Orexinergic bouton density is lower in the cerebral cortex of cetaceans compared to artiodactyls

Highlights

• The orexinergic innervation of the cerebral cortex of Cetartiodactyla is described.

• All species show a similar innervation pattern.

• The density of innervation is far less in cetaceans than artiodactyls.

Abstract

The species of the cetacean and artiodactyl suborders, which constitute the order Cetartiodactyla, exhibit very different sleep phenomenology, with artiodactyls showing typical bihemispheric slow wave and REM sleep, while cetaceans show unihemispheric slow wave sleep and appear to lack REM sleep. The aim of this study was to determine whether cetaceans and artiodactyls have differently organized orexinergic arousal systems by examining the density of orexinergic innervation to the cerebral cortex, as this projection will be involved in various aspects of cortical arousal. This study provides a comparison of orexinergic bouton density in the cerebral cortex of twelve Cetartiodactyla species (ten artiodactyls and two cetaceans) by means of immunohistochemical staining and stereological analysis. It was found that the morphology of the axonal projections of the orexinergic system to the cerebral cortex was similar across all species, as the presence, size and proportion of large and small orexinergic boutons were similar. Despite this, orexinergic bouton density was lower in the cerebral cortex of the cetaceans studied compared to the artiodactyls studied, even when corrected for brain mass, neuron density, glial density and glial:neuron ratio. Results from correlational and principal component analyses indicate that glial density is a major determinant of the observed differences between artiodactyl and cetacean cortical orexinergic bouton density.

Introduction

Orexin/hypocretin is a neuropeptide synthesized by neurons located within the hypothalamus that give rise to axonal projections found throughout the brain (Horvath et al., 1999). In most mammals the majority of these orexinergic neurons are found within the lateral hypothalamus and perifornical area, but have also been observed in the zona incerta region and the ventrolateral hypothalamus near the optic tract (Peyron et al., 1998, van den Pol, 1999, Wagner et al., 2000, Iqbal et al., 2001, Moore et al., 2001, Yoshida et al., 2006, Nixon and Smale, 2007, Datta and MacLean, 2007, Ettrup et al., 2010, Kruger et al., 2010, Bhagwandin et al., 2011a, Bhagwandin et al., 2011b, Gravett et al., 2011, Calvey et al., 2013, Dell et al., 2013, Maseko et al., 2013). A study by Dell et al. (2012) comparing the distribution and number of orexinergic neurons in the brain of the giraffe and harbour porpoise revealed an additional novel medially located parvocellular cluster of orexinergic neurons in the hypothalamus of these two species, which has since also been reported in the African elephant (Maseko et al., 2013). Additionally, for a comparable brain mass, the harbour porpoise had significantly more orexinergic neurons (around 6000) in the hypothalamus than the giraffe, but the giraffe had significantly larger orexinergic neuronal somata (by approximately 1000 μm³ for both magnocellular and parvocellular neurons) than that of the harbour porpoise. As this parvocellular orexinergic cluster had not been identified in other species, nor had the differences in neuronal numbers and size been reported, Dell et al. (2012) concluded that the orexinergic system is potentially more complex in Cetartiodactyla compared to other mammals. Despite this, it is currently unclear whether these novel findings are associated with differences in the terminal networks of the orexinergic system in these species and if differences do occur, whether they have any functional consequences.

Physiological studies demonstrate that orexinergic neuronal firing patterns are greatest during periods of wakefulness, motor activation and sustained attentiveness to external stimuli and the environment (Datta and MacLean, 2007, Alexandre et al., 2013). Thus, the orexinergic system has been implicated in several functions including the promotion and maintenance of arousal and wakefulness (Alexandre et al., 2013), the regulation of food consumption (Edwards et al., 1999), energy metabolism, thermoregulation and locomotion (Peyron et al., 1998, Mintz et al., 2001, Spinazzi et al., 2006), with an overall involvement in “work for reward” behaviours, or in the case of humans, for alertness linked to being happy (Mileykovskiy et al., 2005, McGregor et al., 2011, Blouin et al., 2013). While cetaceans and artiodactyls both belong to the order Cetartiodactyla (Price et al., 2005), there are substantial differences in their life history, all of which appear to be related to functions associated with the orexinergic system. Cetaceans are known to exhibit unihemispheric slow waves and little to no REM sleep (Lyamin et al., 2008), while artiodactyls show the standard bihemispheric mammalian sleep patterns, including REM (Bell and Itabisashi, 1973, Tobler and Schwierin, 1996). Cetacean limbs and necks have been reduced so much that they no longer have a significant locomotor function, whereas in artiodactyls a range of neck and limb proportions are evident (Badlangana et al., 2009). While both groups require substantial food ingestion, cetaceans are carnivorous, whereas the artiodactyls are herbivorous. Cetaceans are under constant, significant thermal pressures due to their aquatic environment (Manger, 2006), whereas the artiodactyls have mechanisms in place to prevent overheating (Kuhnen, 1997, Jessen, 1998, Jessen, 2001, Mitchell et al., 2002). Additionally, it has been demonstrated that dolphins can continuously maintain vigilant behaviour for at least 15 days without any signs of sleep deprivation (Ridgway et al., 2006, Ridgway et al., 2009, Branstetter et al., 2012), an observation not made, but unlikely to be present, in artiodactyls.

Given these variations in the life histories, environments and behaviour of the members of the order Cetartiodactyla, these species may provide an important model for understanding the role of orexinergic neurons and their terminal networks (Dell et al., 2012). It has been reported that sustained arousal, vigilance and cognitive function may be mediated via orexinergic projections to the cortex (Bayer et al., 2004, Yamada et al., 2008). Specifically, in the rat prefrontal cortex it has been demonstrated that orexin has a direct excitatory postsynaptic effect on pyramidal neurons (Song et al., 2006, Yan et al., 2012). Thus, the density of orexin boutons in the cerebral cortex may provide an indication of the degree to which orexin innervation modulates the activity of cortical neurons to support the behaviours associated with activation of hypothalamic orexinergic neurons. Given the higher number of orexinergic neurons in the harbour porpoise hypothalamus when compared to the giraffe hypothalamus (Dell et al., 2012), it is possible that cetaceans as a whole have an increased bouton density in the cerebral cortex when compared to artiodactyls; however, this possibility needs to be balanced with the observation that the orexinergic neurons in the giraffe are larger than those in harbour porpoise, and thus may support larger and more branched axons than the cetaceans in general, leading to a higher orexin bouton density in the artiodactyl cerebral cortex in general. In order to determine which of these possibilities, if either, occurs, the current study provides a comparison of orexinergic bouton density in the anterior cingulate and occipital cerebral cortex of a number of Cetartiodactyl species by means of immunohistochemical staining and stereological analysis.