The evolution of mammalian brain size.

Relative brain size has long been considered a reflection of cognitive capacities and has played a fundamental role in developing core theories in the life sciences. Yet, the notion that relative brain size validly represents selection on brain size relies on the untested assumptions that brain-body allometry is restrained to a stable scaling relationship across species and that any deviation from this slope is due to selection on brain size. Using the largest fossil and extant dataset yet assembled, we find that shifts in allometric slope underpin major transitions in mammalian evolution and are often primarily characterized by marked changes in body size. Our results reveal that the largest-brained mammals achieved large relative brain sizes by highly divergent paths. These findings prompt a reevaluation of the traditional paradigm of relative brain size and open new opportunities to improve our understanding of the genetic and developmental mechanisms that influence brain size.

Is home-based monitoring of ovulation to time frozen embryo transfer a cost-effective alternative for hospital-based monitoring of ovulation? Study protocol of the multicentre, non-inferiority Antarctica-2 randomised controlled trial.

STUDY QUESTION: The objective of this trial is to compare the effectiveness and costs of true natural cycle (true NC-) frozen embryo transfer (FET) using urinary LH tests to modified NC-FET using repeated ultrasound monitoring and ovulation trigger to time FET in the NC. Secondary outcomes are the cancellation rates of FET (ovulation before hCG or no dominant follicle, no ovulation by LH urine test, poor embryo survival), pregnancy outcomes (miscarriage rate, clinical pregnancy rates, multiple ongoing pregnancy rates, live birth rates, costs) and neonatal outcomes (including gestational age, birthweight and sex, congenital abnormalities or diseases of babies born). WHAT IS KNOWN ALREADY: FET is at the heart of modern IVF. To allow implantation of the thawed embryo, the endometrium must be prepared either by exogenous oestrogen and progesterone supplementation (artificial cycle (AC)-FET) or by using the NC to produce endogenous oestradiol before and progesterone after ovulation to time the transfer of the thawed embryo (NC-FET). During an NC-FET, women visit the hospital repeatedly and receive an ovulation trigger to time FET (i.e. modified (m)NC-FET or hospital-based monitoring). From the woman's point of view, a more natural approach using home-based monitoring of the ovulation with LH urine tests to allow a natural ovulation to time FET may be desired (true NC-FET or home-based monitoring). STUDY DESIGN SIZE DURATION: This is a multicentre, non-inferiority prospective randomised controlled trial design. Consenting women will undergo one FET cycle using either true NC-FET or mNC-FET based on randomisation. PARTICIPANTS/MATERIALS SETTING METHODS: Based on our sample size calculation, the study group will consist of 1464 women between 18 and 45 years old who are scheduled for FET. Women with anovulatory cycles, women who need ovulation induction and women with a contra indication for pregnancy will be excluded. The primary outcome is ongoing pregnancy. Secondary outcomes are cancellation rates of FET, pregnancy outcomes (including miscarriage rate, clinical pregnancy, multiple pregnancy rate and live birth rate). Costs will be estimated by counting resource use and calculating unit prices. STUDY FUNDING/COMPETING INTERESTS: The study received a grant from the Dutch Organisation for Health Research and Development (ZonMw 843002807; www.zonmw.nl). ZonMw has no role in the design of the study, collection, analysis, and interpretation of data or writing of the manuscript. F.B. reports personal fees from member of the external advisory board for Merck Serono, grants from Research support grant Merck Serono, outside the submitted work. A.E.P.C. reports and Unrestricted grant of Ferring B.V. to the Center for Reproductive medicine, no personal fee. Author up-to-date on Hyperthecosis. Congress meetings 2019 with Ferring B.V. and Theramex B.V. M.G. reports Department research and educational grants from Guerbet, Merck and Ferring (location VUMC) outside the submitted work. E.R.G. reports personal fees from Titus Health Care, outside the submitted work. C.B.L. reports grants from Ferring, grants from Merck, from Guerbet, outside the submitted work. The other authors have none to declare. TRIAL REGISTRATION NUMBER: Dutch Trial Register (Trial NL6414 (NTR6590), https://www.trialregister.nl/). TRIAL REGISTRATION DATE: 23 July 2017. DATE OF FIRST PATIENT'S ENROLMENT: 10 April 2018.

A test of the lateral semicircular canal correlation to head posture, diet and other biological traits in "ungulate" mammals.

For over a century, researchers have assumed that the plane of the lateral semicircular canal of the inner ear lies parallel to the horizon when the head is at rest, and used this assumption to reconstruct head posture in extinct species. Although this hypothesis has been repeatedly questioned, it has never been tested on a large sample size and at a broad taxonomic scale in mammals. This study presents a comprehensive test of this hypothesis in over one hundred "ungulate" species. Using CT scanning and manual segmentation, the orientation of the skull was reconstructed as if the lateral semicircular canal of the bony labyrinth was aligned horizontally. This reconstructed cranial orientation was statistically compared to the actual head posture of the corresponding species using a dataset of 10,000 photographs and phylogenetic regression analysis. A statistically significant correlation between the reconstructed cranial orientation and head posture is found, although the plane of the lateral semicircular canal departs significantly from horizontal. We thus caution against the use of the lateral semicircular canal as a proxy to infer precisely the horizontal plane on dry skulls and in extinct species. Diet (browsing or grazing) and head-butting behaviour are significantly correlated to the orientation of the lateral semicircular canal, but not to the actual head posture. Head posture and the orientation of the lateral semicircular canal are both strongly correlated with phylogenetic history.

Living on the edge: Daily, seasonal and annual body temperature patterns of Arabian oryx in Saudi Arabia.

Heterothermy, the ability to allow body temperature (Tb) to fluctuate, has been proposed as an adaptive mechanism that enables large ungulates to cope with the high environmental temperatures and lack of free water experienced in arid environments. By storing heat during the daytime and dissipating it during the night, arid-adapted ungulates may reduce evaporative water loss and conserve water. Adaptive heterothermy in large ungulates should be particularly pronounced in hot environments with severely limited access to free water. In the current study we investigated the effects of environmental temperature (ambient, Ta and soil, Ts) and water stress on the Tb of wild, free-ranging Arabian oryx (Oryx leucoryx) in two different sites in Saudi Arabia, Mahazat as-Sayd (MS) and Uruq Bani Ma'arid (UBM). Using implanted data loggers wet took continuous Tb readings every 10 minutes for an entire calendar year and determined the Tb amplitude as well as the heterothermy index (HI). Both differed significantly between sites but contrary to our expectations they were greater in MS despite its lower environmental temperatures and higher rainfall. This may be partially attributable to a higher activity in an unfamiliar environment for translocated animals in UBM. As expected Tb amplitude and HI were greatest during summer. Only minor sex differences were apparent that may be attributable to sex-specific investment into reproduction (e.g. male-male competition) during rut. Our results suggest that the degree of heterothermy is not only driven by extrinsic factors (e.g. environmental temperatures and water availability), but may also be affected by intrinsic factors (e.g. sex and/or behaviour).

Palaeoneurological clues to the evolution of defining mammalian soft tissue traits.

A rich fossil record chronicles the distant origins of mammals, but the evolution of defining soft tissue characters of extant mammals, such as mammary glands and hairs is difficult to interpret because soft tissue does not readily fossilize. As many soft tissue features are derived from dermic structures, their evolution is linked to that of the nervous syutem, and palaeoneurology offers opportunities to find bony correlates of these soft tissue features. Here, a CT scan study of 29 fossil skulls shows that non-mammaliaform Prozostrodontia display a retracted, fully ossified, and non-ramified infraorbital canal for the infraorbital nerve, unlike more basal therapsids. The presence of a true infraorbital canal in Prozostrodontia suggests that a motile rhinarium and maxillary vibrissae were present. Also the complete ossification of the parietal fontanelle (resulting in the loss of the parietal foramen) and the development of the cerebellum in Probainognathia may be pleiotropically linked to the appearance of mammary glands and having body hair coverage since these traits are all controlled by the same homeogene, Msx2, in mice. These suggest that defining soft tissue characters of mammals were already present in their forerunners some 240 to 246 mya.

Microbats appear to have adult hippocampal neurogenesis, but post-capture stress causes a rapid decline in the number of neurons expressing doublecortin.

A previous study investigating potential adult hippocampal neurogenesis in microchiropteran bats failed to reveal a strong presence of this neural trait. As microchiropterans have a high field metabolic rate and a small body mass, it is possible that capture/handling stress may lead to a decrease in the detectable presence of adult hippocampal neurogenesis. Here we looked for evidence of adult hippocampal neurogenesis using immunohistochemical techniques for the endogenous marker doublecortin (DCX) in 10 species of microchiropterans euthanized and perfusion fixed at specific time points following capture. Our results reveal that when euthanized and perfused within 15 min of capture, abundant putative adult hippocampal neurogenesis could be detected using DCX immunohistochemistry. Between 15 and 30 min post-capture, the detectable levels of DCX dropped dramatically and after 30 min post-capture, immunohistochemistry for DCX could not reveal any significant evidence of putative adult hippocampal neurogenesis. Thus, as with all other mammals studied to date apart from cetaceans, bats, including both microchiropterans and megachiropterans, appear to exhibit substantial levels of adult hippocampal neurogenesis. The present study underscores the concept that, as with laboratory experiments, studies conducted on wild-caught animals need to be cognizant of the fact that acute stress (capture/handling) may induce major changes in the appearance of specific neural traits.

Questioning the interpretations of behavioral observations of cetaceans: is there really support for a special intellectual status for this mammalian order?

This review evaluates and contextualizes the behavioral studies undertaken on cetaceans in terms of the relationship of these behaviors to special levels of intelligence associated with these marine mammals and the evolution of their relatively and absolutely large brain size. Many believe that the large size of the cetacean brain and reported behaviors indicate the need to create a special status for these animals in terms of their intellect, positing that they are second to humans in terms of general intelligence. Cetacean brains became relatively large approximately 32millionyearsago, at the Archaeocete-Neocete faunal transition, and have since remained stable in relative size. The behaviors reported for modern cetaceans are thought to parallel those of great apes, to the exclusion of other mammals. By creating an autocatalytic model of cetacean brain evolution, the behaviors thought to be indicative of sophisticated cognitive processes can be assessed as to their potential involvement in the evolution of larger brains in cetaceans. By contextualizing these behaviors in a broader comparative framework, and not the limited cetacean - great ape comparisons mostly used, it is evident that the behaviors used to argue for high levels of intelligence in cetaceans are found commonly across mammals and other vertebrates, and are often observed in invertebrates. This contextualization indicates that cetacean intelligence is qualitatively no different to other vertebrates. In addition, the inability of cetaceans to surpass Piaget stage 4/5 on object permanence tests and to solve an "if and only if, then" abstract task indicates the possibility that their levels of general intelligence may be less than that seen in other vertebrates. Sophisticated cognitive abilities appear to play no role in the evolution of large brain size in cetaceans, indicating that alternative theories of large brain size evolution in cetaceans should be considered in more detail.

Adult neurogenesis in eight Megachiropteran species.

The present study evaluated, using immunohistochemical methods, the presence and characteristics of proliferating and newly generated neurons in the brain of eight wild-caught adult Megachiropteran species. For the neurogenic patterns observed, direct homologies are evident in other mammalian species; however, there were several distinctions in the presence or absence of proliferating and immature neurons, and migratory streams that provide important clues regarding the use of the brain in the analysis of Chiropteran phylogenetic affinities. In all eight species studied, numerous Ki-67- and doublecortin (DCX)-immunopositive cells were identified in the subventricular zone (SVZ). These cells migrated to the olfactory bulb through a Primate-like rostral migratory stream (RMS) that is composed of dorsal and ventral substreams which merge before entering the olfactory bulb. Some cells were observed emerging from the RMS coursing caudally and dorsally to the rostral neocortex. In the dentate gyrus of all species, Ki-67- and DCX-expressing cells were observed in the granular cell layer and hilus. Similar to Primates, proliferating cells and immature neurons were identified in the SVZ of the temporal horn of Megachiropterans. These cells migrated to the rostral and caudal piriform cortex through a Primate-like temporal migratory stream. Sparsely distributed Ki-67-immunopositive, but DCX-immunonegative, cells were identified in the tectum, brainstem and cerebellum. The observations from this study add to a number of neural characteristics that phylogenetically align Megachiropterans to Primates.

Adult neurogenesis in a giant otter shrew (Potamogale velox).

Adult neurogenesis in mammals is typically observed in the subgranular zone of the hippocampal dentate gyrus and the subventricular zone. We investigated adult neurogenesis in the brain of a giant otter shrew (Potamogale velox), a semi-aquatic, central African rainforest mammal of the family Tenrecidae that belongs to the superorder Afrotheria. We examined neurogenesis immunohistochemically, using the endogenous marker doublecortin (DCX), which stains neuronal precursor cells and immature neurons. Our results revealed densely packed DCX-positive cells in the entire extent of the subventricular zone from where cells migrated along the rostral migratory stream to the olfactory bulb. In the olfactory bulb, DCX-expressing cells were primarily present in the granular cell layer with radially orientated dendrites and in the glomerular layer representing periglomerular cells. In the hippocampus, DCX-positive cells were identified in the subgranular and granular layers of the dentate gyrus and strongly labelled DCX-positive processes, presumably dendrites and axons of the newly generated granular cells, were observed in the CA3 regions. In addition, DCX immunoreactive cells were present in the olfactory tubercle, the piriform cortex and the endopiriform nucleus. While DCX-positive fibres have been previously observed in the anterior commissure of the hedgehog and mole, we were able to demonstrate the presence of DCX-positive cells presumably migrating across the anterior commissure. Taken together, the giant otter shrew reveals patterns of neurogenesis similar to that seen in other mammals; however, the appearance of possible neuronal precursor cells in the anterior commissure is a novel observation.

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, Ansell's 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.

Cross-sectional area of the elephant corpus callosum: comparison to other eutherian mammals.

The current study reports our findings of the relationship between cross-sectional area of the corpus callosum and brain mass in over 100 eutherian mammal species. We were specifically interested in determining whether the elephant had a corpus callosum the size that would be expected for eutherian mammal with a brain mass of approximately 5000 g, or whether a different morphology had evolved. To answer this question we first analysed data from primates, other eutherian mammals and cetaceans, finding that primates and other eutherian mammals showed a positive allometric relationship between the two variables, such that larger brains had a relatively larger corpus callosum. Interestingly, primates have a slightly larger corpus callosum than other eutherian mammals, but showed a similar allometric scaling to this group. The cetaceans had a both absolutely and relatively small corpus callosum compared to other mammals and showed isometric scaling with brain mass. The six elephants studied herein had the largest absolute corpus callosums recorded to date; however, relative to the mass of their brain, the size of the corpus callosum was what would be expected of a typical eutherian mammal with a brain mass of approximately 5000 g. The data for elephants hinted at sexual dimorphism in size of the corpus callosum, with female elephants having both an absolute and relatively larger callosum than the males. If this observation is supported in future studies, the elephants will be the first non-primate species to show sexual dimorphism in this neural character. The results are discussed in both an evolutionary and functional context.

[Rest and activity states in the Commerson's dolphin (Cephalorhynchus commersonii)].

The unihemispheric slow-wave sleep, the ability to sleep during swimming with one open eye and the absence of paradoxical sleep in the form of it is observed in all terrestrial mammals are unique features of sleep in cetaceans. Visual observations supplement electrophysiological studies and allow obtaining novel data about sleep of cetaceans. In the present study we examined behavior of 3 adult Commerson's dolphins Cephalorhynchus commersonii which were housed in the oceanarium Sea-World (San Diego, USA). The behavior of the dolphins can be subdivided into 5 swimming types: 1) active swimming marked by variable speed and irregular trajectory of movement (on average for 3 dolphins 35.1 +/- 2.7% of the 24-h period) was scored as active wakefulness; 2) circular swimming was divided into slow and fast swimming and occupied, on average, 44.4 +/- 3.8 and 9.7 +/- 0.8% of the 24-h period, respectively; while in circular swimming, dolphins swam from 1 to 6 circles on one respiration pause; 3) quiet chaotic swimming (3.9 +/- 1.2%) that occurred at the bottom and was not accompanied by signs of activity; 4) floating, and 5) slow swimming at the surface (4.1 +/- 0.5 and 2.8 +/- 0.4%), respectively; the latter two swimming types were accompanied by frequent respiration (hyperventilation). We suggest that sleep in Commerson's dolphins occurred predominantly during the circular and quiet swimming. From time to time the dolphins slowed down their speeds and even stopped for several seconds. Such episodes appeared to be the deepest sleep episodes. In all dolphins muscle jerks as well erections in the male were observed. Jerks and erections occurred during the circular and quiet chaotic swimming. Similar to other studied small cetaceans, Commerson's dolphins are in a state of almost uninterrupted swimming during 24 h per day and they sleep during swimming. Some muscle jerks that we observed in the dolphins in this study might have been episodes of paradoxical sleep.

Primate-like retinotectal decussation in an echolocating megabat, Rousettus aegyptiacus.

The current study was designed to reveal the retinotectal pathway in the brain of the echolocating megabat Rousettus aegyptiacus. The retinotectal pathway of other species of megabats shows the primate-like pattern of decussation in the retina; however, it has been reported that the echolocating Rousettus did not share this feature. To test this prior result we injected fluorescent dextran tract tracers into the right (fluororuby) and left (fluoroemerald) superior colliculi of three adult Rousettus. After a 2-week survival period the animals were killed, fixed via transcardial perfusion, and the retinas whole mounted and examined under fluorescent excitation to reveal the pattern of retrograde transport. Red and green labeled retinotectal ganglion cells were found in side-by-side patches on either side of a vertical decussation line in the temporal retina of all six retinas. The Rousettus examined thus exhibited the same pattern of retinal decussation as reported previously for other megabats and primates, but unlike that seen in other mammals. The current result indicates that the prior study appears to have suffered technical problems leading to an incorrect conclusion. The results of our study indicate that, as may be expected, all megabats share the derived retinotectal pathway once thought to be the exclusive domain of primates. The current study provides additional support for the diphyletic origin of the Chiroptera and aligns the megabats phylogenetically as a sister group to primates.

Observations on the giraffe central nervous system related to the corticospinal tract, motor cortex and spinal cord: what difference does a long neck make?

The mammalian corticospinal tract is known to contain axons that travel from the cerebral cortex to various levels of the spinal cord and its main function is thought to be the mediation of voluntary movement. The current study describes neuroanatomy related to the corticospinal tract of the giraffe. This animal presents a specific morphology that may present challenges to this neural pathway in terms of the metabolism required for correct functioning and maintenance of potentially very long axons. The spinal cord of the giraffe can be up to 2.6 m long and forms the conus medullaris at the level of the sacral vertebrae. Primary motor cortex was found in a location typical of that of other ungulates, and the cytoarchitectonic appearance of this cortical area was similar to that previously reported for sheep, despite the potential distance that the axons emanating from the layer 5 gigantopyramidal neurons must travel. A typically mammalian dorsal striatopallidal complex was transected by a strongly coalesced internal capsule passing through to the pons and forming clearly identifiable but somewhat flattened (in a dorsoventral plane) pyramidal tracts. These tracts terminated in a spinal cord that exhibited no unique anatomical features related to its length. Our results, at least at the level of organization investigated herein, show that the corticospinal tract of the giraffe resembled that of a typical ungulate.

The nigrostriatal DA pathway and Parkinson's disease.

The discovery of the nigrostriatal DA system in the rat was made possible by the highly specific and sensitive histochemical fluorescence method of Falck and Hillarp in combinations with electrolytic lesions in the substantia nigra and removal of major parts of the neostriatum. Recent work on DA neuron evolution shows that in the Bottlenose Dolphin the normal DA cell groups of the substantia nigra are very cell sparse, while there is a substantial expansion of the A9 medial and A10 lateral subdivisions forming an impressive "ventral wing" in the posterior substantia nigra. The nigrostriatal DA pathway mainly operates via Volume Transmission. Thus, DA diffuses along concentration gradients in the ECF to reach target cells with high affinity DA receptors. A novel feature of the DA receptor subtypes is their physical interaction in the plasma membrane of striatal neurons forming receptor mosaics (RM) with the existence of two types of RM. The "functional decoding unit" for DA is not the single receptor, but rather the RM that may affect not only the integration of signals in the DA neurons but also their trophic conditions. In 1991 A2A receptor antagonists were indicated to represent novel antiparkinsonian drugs based on the existence of A2A/D2 receptor-receptor interactions and here P2X receptor antagonists are postulated to be neuroprotective drugs in treatment of Parkinson's Disease.

Specialization in pyramidal cell structure in the sensory-motor cortex of the vervet monkey (Cercopethicus pygerythrus).

Recent studies have revealed systematic differences in the pyramidal cell structure between functionally related cortical areas of primates. Trends for a parallel in pyramidal cell structure and functional complexity have been reported in visual, somatosensory, motor, cingulate and prefrontal cortex in the macaque monkey cortex. These specializations in structure have been interpreted as being fundamental in determining cellular and systems function, endowing circuits in these different cortical areas with different computational power. In the present study we extend our initial finding of systematic specialization of pyramidal cell structure in sensory-motor cortex in the macaque monkey [Cereb Cortex 12 (2002) 1071] to the vervet monkey. More specifically, we investigated pyramidal cell structure in somatosensory and motor areas 1/2, 5, 7, 4 and 6. Neurones in fixed, flat-mounted, cortical slices were injected intracellularly with Lucifer Yellow and processed for a light-stable 3,3'-diaminobenzidine reaction product. The size of, number of branches in, and spine density of the basal dendritic arbors varied systematically such that there was a trend for increasing complexity in arbor structure with progression through 1/2, 5 and 7. In addition, cells in area 6 were larger, more branched, and more spinous than those in area 4.

Visual thalamocortical projections in the flying fox: parallel pathways to striate and extrastriate areas.

We studied thalamic projections to the visual cortex in flying foxes, animals that share neural features believed to resemble those present in the brains of early primates. Neurones labeled by injections of fluorescent tracers in striate and extrastriate cortices were charted relative to the architectural boundaries of thalamic nuclei. Three main findings are reported: First, there are parallel lateral geniculate nucleus (LGN) projections to striate and extrastriate cortices. Second, the pulvinar complex is expansive, and contains multiple subdivisions. Third, across the visual thalamus, the location of cells labeled after visual cortex injections changes systematically, with caudal visual areas receiving their strongest projections from the most lateral thalamic nuclei, and rostral areas receiving strong projections from medial nuclei. We identified three architectural layers in the LGN, and three subdivisions of the pulvinar complex. The outer LGN layer contained the largest cells, and had strong projections to the areas V1, V2 and V3. Neurones in the intermediate LGN layer were intermediate in size, and projected to V1 and, less densely, to V2. The layer nearest to the origin of the optic radiation contained the smallest cells, and projected not only to V1, V2 and V3, but also, weakly, to the occipitotemporal area (OT, which is similar to primate middle temporal area) and the occipitoparietal area (OP, a "third tier" area located near the dorsal midline). V1, V2 and V3 received strong projections from the lateral and intermediate subdivisions of the pulvinar complex, while OP and OT received their main thalamic input from the intermediate and medial subdivisions of the pulvinar complex. These results suggest parallels with the carnivore visual system, and indicate that the restriction of the projections of the large- and intermediate-sized LGN layers to V1, observed in present-day primates, evolved from a more generalized mammalian condition.

The distribution and morphological characteristics of catecholaminergic cells in the brain of monotremes as revealed by tyrosine hydroxylase immunohistochemistry.

The present study describes the distribution and cellular morphology of catecholaminergic neurons in the CNS of two species of monotreme, the platypus (Ornithorhynchus anatinus) and the short-beaked echidna (Tachyglossus aculeatus). Tyrosine hydroxylase immunohistochemistry was used to visualize these neurons. The standard A1-A17, C1-C3 nomenclature was used for expediency, but the neuroanatomical names of the various nuclei have also been given. Monotremes exhibit catecholaminergic neurons in the diencephalon (A11, A12, A13, A14, A15), midbrain (A8, A9, A10), rostral rhombencephalon (A5, A6, A7), and medulla (A1, A2, C1, C2). The subdivisions of these neurons are in general agreement with those of other mammals, and indeed other amniotes. Apart from minor differences, those being a lack of A4, A3, and C3 groups, the catecholaminergic system of monotremes is very similar to that of other mammals. Catecholaminergic neurons outside these nuclei, such as those reported for other mammals, were not numerous with occasional cells observed in the striatum. It seems unlikely that differences in the sleep phenomenology of monotremes, as compared to other mammals, can be explained by these differences. The similarity of this system across mammalian and amniote species underlines the evolutionary conservatism of the catecholaminergic system.

The distribution and morphological characteristics of serotonergic cells in the brain of monotremes.

The distribution and cellular morphology of serotonergic neurons in the brain of two species of monotremes are described. Three clusters of serotonergic neurons were found: a hypothalamic cluster, a cluster in the rostral brainstem and a cluster in the caudal brainstem. Those in the hypothalamus consisted of two groups, the periventricular hypothalamic organ and the infundibular recess, that were intimately associated with the ependymal wall of the third ventricle. Within the rostral brainstem cluster, three distinct divisions were found: the dorsal raphe nucleus (with four subdivisions), the median raphe nucleus and the cells of the supralemniscal region. The dorsal raphe was within and adjacent to the periaqueductal gray matter, the median raphe was associated with the midline ventral to the dorsal raphe, and the cells of the supralemniscal region were in the tegmentum lateral to the median raphe and ventral to the dorsal raphe. The caudal cluster consisted of three divisions: the raphe obscurus nucleus, the raphe pallidus nucleus and the raphe magnus nucleus. The raphe obscurus nucleus was associated with the dorsal midline at the caudal-most part of the medulla oblongata. The raphe pallidus nucleus was found at the ventral midline of the medulla around the inferior olive. Raphe magnus was associated with the midline of the medulla and was found rostral to both the raphe obscurus and raphe pallidus. The results of our study are compared in an evolutionary context with those reported for other mammals and reptiles.

The distribution and morphological characteristics of cholinergic cells in the brain of monotremes as revealed by ChAT immunohistochemistry.

The present study employs choline acetyltransferase (ChAT) immunohistochemistry to identify the cholinergic neuronal population in the central nervous system of the monotremes. Two of the three extant species of monotreme were studied: the platypus (Ornithorhynchus anatinus) and the short-beaked echidna (Tachyglossus aculeatus). The distribution of cholinergic cells in the brain of these two species was virtually identical. Distinct groups of cholinergic cells were observed in the striatum, basal forebrain, habenula, pontomesencephalon, cranial nerve motor nuclei, and spinal cord. In contrast to other tetrapods studied with this technique, we failed to find evidence for cholinergic cells in the hypothalamus, the parabigeminal nucleus (or nucleus isthmus), or the cerebral cortex. The lack of hypothalamic cholinergic neurons creates a hiatus in the continuous antero-posterior aggregation of cholinergic neurons seen in other tetrapods. This hiatus might be functionally related to the phenomenology of monotreme sleep and to the ontogeny of sleep in mammals, as juvenile placental mammals exhibit a similar combination of sleep elements to that found in adult monotremes.