Nadine Gravett

Nuclear organization and morphology of cholinergic, putative catecholaminergic and serotonergic neurons in the brain of the rock hyrax, Procavia capensis

The nuclear subdivisions of the cholinergic, putative catecholaminergic and serotonergic systems within the brain of the rock hyrax (Procavia capensis) were identified following immunohistochemistry for acetylcholinesterase, tyrosine hydroxylase and serotonin. The aim of the present study was to investigate possible differences in the complement of nuclear subdivisions of these systems by comparing those of the rock hyrax to published studies of other mammals. The rock hyrax belongs to the order Hyracoidea and forms part of the Afroplacentalia mammalian cohort. For the most part, the nuclear organization of these three systems closely resembled that described for many other mammalian species. The nuclear organization of the serotonergic system was identical to that seen in all eutherian mammals. The nuclear organization of the putative catecholaminergic system was very similar to that seen in rodents except for the lack of a C3 nucleus and the compact division of the locus coeruleus (A6c). In addition, the diffuse locus coeruleus (A6d) appeared to contain very few tyrosine hydroxylase immunoreactive (TH+) neurons. The cholinergic system showed many features in common with that seen in both rodents and primates; however, there were three differences of note: (1) cholinergic neurons were observed in the anterior nuclei of the dorsal thalamus; (2) cholinergic parvocellular nerve cells, probably representing interneurons, forming subdivisions of the laterodorsal and pedunculopontine tegmental nuclei were observed at the midbrain/pons interface; and (3) a large number of cholinergic nerve cells in the periventricular grey of the medulla oblongata were observed. Thus, while there are many similarities to other mammalian species, the nuclear organization of these systems in the rock hyrax shows specific differences to what has been observed previously in other mammals. These differences are discussed in both a functional and phylogenetic perspective.

Cellular scaling rules for the brain of afrotherians

Quantitative analysis of the cellular composition of rodent, primate and eulipotyphlan brains has shown that non-neuronal scaling rules are similar across these mammalian orders that diverged about 95 million years ago, and therefore appear to be conserved in evolution, while neuronal scaling rules appear to be free to vary in evolution in a clade-specific manner. Here we analyze the cellular scaling rules that apply to the brain of afrotherians, believed to be the first clade to radiate from the common eutherian ancestor. We find that afrotherians share non-neuronal scaling rules with rodents, primates and eulipotyphlans, as well as the coordinated scaling of numbers of neurons in the cerebral cortex and cerebellum. Afrotherians share with rodents and eulipotyphlans, but not with primates, the scaling of number of neurons in the cortex and in the cerebellum as a function of the number of neurons in the rest of the brain. Afrotheria also share with rodents and eulipotyphlans the neuronal scaling rules that apply to the cerebral cortex. Afrotherians share with rodents, but not with eulipotyphlans nor primates, the neuronal scaling rules that apply to the cerebellum. Importantly, the scaling of the folding index of the cerebral cortex with the number of neurons in the cerebral cortex is not shared by either afrotherians, rodents, or primates. The sharing of some neuronal scaling rules between afrotherians and rodents, and of some additional features with eulipotyphlans and primates, raise the interesting possibility that these shared characteristics applied to the common eutherian ancestor. In turn, the clade-specific characteristics that relate to the distribution of neurons along the surface of the cerebral cortex and to its degree of gyrification suggest that these characteristics compose an evolutionarily plastic suite of features that may have defined and distinguished mammalian groups in evolution.

Nuclear organization of cholinergic, putative catecholaminergic and serotonergic nuclei in the brain of the eastern rock elephant shrew, Elephantulus myurus

The organization of the nuclear subdivisions of the cholinergic, putative catecholaminergic and serotonergic systems of the brain of the elephant shrew (Elephantulus myurus) were determined following immunohistochemistry for choline acetyltransferase, tyrosine hydroxylase and serotonin, respectively. This was done in order to determine if differences in the nuclear organization of these systems in comparison to other mammals were evident and how any noted differences may relate to specialized behaviours of the elephant shrew. The elephant shrew belongs to the order Macroscelidea, and forms part of the Afrotherian mammalian cohort. In general, the organization of the nuclei of these systems resembled that described in other mammalian species. The cholinergic system showed many features in common with that seen in the rock hyrax, rodents and primates; however, specific differences include: (1) cholinergic neurons were observed in the superior and inferior colliculi, as well as the cochlear nuclei; (2) cholinergic neurons were not observed in the anterior nuclei of the dorsal thalamus as seen in the rock hyrax; and (3) cholinergic parvocellular nerve cells forming subdivisions of the laterodorsal and pedunculopontine tegmental nuclei were not observed at the midbrain/pons interface as seen in the rock hyrax. The organization of the putative catecholaminergic system was very similar to that seen in the rock hyrax and rodents except for the lack of the rodent specific C3 nucleus, the dorsal division of the anterior hypothalamic group (A15d) and the compact division of the locus coeruleus (A6c). The nuclear organization of the serotonergic system was identical to that seen in all eutherian mammals studied to date. The additional cholinergic neurons found in the cochlear nucleus and colliculi may relate to a specific acoustic signalling system observed in elephant shrews expressed when the animals are under stress or detect a predator. These neurons may then function to increase attention to this type of acoustic signal termed foot drumming.

Distribution of orexin-A immunoreactive neurons and their terminal networks in the brain of the rock hyrax, Procavia capensis.

The present study describes the distribution of orexin-A immunoreactive neurons and terminal networks in relation to the previously described catecholaminergic, cholinergic and serotonergic systems within the brain of the rock hyrax, Procavia capensis. Adult female rock hyrax brains were sectioned and immunohistochemically stained with an antibody to orexin-A. The staining revealed that the neurons were mainly located within the hypothalamus as with other mammals. The orexinergic terminal network distribution also resembled the typical mammalian plan. High-density orexinergic terminal networks were located within regions of the diencephalon (e.g. paraventricular nuclei), midbrain (e.g. serotonergic nuclei) and pons (locus coeruleus), while medium density orexinergic terminal networks were evident in the telencephalic (e.g. basal forebrain), diencephalic (e.g. hypothalamus), midbrain (e.g. periaqueductal gray matter), pontine (e.g. serotonergic nuclei) and medullary regions (e.g. serotonergic and catecholaminergic nuclei). Although the distribution of the orexinergic terminal networks was typically mammalian, the rock hyrax did show one atypical feature, the presence of a high-density orexinergic terminal network within the anterodorsal nucleus of the dorsal thalamus (AD). The dense orexinergic innervation of the AD nucleus has only been reported previously in the Nile grass rat, Arvicanthis niloticus and Syrian hamster, Mesocricetus auratus, both diurnal mammals. It is possible that orexinergic innervation of the AD nucleus might be a unique feature associated with diurnal mammals. It was also noted that the dense orexinergic innervation of the AD nucleus coincided with previously identified cholinergic neurons and terminal networks in this particular nucleus of the rock hyrax brain. It is possible that this dense orexinergic innervation of the AD nucleus in the brain of the rock hyrax may act in concert with the cholinergic neurons and/or the cholinergic axonal terminals, which in turn may influence arousal states and motivational processing.

Arabian Oryx (Oryx leucoryx) Respond to Increased Ambient Temperatures with a Seasonal Shift in the Timing of Their Daily Inactivity Patterns

The Arabian oryx inhabits an environment where summer ambient temperatures can exceed 40 °C for extended periods of time. While the oryx uses a suite of adaptations that aid survival, the effects of this extreme environment on inactivity are unknown. To determine how the oryx manages inactivity seasonally, we measured the daily rhythm of body temperature and used fine-grain actigraphy, in 10 animals, to reveal when the animals were inactive in relation to ambient temperature and photoperiod. We demonstrate that during the cooler winter months, the oryx was inactive during the cooler parts of the 24-h day (predawn hours), showing a nighttime (nocturnal) inactivity pattern. In contrast, in the warmer summer months, the oryx displayed a bimodal inactivity pattern, with major inactivity bouts (those greater than 1 h) occurring equally during both the coolest part of the night (predawn hours) and the warmest part of the day (afternoon hours). Of note, the timing of the daily rhythm of body temperature did not vary seasonally, although the amplitude did change, leading to a seasonal alteration in the phase relationship between inactivity and the body temperature rhythm. Because during periods of inactivity the oryx were presumably asleep for much of the time, we speculate that the daytime shift in inactivity may allow the oryx to take advantage of the thermoregulatory physiology of sleep, which likely occurs when the animal is inactive for more than 1 h, to mitigate environmentally induced increases in body temperature.

Distribution of parvalbumin, calbindin and calretinin containing neurons and terminal networks in relation to sleep associated nuclei in the brain of the giant Zambian mole-rat (Fukomys mechowii)

To broaden the understanding of the neural control and evolution of the sleep-wake cycle in mammals, the distribution and interrelations of sleep associated nuclei with neurons and terminal networks expressing the calcium-binding proteins parvalbumin, calbindin and calretinin were explored in a rodent that lacks a significant visual system. The sleep-associated nuclei explored include the cholinergic basal forebrain and pontine nuclei, the catecholaminergic locus coeruleus complex, the serotonergic dorsal raphe nuclear complex, the hypothalamic orexinergic nuclei, and the thalamic reticular nucleus. Zambian mole-rat brains were sectioned and stained in a one in nine series for Nissl, myelin, choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), serotonin (5HT), orexin (OrxA), calbindin (CB), calretinin (CR) and parvalbumin (PV). We observed that while the density of immunopositive calbindin (CB+) neurons and terminal networks varied in the different sleep related nuclei, they were found in all nuclei apart from the compact and diffuse subdivisions of the subcoeruleus, which lacked CB+ neurons but evinced a CB+ terminal network. The density of calretinin immunopositive (CR+) neurons and terminal networks varied between the sleep related nuclei, but was present in all nuclei examined. Neurons and terminal networks associated with PV immunoreactivity were the most sparsely distributed in these nuclei, but were present in the majority of nuclei. The thalamic reticular nucleus had the highest density of PV+ neurons and terminal networks, while PV+ neurons were absent in the cholinergic pontine nuclei, and PV+ neurons and terminal networks were absent in the orexinergic nuclei. The increased presence of neurons and terminal networks expressing the calcium binding proteins in comparison to that seen in the laboratory rat, specifically in the brainstem, may account for the prominent muscle twitches during REM sleep previously observed in this subterranean African rodent.

Inactivity/sleep in two wild free-roaming African elephant matriarchs – Does large body size make elephants the shortest mammalian sleepers?

The current study provides details of sleep (or inactivity) in two wild, free-roaming African elephant matriarchs studied in their natural habitat with remote monitoring using an actiwatch subcutaneously implanted in the trunk, a standard elephant collar equipped with a GPS system and gyroscope, and a portable weather station. We found that these two elephants were polyphasic sleepers, had an average daily total sleep time of 2 h, mostly between 02:00 and 06:00, and displayed the shortest daily sleep time of any mammal recorded to date. Moreover, these two elephants exhibited both standing and recumbent sleep, but only exhibited recumbent sleep every third or fourth day, potentially limiting their ability to enter REM sleep on a daily basis. In addition, we observed on five occasions that the elephants went without sleep for up to 46 h and traversed around 30 km in 10 h, possibly due to disturbances such as potential predation or poaching events, or a bull elephant in musth. They exhibited no form of sleep rebound following a night without sleep. Environmental conditions, especially ambient air temperature and relative humidity, analysed as wet-bulb globe temperature, reliably predict sleep onset and offset times. The elephants selected novel sleep sites each night and the amount of activity between sleep periods did not affect the amount of sleep. A number of similarities and differences to studies of elephant sleep in captivity are noted, and specific factors shaping sleep architecture in elephants, on various temporal scales, are discussed.

The distribution of doublecortin-immunopositive cells in the brains of four afrotherian mammals : the Hottentot golden mole (Amblysomus hottentotus), the rock hyrax (Procavia capensis), the eastern rock sengi (Elephantulus myurus) and the four-toed sengi (Petrodromus tetradactylus)

Adult neurogenesis in the mammalian brain is now a widely accepted phenomenon, typically occurring in two forebrain structures: the subgranular zone (SGZ) of the hippocampal dentate gyrus and the subventricular zone (SVZ). Until recently, the majority of studies have focused on laboratory rodents, and it is under debate whether the process of adult neurogenesis occurs outside of the SGZ and the SVZ in other mammalian species. In the present study, we investigated potential adult neurogenetic sites in the brains of two elephant shrews/sengis, a golden mole and a rock hyrax, all members of the superorder Afrotheria. Doublecortin (DCX) immunoreactivity was used as a proxy to visualise adult neurogenesis, which is expressed in neuronal precursor cells and immature neurons. In all four species, densely packed DCX-positive cells were present in the SVZ, from where cells appear to migrate along the rostral migratory stream towards the olfactory bulb (OB). DCX-immunopositive cells were present in the granular cell layer and the glomerular layer of the OB. In the hippocampus, DCX-immunopositive cells were observed in the SGZ and in the granular layer of the dentate gyrus, with DCX-immunopositive processes extending into the molecular layer. In addition to these well-established adult neurogenic regions, DCX-immunopositive cells were also observed in layer II of the neocortex and the piriform cortex. While the present study reveals a similar pattern of adult neurogenesis to that reported previously in other mammals, further studies are needed to clarify if the cortical DCX-immunopositive cells are newly generated neurons or cells undergoing cortical remodelling.

Sleep in the Rock Hyrax, Procavia capensis

We investigated sleep in therock hyrax, Procavia capensis, a social mammal that typically lives in colonies on rocky outcrops throughout most parts of Southern Africa. The sleep of 5 wild-captured, adult rock hyraxes was recorded continuously for 72 h using telemetric relay of signals and allowing unimpeded movement. In addition to waking, slow wave sleep (SWS) and an unambiguous rapid eye movement (REM) state, a sleep state termed somnus innominatus (SI), characterized by low-voltage, high-frequency electroencephalogram, an electromyogram that stayed at the same amplitude as the preceding SWS episode and a mostly regular heart rate, were identified. If SI can be considered a form of low-voltage non-REM, the implication would be that the rock hyrax exhibits the lowest amount of REM recorded for any terrestrial mammal studied to date. Conversely, if SI is a form of REM sleep, it would lead to the classification of a novel subdivision of this state; however, further investigation would be required. The hyraxes spent on average 15.89 h (66.2%) of the time awake, 6.02 h (25.1%) in SWS, 43 min (3%) in SI and 6 min (0.4%) in REM. The unambiguous REM sleep amounts were on average less than 6 min/day. The most common state transition pathway in these animals was found to be wake → SWS → wake. No significant differences were noted with regard to total sleep time, number of episodes and episode duration for all states between the light and dark periods.Thus, prior classification of the rock hyrax as strongly diurnal does not appear to hold under controlled laboratory conditions.

Orexinergic neuron numbers in three species of African mole rats with rhythmic and arrhythmic chronotypes

In the present study, orexinergic cell bodies within the brains of rhythmic and arrhythmic circadian chronotypes from three species of African mole rat (Highveld mole rat-Cryptomys hottentotus pretoriae, Ansells mole rat--Fukomys anselli and the Damaraland mole rat--Fukomys damarensis) were identified using immunohistochemistry for orexin-A. Immunopositive orexinergic (Orx+) cell bodies were stereologically assessed and absolute numbers of orexinergic cell bodies were determined for the distinct circadian chronotypes of each species of mole rat examined. The aim of the study was to investigate whether the absolute numbers of identified orexinergic neurons differs between distinct circadian chronotypes with the hypothesis of elevated hypothalamic orexinergic neurons in the arrhythmic chronotypes compared with the rhythmic chronotypes. We found statistically significant differences between the circadian chronotypes ofF. anselli, where the arrhythmic group had higher mean numbers of hypothalamic orexin neurons compared with the rhythmic group. These differences were observed when the raw data was compared and when the raw data was corrected for body mass (M(b)) and brain mass (M(br)). For the two other species investigated, no significant differences were noted between the chronotypes, although a statistically significant difference was noted between all rhythmic and arrhythmic individuals of the current study when the counts of orexin neurons were corrected for M(b)--the arrhythmic individuals had larger numbers of orexin cells.