Nuclear organization and morphology of serotonergic neurons in the brain of the Nile crocodile, Crocodylus niloticus

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Abstract

The present study describes the location and nuclear organization of the serotonergic system in a representative of the order Crocodylia, the Nile crocodile (Crocodylus niloticus). We found evidence for serotonergic neurons in three regions of the brain, including the diencephalon, rostral and caudal brainstem, as previously reported in several other species of reptile. Within the diencephalon we found neurons in the periventricular organ of the hypothalamus, but not in the infundibular recess as noted in some other reptilian species. In addition we found serotonergic neurons in the pretectal nucleus, this being the first description of these neurons in any species. Within the rostral brainstem we found medial and lateral divisions of the superior raphe nucleus and a widely dispersed group of neurons in the tegmentum, the superior reticular nucleus. In the caudal brainstem we observed the inferior raphe nucleus and the inferior reticular nucleus. While much of the serotonergic system of the Nile crocodile is similar to that seen in other reptiles the entire suite of features appears to distinguish the crocodile studied from the members of the Squamate (lizards and snakes) and Testudine (turtles, tortoises and terrapins) reptiles previously studied. The observations are suggestive of order-specific patterns of nuclear organization of this system in the reptiles, reflecting potential evolutionary constraints in the mutability of the nuclear organization as seen for similar systems in mammals.

Introduction

Almost the entire central nervous system of vertebrates receives innervation from the serotonergic system. The neurons producing serotonin are for the most part found within the brainstem in distinct rostral and caudal nuclear clusters, exclusively so in therian mammals (Dahlström and Fuxe, 1964, Steinbusch, 1981, Törk, 1990, Jacobs and Azmitia, 1992). The only mammals reported to have serotonergic neurons located outside of the brainstem are the monotremes, which exhibit serotonergic immunoreactive neurons in the periventricular organ (Manger et al., 2002c). In other amniotes (birds and reptiles), serotonergic neurons have been found in other regions of the brain in addition to the brainstem, but for the most part are located within the hypothalamus (e.g. Marschall, 1980, Smeets and Steinbusch, 1988, Cozzi et al., 1991, Challet et al., 1991, Challet et al., 1996).

Reports detailing aspects of the location and morphology of the serotonergic system within the brain of reptiles are not numerous, and the species investigated include: Chrysemys picta (Parent and Poirier, 1971, Parent and Poitras, 1974); Lacerta sicula and Lacerta muralis (Marschall, 1980); Clemmys japonica (Ueda et al., 1983); Varanus exanthematicus (Wolters et al., 1985); Gekko gecko (Smeets and Steinbusch, 1988); Psammodromus algirus and Podarcis hispanica (Guirado et al., 1989); Lacerta agilis (Petko and Ihionvien, 1989); Chameleo chameleo (Bennis et al., 1990); Vipera aspis (Challet et al., 1991); and Pseudomys scripta (Kiehn et al., 1992). All these previous studies have examined members of the orders Testudines (turtles, tortoises and terrapins) or Squamata (lizards and snakes), but to date no studies have described the serotonergic system within the brain of a member of the order Crocodilia, even though the existence of serotonin has been demonstrated in the brain of Caiman sclerops (Doshi and Huggins, 1977). This latter study demonstrated differential distribution in the levels of serotonin in the Caiman brain, with the greatest concentrations being found in the midbrain tegmentum, pons and medulla (Doshi and Huggins, 1977).

Given that all vertebrates examined with either formaldehyde fluorescence or immunohistochemical techniques have revealed serotonergic neurons and nuclei within the brain it would be expected that serotonergic neurons would be found in the brain of the crocodile; however, there is variation in the nuclear organization and, at least in mammals, this variation appears to be related to phylogeny (Manger, 2005, Da Silva et al., 2006, Maseko et al., 2007). Given the variation in nuclear organization reported in reptiles (see above references for details and also Table 1), and the close relationship of the crocodiles to birds (Hedges and Poling, 1999), a study of the serotonergic system of the crocodile brain may reveal insights into the evolutionary patterns of the nuclear organization of serotonergic system in reptiles and/or birds.

Studies within mammals have shown that the serotonergic system is involved in a range of behavioural and physiological actions, including the sleep–wake cycle, motor activity, thermoregulation, food consumption, sexual behaviour and nociception (e.g. Fuxe et al., 1970, Jacobs and Azmitia, 1992, Sakai and Crochet, 2001). A series of studies in a reptile, Anolis carolinensis, have shown that for this lizard the serotonergic system is involved in the modulation of aggressive social interactions and is modulated by social signals (Summers and Greenberg, 1995, Summers et al., 1997, Summers et al., 1998, Summers et al., 2003a, Summers et al., 2003b, Summers et al., 2005a, Summers et al., 2005b, Emerson et al., 2000, Korzan et al., 2000, Korzan et al., 2001, Larson and Summers, 2001, Summers, 2001, Korzan and Summers, 2004). Given the important role serotonin plays in the mediation of aggression and sociality in Anolis, it is of interest to establish a baseline comparative anatomical description for this system in the brain of the Nile crocodile, a reptilian species that also demonstrates social hierarchies and significant aggressive behaviour (Morpurgo et al., 1993).

Section snippets

Materials and methods

Six juvenile male specimens of Crocodylus niloticus, the Nile crocodile, were used in the current study. The guidelines of the University of the Witwatersrand Animal Ethics Committee, which parallel those set down by the NIH, for the use of animals in scientific experiments were followed. The crocodiles used in this study were approximately 1 m in length, ranged in weight from 4.3 to 5.6 kg, and had brain weights ranging from 4.9 to 5.3 g. Intramuscular injections of ketamine (80 mg/kg) and

Results

Immunoreactive serotonergic neurons were observed in the brain of the Nile crocodile from the level of the hypothalamus through to the spinomedullary junction. In the crocodile brain we could readily identify three distinct serotonergic nuclear clusters and these include the diencephalic/pretectal (2 nuclei), rostral brainstem (3 nuclei) and caudal brainstem (2 nuclei) nuclear clusters (Fig. 1). Within the pretectal region we describe a previously unrecorded serotonergic immunoreactive group of

Discussion

The current study of the serotonergic system in the crocodile revealed much in common with that of previous studies of reptiles, but certain differences of interest were noted. While we found serotonergic nuclei in the hypothalamic periventricular organ of the diencephalon, and in the rostral and caudal brainstem, we did not find evidence for serotonergic nuclei in the ependymal wall of the infundibular recess. Interestingly, we found a previously undescribed serotonergic neuronal cluster

Acknowledgments

This work was supported by funding from the South African National Research Foundation (PRM) and SIDA (KF). The authors wish to extend their gratitude to the members of the Central Animal Services of the University of the Witwatersrand for their enthusiastic and expert assistance with animal handling and care during this project.

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