 mm in the rat, 0.76 mm in the guinea pig, 0.76 mm in the mouse, 0.80–1.0 mm in the squirrel monkey, 0.90 mm in the macaque monkey, 0.95–1.65 mm in five marsupial species, 1.74 mm in the domestic pig and 2.15 mm in the domestic cat) (van den Pol, 1980; Lydic et al., 1982; Cassone et al., 1988).

There are great differences in the general topography and cytoarchitecture of the SCN across species. It seems reasonable to argue that mammals can be divided in two groups concerning the morphology of the SCN: camel, sheep, cow and human (and maybe other species) with a relatively long and large SCN, protruding out of the hypothalamus in its rostral division, and other mammals (including rodents) with the SCN corresponding to more “classical” descriptions.

Based on retinal projections, size and shape criteria as well as neurochemical phenotypes, the SCN, especially in rodents, has been divided in two dorso-ventral subdivisions: the dorsomedial SCN or ‘shell’ and the ventrolateral SCN or ‘core’ (Abrahamson and Moore, 2001; Morin et al., 2006). In the present histological study, no such subdivisions were evident in the camel SCN. However, clear cytoarchitectural differences were identified with respect to the size of SCN neurons, with populations of dorsal parvicellular and ventral magnocellular neurons. This organization is largely confirmed by the immunofluorescence studies. In sheep, there is also no clear dorso-ventral subdivision of the SCN, but neurons are smaller in the ventral than in the dorsal region of the SCN (Tessonneaud et al., 1994). Neurons of the camel SCN are the smallest cells among those observed within the hypothalamus and adjacent areas. Nevertheless, the camel SCN contains neurons larger than those reported in sheep and other species, especially in the ventral magnocellular part, in which neurons are larger than 20 μm in diameter and can reach up to 50 μm. The largest somal diameters in the SCN of many species (including the domestic pig, domestic cat, mouse, guinea pig, rat, hamster and five marsupial species) do not exceed 12.5 ± 3.1 μm (measured in the ventrolateral division of the cat SCN), while in other species the diameter of SCN neurons ranges between 7.0 and 10.7 μm (van den Pol, 1980; Cassone et al., 1988).

The neurochemical organization of the camel SCN is different from that of the other species studied so far. In camel SCN, two populations of TH neurons were identified, ventral magnocellular or dorsal parvicellular. Their location, number, size and immunoreactivity intensity, suggests that they could have different roles. In the absence of a precise additional phenotyping of these neurons, it is difficult to further speculate on their role (s). To our knowledge, no similar findings were reported in the SCN of other mammals. Very few TH-ir neurons were observed to be homogeneously distributed in the SCN of sheep (Tillet et al., 1994). Moreover, in most rodents, this nucleus either lacks or contains only a few TH neurons. For example, in the rat, a few TH neurons were reported to be present only transiently in the developing brain (Battaglia et al., 1995; Ugrumov, 2013). In the Syrian hamster, only sparse TH-ir round-shaped cells were observed in the SCN, mostly outside the nucleus and lining its borders (Vincent, 1988; Novak and Nunez, 1998; Strother et al., 1998).

Tyrosine hydroxylase is the rate-limiting enzyme for the biosynthesis of catecholamines (dopamine, epinephrine and norepinephrine) and catalyzes the transformation of the amino acid L-tyrosine into L-DOPA (L-3, 4 Dihydroxyphenylalanine), which is then converted to dopamine by AADC (also called Dopa-decarboxylase). Within various animals, the AADC-ir neurons in the SCN seem to be more abundant than the TH neurons (Sheep: Tillet et al., 1994; rat: Jaeger et al., 1984; Inatomi, 1994; cat: Kitahama et al., 1988; house-shrew: Karasawa et al., 1992).

Regarding the puzzle of the relatively high density of TH-ir neurons we observed in the camel SCN, we have investigated their possible dopaminergic phenotype by testing colocalization with AADC. The double immunostaining showed that AADC-ir neurons are very rare and sparse within the nucleus, large-sized and never coexpress TH. This indicates that there are no catecholaminergic, especially no dopaminergic, neurons in the SCN of the camel. The lack of AADC expression within the TH-ir neurons indicates the synthesis of L-DOPA, but not dopamine, in these neurons. This raises several questions. L-DOPA itself has a role in mediating the release of neurotrophic factors that are important for the growth, survival and differentiation of neurons (Lopez et al., 2008; Malenka et al., 2009; Zigmond et al., 2012; Hiroshima et al., 2014). In the camel SCN, given the relatively high number of TH-neurons, L-DOPA could be involved in the survival and differentiation of neurons. A possible involvement of TH neurons in mediating circadian activity is also suggested by the colocalization of TH with VIP especially in rSCN neurons.

The presence of OT-ir neurons in the SCN has been reported briefly by El May et al. (1987) when studying the hypothalamo-hypophyseal axis of the camel. However, their given localization of the SCN and their description as a large extension from the optic chiasm to the pituitary stalk attachment were not very precise. In the present work, the camel SCN cartography is performed at high spatial resolution and results confirmed clearly that OT neurons are found within the mSCN and are either scattered throughout the nucleus or grouped ventro-laterally. The presence of OT-ir neurons within the SCN is unusual, and represents a peculiarity of the nucleus in the camel. Sofroniew and Weindl (1980) studied the neuropeptidergic content of the SCN in 13 species belonging to 6 mammalian orders (marsupials, rodents, lagomorphs, artiodactyls, carnivores and primates). In all of these animals, the SCN lacks OT-ir neurons, as confirmed in other studies (Sofroniew and Glasmann, 1981; Reuss et al., 1989; Caba et al., 1996). For the camel, a possible role of OT neurons in the modulation of the circadian clock activity cannot be excluded. This could be of particular interest in species such as the dromedary, that faces the problem of adaptation to the harsh environment of the desert and which has to adapt its physiology and circadian rhythms to this biotope.

The existence of vasopressinergic neurons in the camel SCN is consistent with reports in other mammalian species (Vandesande et al., 1975; Vandesande and Dierickx, 1975; Sofroniew and Weindl, 1980; Card et al., 1981; Tillet et al., 1989; Kalsbeek and Buijs, 1992; Kikusui et al., 1997; Abrahamson and Moore, 2001; Kalsbeek and Buijs, 2002). However, the distribution of these neurons is different in the camel SCN compared to the animals studied thus far. They occupy different locations on the rostrocaudal extension of the nucleus and appear more caudally in a dorsomedial position forming a population equivalent to the “shell” described in rodents (Ibata et al., 1999; Moore et al., 2002; Morin et al., 2006; Nascimento et al., 2010). The distribution of AVP-ir neurons in the camel SCN, as well as their relatively high number, seem to be a peculiarity of a species living in arid and desert biotopes. In the jerboa (Jaculus orientalis), a semi-desert rodent living in the same latitudes as the dromedary, AVP neurons in the SCN are intensely immunostained, and are located in the dorsomedial and ventromedial but also dorsal, dorsolateral and ventral portions of the SCN (Lakhdar-Ghazal et al., 1995a). Across all species studied to date, the AVP neurons located in the dorsomedial SCN play a crucial role in the development and distribution of circadian signals (for a review, see Reghunandanan and Reghunandanan, 2006) and this could also be the case for the camel SCN. Furthermore, studies have shown that the amount of vasopressin release in the SCN (Kalsbeek et al., 1995) and its mRNA levels (rat: Larsen et al., 1994, mice: Smith and Carter, 1996, Siberian hamster: Duncan et al., 1995) show daily variations with a diurnal acrophase. This rhythmicity is maintained under constant conditions (Yamase et al., 1991; Cagampang et al., 1994), demonstrating its circadian origin.

A relatively large number of VIP-ir neurons were found in the camel SCN. In the present investigation, the highest density of these neurons within the camel hypothalamus was observed in the SCN. Similar observations have been made in other mammalian species (Card et al., 1981; Stopa et al., 1984; Cassone et al., 1988; Tessonneaud et al., 1994; Abrahamson and Moore, 2001; Moore et al., 2002). It is well known that the ventrolateral subdivision of the SCN receives direct retinal afferents (Moore, 1973; Cassone et al., 1988; Ibata et al., 1989; Abrahamson and Moore, 2001), contacting VIP neurons (Ibata et al., 1989). Moreover, VIP expression (mRNA and peptide) in the SCN exhibits a nycthemeral rhythm depending on the LD cycle (Albers et al., 1990; Ibata et al., 1993; Shinohara et al., 1993; Yang et al., 1993; Larsen et al., 1994). In the camel SCN, the most rostral VIP-ir neuronal population, forming a cluster of cells in the ventral part of the mSCN, is similar in its shape and location to the findings reported in other species. Although no data on retinal projections is available in the camel, due to its location this group of VIP neurons could serve as the target of such projections also in the camel.

In rodents, VIP neurons project to the dorsal AVP neurons in the SCN (Jacomy et al., 1999) to regulate the activity of the clock by light (Harmar et al., 2002). Our findings suggest that VIP neurons in the camel SCN also project to the AVP neurons located dorsally. We also observed a dense plexus of VIP-ir fibers directed to the hypothalamic area above the SCN, which seem to reach the PVN and the dorsomedial nucleus, as reported in several other species (Kalsbeek et al., 1993; Saper et al., 2005). The neurophysiological significance of such dorsal projections could be related to the well-known modulatory role of VIP on AVP neurons (Watanabe et al., 1998; Jacomy et al., 1999; Maywood et al., 2006) and thus on clock activity. AVP neurons, in turn, would distribute the modulated circadian message to other structures in the brain.

In the jerboa, the SCN content of VIP shows seasonal variations (Lakhdar-Ghazal et al., 1992) which seems to be related to the photoperiod and the effect of sex hormones (Oukouchoud et al., 2003). Such mechanisms remain to be investigated in the camel. However, in view of data on the variation in the duration of melatonin secretion in this species (El Allali et al., 2005, 2008) and the existence of seasonal breeding activity, it seems reasonable to suppose that the photoperiod could modulate VIP expression in the camel SCN.

The present results show that the NPY-ir fibers were the densest in the camel SCN among the other neurochemically identified fibers investigated in this study. The NPY fibers form a dense plexus in different parts of the camel SCN, and especially in the mSCN. Such a high density of NPY fibers coming from the IGL has been previously described in the rodent SCN (Moore et al., 1984; Ueda et al., 1986; Sabatino et al., 1987; Card and Moore, 1989; Morin et al., 1992; Lakhdar-Ghazal et al., 1995b; Jacob et al., 1999; Menet et al., 2001; Abrahamson and Moore, 2001) and these fibers contact VIP neurons (Ibata et al., 1988; François-Bellan and Bosler, 1992). This innervation is reportedly involved in non-photic synchronization mechanisms (Challet et al., 1996, 1997; Juhl et al., 2007). The neuroanatomical pathways and the entrainment by the IGL are thus well demonstrated in rodents, but remain to be fully understood in the camel, sheep and most non-human primates. In non-human primates, a complex of NPY neurons, the pregeniculate nucleus, is equivalent to the IGL of rodents, but does not send efferents to the SCN (Moore, 1989; Chevassus-au-Louis and Cooper, 1998). Likewise, the SCN of the sheep harbors only sparse NPY-ir fibers (Tillet et al., 1989). The present findings show a dense plexus of NPY fibers in the camel SCN, but a combination of immunohistochemistry and tract tracing is necessary to address its origin from the IGL.

The camel has to adapt its physiology and to anticipate changes in its harsh environment by integrating the most important environmental cues, mainly the environmental temperature and LD cycles. The density of NPY-ir fibers in the camel SCN may reflect the integration of non-photic signals, correlated with an important non-photic entrainment of the circadian clock represented by the daily cycle of environmental temperature (El Allali et al., 2013).

The present findings also demonstrate a dense plexus of Met-Enk-ir fibers in the camel SCN. Met-Enk innervation of the SCN has also been reported in others species, including: mouse (Abrahamson and Moore, 2001), sheep (Tillet et al., 1989; Tessonneaud et al., 1994) and Syrian hamster (Morin et al., 1992). In this latter species, Met-Enk fibers originate from neurons located in the IGL (Morin and Blanchard, 1995) and participate in photic transmission and clock synchronization (Harrington and Rusak, 1986; Pickard et al., 1987; Edelstein and Amir, 1999; Juhl et al., 2007).

Our data for the camel SCN demonstrate that 5-HT immunopositive fibers cross the hypothalamus, especially the periventricular areas, toward the SCN. A high density of 5-HT fibers in the SCN has been reported for several species (Ueda et al., 1983; Abrahamson and Moore, 2001). These fibers constitute a third major set of afferents to the SCN and are involved in the transmission of non-photic stimuli to this nucleus. Serotonergic modulation of the SCN is well characterized in the Syrian hamster (Azmitia and Segal, 1978; Meyer-Bernstein and Morin, 1996; Hay-Schmidt et al., 2003; Vrang et al., 2003). Moreover, in some species (rat and cat), direct retinal projections to the raphe nuclei were identified. Photic information could therefore also reach the SCN indirectly through 5-HT fibers. The existence of a significant 5-HT innervation in the camel SCN could be related to the particular adaptation of this species to its biotope thus requiring pathways combining non-photic and photic entrainment.

In most mammals, the relative importance of photic versus non-photic entrainment of the circadian clock is not fully understood. In addition to LD entrainment, several non-photic factors can synchronize the circadian clock. These stimuli may be behavioral, dietary or other environmental factors such as environmental temperature, for which little information is available to date. It appears that the unusual morphology of the camel SCN (both in terms of its length and shape) and the uncommon existence of TH and OT neurons, in addition to the presence of a dense innervation of NPY, Met-Enk and 5-HT fibers, reflect collectively the importance of this nucleus in the circadian adaptation of the camel to its harsh biotope. The synchronization of the circadian clock in this species both by photic and non-photic cues deserves special study. Under experimental conditions (El Allali et al., 2013), we demonstrated that both entrainments occur in the camel: synchronization by the LD cycle and by the daily cycle of environmental temperature. Neuroanatomical interactions in the camel brain between different pathways for photic and non-photic entrainment are also very likely and warrant further investigation.

There were no animal sacrifices to carry out this study. The work was conducted by using camels’s brains. Samples were taken as animals were slaughtered to provide meat for public consumption. The study was in conformation with the Hassan II Agronomy and Veterinary Institute of Rabat and Moroccan Ministry of Agriculture recommendations which are in accordance with international ethical standards (Touitou et al., 2006).

KEA, AC, PP, and NL-G conceived and designed the work; KEA, MA, and MO performed brains sampling; KEA performed immunohistochemical labeling experiments; KEA and MP performed image acquisition and quantification of immunofluorescence; KEA, EC, and PP prepared the manuscript; MA, MP, MO, EC, ME, NL-G, AC, and PP revised and approved the final review.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.