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Author(s): M. Reibold [1, 2]; P. Paufler (corresponding author) [1]; A. A. Levin [1]; W. Kochmann [1]; N. Pätzke [1]; D. C. Meyer [1] The steel of Damascus blades, which [...]

The Guiana dolphin (Sotalia guianensis) is a common species along Central and South American coastal waters. Although much effort has been made to understand its behavioral ecology and evolution, very little is known about its brain. The use of ultra-high field MRI in anatomical descriptions of cetacean brains is a very promising approach that is still uncommon. In this study, we present for the first time a full anatomical description of the Guiana dolphin's brain based on high-resolution ultra-high-field magnetic resonance imaging, providing an exceptional level of brain anatomical details, and enriching our understanding of the species. Brain structures were labeled and volumetric measurements were delineated for many distinguishable structures, including the gray matter and white matter of the cerebral cortex, amygdala, hippocampus, superior and inferior colliculi, thalamus, corpus callosum, ventricles, brainstem and cerebellum. Additionally, we provide the surface anatomy of the Guiana dolphin brain, including the labeling of main sulci and gyri as well as the calculation of its gyrification index. These neuroanatomical data, absent from the literature to date, will help disentangle the history behind cetacean brain evolution and consequently, mammalian evolution, representing a significant new source for future comparative studies., (© 2024. The Author(s).)

Brain reconstruction, specially of the cerebral cortex, is a challenging task and even more so when it comes to highly gyrified brained animals. Here, we present Stitcher, a novel tool capable of generating such surfaces utilizing MRI data and manual segmentation. Stitcher makes a triangulation between consecutive brain slice segmentations by recursively adding edges that minimize the total length and simultaneously avoid self-intersection. We applied this new method to build the cortical surfaces of two dolphins: Guiana dolphin (Sotalia guianensis), Franciscana dolphin (Pontoporia blainvillei); and one pinniped: Steller sea lion (Eumetopias jubatus). Specifically in the case of P. blainvillei, two reconstructions at two different resolutions were made. Additionally, we also performed reconstructions for sub and non-cortical structures of Guiana dolphin. All our cortical mesh results show remarkable resemblance with the real anatomy of the brains, except P. blainvillei with low-resolution data. Sub and non-cortical meshes were also properly reconstructed and the spatial positioning of structures was preserved with respect to S. guianensis cerebral cortex. In a comparative perspective between methods, Stitcher presents compatible results for volumetric measurements when contrasted with other anatomical standard tools. In this way, Stitcher seems to be a viable pipeline for new neuroanatomical analysis, enhancing visualization and descriptions of non-primates species, and broadening the scope of compared neuroanatomy., Competing Interests: Declarations. Competing Interest: The authors declare no competing interests., (© 2024. The Author(s).)

The brain is a complex organ demonstrated by the occurrence of specific types of functional lipids. Despite some studies focusing on providing the animal brain lipid signature, there are limited studies focusing on the comprehensive and regiospecific characterization of multiple animal brain lipidome. Herein we characterized about 294 lipid molecular species from six different lipid classes in different portions of the brain after fixation from mammals of different habitats, fully-aquatic (n = 6), semi-aquatic (n = 6), and terrestrial (n = 4), using liquid chromatography-mass spectrometry. The untargeted brain lipid profiling revealed a significant difference in total lipid levels between fully-aquatic, semi-aquatic, and terrestrial mammals. The polyunsaturated fatty acids and cholesterol esters are abundant in brain tissue of semi-aquatic followed by fully-aquatic mammals whereas phosphatidylethanolamines are profoundly high in terrestrial species. The regiospecific analysis revealed a predominance of sphingolipids in all the groups but no significant differences were observed between the different portions of the brain such as the cerebellum, cortex, pons, spinal cord, and thalamus. Interestingly the multivariate analysis showed almost the same lipid compositions in the spinal cord and thalamus of terrestrial mammals. Overall, this is the first report to compare the comprehensive brain-lipidome among different mammalian groups inhabiting three distinct habitats. These results indicate that the brain lipid composition is specific to the animal habitat., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Several environmental chemicals are suspected risk factors for autism spectrum disorder (ASD), including valproic acid (VPA) and pesticides acting on nicotinic acetylcholine receptors (nAChRs), if administered during pregnancy. However, their target processes in fetal neuro-development are unknown. We report that the injection of VPA into the fetus impaired imprinting to an artificial object in neonatal chicks, while a predisposed preference for biological motion (BM) remained intact. Blockade of nAChRs acted oppositely, sparing imprinting and impairing BM preference. Beside ketamine and tubocurarine, significant effects of imidacloprid (a neonicotinoid insecticide) appeared at a dose ≤1 ppm. In accord with the behavioral dissociations, VPA enhanced histone acetylation in the primary cell culture of fetal telencephalon, whereas ketamine did not. VPA reduced the brain weight and the ratio of NeuN-positive cells (matured neurons) in the telencephalon of hatchlings, whereas ketamine/tubocurarine did not. Despite the distinct underlying mechanisms, both VPA and nAChR blockade similarly impaired imprinting to biological image composed of point-light animations. Furthermore, both impairments were abolished by postnatal bumetanide treatment, suggesting a common pathology underlying the social attachment malformation. Neurotransmission via nAChR is thus critical for the early social bond formation, which is hindered by ambient neonicotinoids through impaired visual predispositions for animate objects., (© The Author(s) 2022. Published by Oxford University Press.)

To elucidate factors underlying the evolution of large brains in cetaceans, we examined 16 brains from 14 cetartiodactyl species, with immunohistochemical techniques, for evidence of non-shivering thermogenesis. We show that, in comparison to the 11 artiodactyl brains studied (from 11 species), the 5 cetacean brains (from 3 species), exhibit an expanded expression of uncoupling protein 1 (UCP1, UCPs being mitochondrial inner membrane proteins that dissipate the proton gradient to generate heat) in cortical neurons, immunolocalization of UCP4 within a substantial proportion of glia throughout the brain, and an increased density of noradrenergic axonal boutons (noradrenaline functioning to control concentrations of and activate UCPs). Thus, cetacean brains studied possess multiple characteristics indicative of intensified thermogenetic functionality that can be related to their current and historical obligatory aquatic niche. These findings necessitate reassessment of our concepts regarding the reasons for large brain evolution and associated functional capacities in cetaceans.

Employing cyto-, myelo-, and chemoarchitectural staining techniques, we analyzed the structure of the hippocampal formation in the banded mongoose and domestic ferret, species belonging to the two carnivoran superfamilies, which have had independent evolutionary trajectories for the past 55 million years. Our observations indicate that, despite the time since sharing a last common ancestor, these species show extensive similarities. The four major portions of the hippocampal formation (cornu Ammonis, dentate gyrus, subicular complex, and entorhinal cortex) were readily observed, contained the same internal subdivisions, and maintained the topological relationships of these subdivisions that could be considered typically mammalian. In addition, adult hippocampal neurogenesis was observed in both species, occurring at a rate similar to that observed in other mammals. Despite the overall similarities, several differences to each other, and to other mammalian species, were observed. We could not find evidence for the presence of the CA2 and CA4 fields of the cornu Ammonis region. In the banded mongoose the dentate gyrus appears to be comprised of up to seven lamina, through the sublamination of the molecular and granule cell layers, which is not observed in the domestic ferret. In addition, numerous subtle variations in chemoarchitecture between the two species were observed. These differences may contribute to an overall variation in the functionality of the hippocampal formation between the species, and in comparison to other mammalian species. These similarities and variations are important to understanding to what extent phylogenetic affinities and constraints affect potential adaptive evolutionary plasticity of the hippocampal formation., (© 2020 Wiley Periodicals LLC.)

The current study provides an analysis of the cytoarchitecture, myeloarchitecture, and chemoarchitecture of the amygdaloid body of the banded mongoose (Mungos mungo) and domestic ferret (Mustela putorius furo). Using architectural and immunohistochemical stains, we observe that the organization of the nuclear and cortical portions of the amygdaloid complex is very similar in both species. The one major difference is the presence of a cortex-amygdala transition zone observed in the domestic ferret that is absent in the banded mongoose. In addition, the chemoarchitecture is, for the most part, quite similar in the two species, but several variances, such as differing densities of neurons expressing the calcium-binding proteins in specific nuclei are noted. Despite this, certain aspects of the chemoarchitecture, such as the cholinergic innervation of the magnocellular division of the basal nuclear cluster and the presence of doublecortin expressing neurons in the shell division of the accessory basal nuclear cluster, appear to be consistent features of the Eutherian mammal amygdala. The domestic ferret presented with an overall lower myelin density throughout the amygdaloid body than the banded mongoose, a feature that may reflect artificial selection in the process of domestication for increased juvenile-like behavior in the adult domestic ferret, such as a muted fear response. The shared, but temporally distant, ancestry of the banded mongoose and domestic ferret allows us to generate observations relevant to understanding the relative influence that phylogenetic constraints, adaptive evolutionary plasticity, and the domestication process may play in the organization and chemoarchitecture of the amygdaloid body., (© 2020 Wiley Periodicals LLC.)

This study provides an analysis of the cytoarchitecture, myeloarchitecture, and chemoarchitecture of the diencephalon (dorsal thalamus, ventral thalamus, and epithalamus) of the banded mongoose (Mungos mungo) and domestic ferret (Mustela putorius furo). Using architectural and immunohistochemical stains, we observe that the nuclear organization of the diencephalon is very similar in the two species, and similar to that reported in other carnivores, such as the domestic cat and dog. The same complement of putatively homologous nuclei were identified in both species, with only one variance, that being the presence of the perireticular nucleus in the domestic ferret, that was not observed in the banded mongoose. The chemoarchitecture was also mostly consistent between species, although there were a number of minor variations across a range of nuclei in the density of structures expressing the calcium-binding proteins parvalbumin, calbindin, and calretinin. Thus, despite almost 53 million years since these two species of carnivores shared a common ancestor, strong phylogenetic constraints appear to limit the potential for adaptive evolutionary plasticity within the carnivore order. Apart from the presence of the perireticular nucleus, the most notable difference between the species studied was the physical inversion of the dorsal lateral geniculate nucleus, as well as the lateral posterior and pulvinar nuclei in the domestic ferret compared to the banded mongoose and other carnivores, although this inversion appears to be a feature of the Mustelidae family. While no functional sequelae are suggested, this inversion is likely to result from the altricial birth of Mustelidae species., (© 2020 Wiley Periodicals LLC.)

We quantified both proliferative (Ki-67 immunohistochemistry) and immature (doublecortin immunohistochemistry) cells within the dentate gyrus of adult Egyptian fruit bats from three distinct environments: (a) primary rainforest, (b) subtropical woodland, and (c) fifth-generation captive-bred. We used four different previously reported methods to assess the effect of the environment on proliferative and immature cells: (a) the comparison of raw totals of proliferative and immature cells; (b) these totals standardized to brain mass; (c) these totals expressed as a density using the volume of the granular cell layer (GCLv) for standardization; and (d) these totals expressed as a percentage of the total number of granule cells. For all methods, the numbers of proliferative cells did not differ statistically among the three groups, indicating that the rate of proliferation, while malleable to experimental manipulation or transiently in response to events of importance in the natural habitat, appears to occur, for the most part, at a predetermined rate within a species. For the immature cells, raw numbers and standardizations to brain mass and GCLv revealed no difference between the three groups studied; however, standardization to total granule cell numbers indicated that the two groups of wild-caught bats had significantly higher numbers of immature neurons than the captive-bred bats. These contrasting results indicate that the interpretation of the effect of the environment on the numbers of immature neurons appears method dependent. It is possible that current methods are not sensitive enough to reveal the effect of different environments on proliferative and immature cells., (© 2020 Wiley Periodicals, Inc.)

In the current study, we use tyrosine hydroxylase (TH) immunohistochemistry to detail the nuclear parcellation and cellular morphology of neurons belonging to the catecholaminergic system in the brain of the Nile crocodile. In general, our results are similar to that found in another crocodilian (the spectacled caiman) and indeed other vertebrates, but certain differences of both evolutionary and functional significance were noted. TH immunopositive (TH+) neurons forming distinct nuclei were observed in the olfactory bulb (A16), hypothalamus (A11, A13-15), midbrain (A8-A10), pons (A5-A7) and medulla oblongata (area postrema, C1, C2, A1, A2), encompassing the more commonly observed nuclear complexes of this system across vertebrates. In addition, TH + neurons forming distinct nuclei not commonly identified in vertebrates were observed in the anterior olfactory nucleus, the pretectal nuclear complex, adjacent to the posterior commissure, and within nucleus laminaris, nucleus magnocellularis lateralis and the lateral vestibular nucleus. Palely stained TH + neurons were observed in some of the serotonergic nuclei, including the medial and lateral divisions of the superior raphe nucleus and the inferior raphe and inferior reticular nucleus, but not in other serotonergic nuclei. In birds, a high density of TH + fibres and pericellular baskets in the dorsal ventricular ridge marks the location of the nidopallium caudolaterale (NCL), a putative avian analogue of mammalian prefrontal cortex. In the dorsal ventricular ridge (DVR) of the crocodile a small region in the caudolateral anterior DVR (ADVRcl) revealed a slightly higher density of TH + fibres and some pericellular baskets (formed by only few TH + fibres). These results are discussed in an evolutionary and functional framework., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Relatively little neuroscience research has been focused on artiodactyls. Recent observations of complex social interactions in domestic and wild species suggest that analyses of artiodactyl brain anatomy would be of comparative value. In this study, we examined how the distribution of cortical neuropil space (a proxy for connectivity) varies across representative members of this diverse clade. Using image analysis techniques, we quantified the neuropil space in the anterior cingulate cortex (ACC) and the occipital (putative primary visual) cortex (OC) of 12 artiodactyl species from adult specimens. Additionally, we conducted a preliminary investigation of variation in ACC neuropil space in a developmental series of five white-tailed deer (Odocoileus virginianus). Results indicate a consistent pattern of greater neuropil space in the ACC in comparison to the OC among all species, and a gradual increase in ACC neuropil space toward maturity in the white-tailed deer. Given the taxa that have the greatest cortical neuropil space, we hypothesize that such enhanced connectivity might be needed to support behaviors such as group foraging and attentiveness to conspecifics. These results help advance a broader understanding of diversity in neural circuitry in artiodactyls and point to the need for more in-depth comparisons of cortical neuron morphology and organization in this relatively understudied taxonomic group. Anat Rec, 301:1871-1881, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

The brain of the crocodile is known to gain in mass allometrically throughout life, and the addition of neurons (as well as non-neurons) appears to play a significant role in this increasing brain mass. We used immunohistochemistry in the brains of 12 Nile crocodiles ranging between 350 g and 86 kg in body mass and 1.99 g to 7.9 g in brain mass to identify the regions of the brain in which neurons immunopositive for doublecortin (DCX), a marker for potential adult neurogenesis, are found. Similar to other reptiles, potential newly born neurons, those immunopositive for DCX, were found throughout the telencephalon, the main and accessory olfactory bulbs and the olfactory tract, and in the cerebellar cortex; however, no DCX immunopositive neurons were observed in the diencephalon or brainstem. An apparent moderate decrease in the density of DCX labeled neurons in the olfactory bulbs and tract as well as the cerebellar cortex was observed with increasing brain mass, but the observed qualitative density of labeled neurons within the telencephalon was maintained irrespective of brain mass. Three potential neurogenic zones, within the sulci of the lateral ventricle, were identified, and these are similar to those seen in other reptiles. This study indicates that at least part of the gain in brain mass with age in the Nile crocodile may be accounted for by the potential addition and integration of new neurons into the existing circuitry, especially so for the olfactory system, telencephalon and cerebellar cortex. Anat Rec, 301:659-672, 2018. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

We examined the effect of chronic prenatal alcohol exposure (PAE) on the process of adult neurogenesis in C57BL/6J mice at early adulthood (PND 56). Pregnant mice, and their in utero litters, were exposed to alcohol, through oral gavage, on gestational days 7-16, with recorded blood alcohol concentrations averaging 184 mg/dL (CA group). Two control groups, sucrose (CAc) and non-treated (NTc) control groups were also examined. The brains of pups at PND 56 from each experimental group were sectioned in a sagittal plane, and stained for Nissl substance with cresyl violet, and immunostained for Ki-67 which labels proliferative cells and doublecortin (DCX) for immature neurons. Morphologically, the neurogenic pattern was identical in all three groups studied. Populations of Ki-67 immunopositive cells in the dentate gyrus were not statistically significantly different between the experimental groups and there were no differences between the sexes. Thus, the PAE in this study does not appear to have a strong effect on the proliferative process in the adult hippocampus. In contrast, the numbers of immature neurons, labeled with DCX, was statistically significantly lower in the prenatal alcohol exposed mice compared with the two control groups. Alcohol significantly lowered the number of DCX hippocampal cells in the male mice, but not in the female mice. This indicates that the PAE appears to lower the rate of conversion of proliferative cells to immature neurons and this effect of alcohol is sexually dimorphic. This lowered number of immature neurons in the hippocampus appears to mirror hippocampal dysfunctions observed in FASD children.

This study investigated the pattern of adult neurogenesis throughout the brains of three prosimian primate species using immunohistochemical techniques for endogenous markers of this neural process. Two species, Galago demidoff and Perodicticus potto, were obtained from wild populations in the primary rainforest of central Africa, while one species, Lemur catta, was captive-bred. Two brains from each species, perfusion-fixed with 4% paraformaldehyde, were sectioned (50 µm section thickness) in sagittal and coronal planes. Using Ki-67 and doublecortin (DCX) antibodies, proliferating cells and immature neurons were identified in the two canonical neurogenic sites of mammals, the subventricular zone of the lateral ventricle (SVZ) giving rise to the rostral migratory stream (RMS), and the subgranular zone of the dentate gyrus of the hippocampus. In addition a temporal migratory stream (TMS), emerging from the temporal horn of the lateral ventricle to supply the piriform cortex and adjacent brain regions with new neurons, was also evident in the three prosimian species. While no Ki-67-immunoreactive cells were observed in the cerebellum, DCX-immunopositive cells were observed in the cerebellar cortex of all three species. These findings are discussed in a phylogenetic context., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

The nuclear organization of the cholinergic, catecholaminergic, serotonergic and orexinergic neurons in the brains of two species of carnivore, the banded mongoose (Mungos mungo) and domestic ferret (Mustela putorius furo), is presented. The banded mongoose belongs to the feliform suborder and the domestic ferret to the caniform suborder, having last shared a common ancestor approximately 53 million years ago; however, they have a very similar overall morphology and life history, presenting an interesting opportunity to examine the extent of evolutionary plasticity in these systems. The brains of the two carnivore species were coronally sectioned and immunohistochemically stained with antibodies against choline acetyltransferase, tyrosine hydroxylase, serotonin and orexin-A. The overall organization and complement of the nuclei of these systems was identical between the two species, although minor differences were noted. Moreover, this overall organization is identical to other studies undertaken in the domestic cat and dog. While for the most part the nuclei forming these systems are similar to those observed in other mammals, two species differences, which appear to be carnivore-specific, were noted. First, cholinergic neurons were observed in the lateral septal nucleus of both species, an apparently carnivore specific feature not recorded previously in other mammals. Second, the serotonergic neurons of the peripheral division of the dorsal raphe complex exhibited a significant caudad expansion, intermingling with the cholinergic and catecholaminergic nuclei of the pons, a carnivore specific feature. These carnivore specific features likely have functional consequences related to coping with stress and the expression of sleep., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This study uses Ki-67 and doublecortin (DCX) immunohistochemistry to delineate potential neurogenic zones, migratory pathways, and terminal fields associated with adult neurogenesis in the brains of three microchiropterans. As with most mammals studied to date, the canonical subgranular and subventricular neurogenic zones were observed. Distinct labeling of newly born cells and immature neurons within the dentate gyrus of the hippocampus was observed in all species. A distinct rostral migratory stream (RMS) that appears to split around the medial aspect of the caudate nucleus was observed. These two rostral stream divisions appear to merge at the rostroventral corner of the caudate nucleus to turn and enter the olfactory bulb, where a large terminal field of immature neurons was observed. DCX immunolabeled neurons were observed mostly in the rostral neocortex, but a potential migratory stream to the neocortex was not identified. A broad swathe of newly born cells and immature neurons was found between the caudoventral division of the RMS and the piriform cortex. In addition, occasional immature neurons were observed in the amygdala and DCX-immunopositive axons were observed in the anterior commissure. While the majority of these features have been found in several mammal species, the large number of DCX immunolabeled cells found between the RMS and the piriform cortex and the presence of DCX immunostained axons in the anterior commissure are features only observed in microchiropterans and insectivores to date. In the diphyletic scenario of chiropteran evolution, these observations align the microchiropterans with the insectivores. Anat Rec, 299:1548-1560, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The present study provides the first systematic immunohistochemical neuroanatomical investigation of the systems involved in the control and regulation of sleep in an odontocete cetacean, the harbor porpoise (Phocoena phocoena). The odontocete cetaceans show an unusual form of mammalian sleep, with unihemispheric slow waves, suppressed REM sleep, and continuous bodily movement. All the neural elements involved in sleep regulation and control found in bihemispheric sleeping mammals were present in the harbor porpoise, with no specific nuclei being absent, and no novel nuclei being present. This qualitative similarity of nuclear organization relates to the cholinergic, noradrenergic, serotonergic, and orexinergic systems and is extended to the γ-aminobutyric acid (GABA)ergic elements involved with these nuclei. Quantitative analysis of the cholinergic and noradrenergic nuclei of the pontine region revealed that in comparison with other mammals, the numbers of pontine cholinergic (126,776) and noradrenergic (122,878) neurons are markedly higher than in other large-brained bihemispheric sleeping mammals. The diminutive telencephalic commissures (anterior commissure, corpus callosum, and hippocampal commissure) along with an enlarged posterior commissure and supernumerary pontine cholinergic and noradrenergic neurons indicate that the control of unihemispheric slow-wave sleep is likely to be a function of interpontine competition, facilitated through the posterior commissure, in response to unilateral telencephalic input related to the drive for sleep. In addition, an expanded peripheral division of the dorsal raphe nuclear complex appears likely to play a role in the suppression of REM sleep in odontocete cetaceans. Thus, the current study provides several clues to the understanding of the neural control of the unusual sleep phenomenology present in odontocete cetaceans. J. Comp. Neurol. 524:1999-2017, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The current study analyzed the nuclear organization of the neural systems related to the control and regulation of sleep and wake in the basal forebrain, diencephalon, midbrain, and pons of the minke whale, a mysticete cetacean. While odontocete cetaceans sleep in an unusual manner, with unihemispheric slow wave sleep (USWS) and suppressed REM sleep, it is unclear whether the mysticete whales show a similar sleep pattern. Previously, we detailed a range of features in the odontocete brain that appear to be related to odontocete-type sleep, and here present our analysis of these features in the minke whale brain. All neural elements involved in sleep regulation and control found in bihemispheric sleeping mammals and the harbor porpoise were present in the minke whale, with no specific nuclei being absent, and no novel nuclei being present. This qualitative similarity relates to the cholinergic, noradrenergic, serotonergic and orexinergic systems, and the GABAergic elements of these nuclei. Quantitative analysis revealed that the numbers of pontine cholinergic (274,242) and noradrenergic (203,686) neurons, and hypothalamic orexinergic neurons (277,604), are markedly higher than other large-brained bihemispheric sleeping mammals. Small telencephalic commissures (anterior, corpus callosum, and hippocampal), an enlarged posterior commissure, supernumerary pontine cholinergic and noradrenergic cells, and an enlarged peripheral division of the dorsal raphe nuclear complex of the minke whale, all indicate that the suite of neural characteristics thought to be involved in the control of USWS and the suppression of REM in the odontocete cetaceans are present in the minke whale. J. Comp. Neurol. 524:2018-2035, 2016. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)

This study provides the first systematic analysis of the nuclear organization of the neural systems related to sleep and wake in the basal forebrain, diencephalon, midbrain, and pons of the river hippopotamus, one of the closest extant terrestrial relatives of the cetaceans. All nuclei involved in sleep regulation and control found in other mammals, including cetaceans, were present in the river hippopotamus, with no specific nuclei being absent, but novel features of the cholinergic system, including novel nuclei, were present. This qualitative similarity relates to the cholinergic, noradrenergic, serotonergic, and orexinergic systems and is extended to the γ-aminobutyric acid (GABA)ergic elements of these nuclei. Quantitative analysis reveals that the numbers of pontine cholinergic (259,578) and noradrenergic (127,752) neurons, and hypothalamic orexinergic neurons (68,398) are markedly higher than in other large-brained mammals. These features, along with novel cholinergic nuclei in the intralaminar nuclei of the dorsal thalamus and the ventral tegmental area of the midbrain, as well as a major expansion of the hypothalamic cholinergic nuclei and a large laterodorsal tegmental nucleus of the pons that has both parvocellular and magnocellular cholinergic neurons, indicates an unusual sleep phenomenology for the hippopotamus. Our observations indicate that the hippopotamus is likely to be a bihemispheric sleeper that expresses REM sleep. The novel features of the cholinergic system suggest the presence of an undescribed sleep state in the hippopotamus, as well as the possibility that this animal could, more rapidly than other mammals, switch cortical electroencephalographic activity from one state to another. J. Comp. Neurol. 524:2036-2058, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The organization of the cholinergic, catecholaminergic, and serotonergic neurons in the brains of five species of insectivores and the orexinergic (hypocretinergic) system in four insectivore species is presented. We aimed to investigate the nuclear complement of these neural systems in comparison to those of other mammalian species. Brains of insectivores were coronally sectioned and immunohistochemically stained with antibodies against choline acetyltransferase, tyrosine hydroxylase, serotonin and orexin-A. The majority of nuclei were similar among the species investigated and to mammals in general, but certain differences in the nuclear complement highlighted potential phylogenetic interrelationships. In the cholinergic system, the three shrew species lacked parabigeminal and Edinger-Westphal nuclei. In addition, the appearance of the laterodorsal tegmental nucleus in all insectivores revealed a mediodorsal arch. All three of these features are the same as those present in microchiropterans. The catecholaminergic system of the three shrew species lacked the A4 and A15d nuclei, as well as having an incipient A9v nucleus, again features found in microchiropteran brains. The serotonergic and orexinergic systems of the insectivores are similar to those seen across most eutherian mammals. The analysis of similarities and differences across mammalian species indicates a potential phylogenetic relationship between the Soricidae (shrews) and the microchiropterans., (Copyright © 2015 Elsevier B.V. All rights reserved.)

It is generally believed that animals with larger bodies require larger brains, composed of more neurons. Across mammalian species, there is a correlation between body mass and the number of brain neurons, albeit with low allometric exponents. If larger bodies imperatively require more neurons to operate them, then such an increase in the number of neurons should be detected across individuals of a continuously growing species, such as the Nile crocodile. In the current study we use the isotropic fractionator method of cell counting to determine how the number of neurons and non-neurons in 6 specific brain regions and the spinal cord change with increasing body mass in the Nile crocodile. The central nervous system (CNS) structures examined all increase in mass as a function of body mass, with allometric exponents of around 0.2, except for the spinal cord, which increases with an exponent of 0.6. We find that numbers of non-neurons increase slowly, but significantly, in all CNS structures, scaling as a function of body mass with exponents ranging between 0.1 and 0.3. In contrast, numbers of neurons scale with body mass in the spinal cord, olfactory bulb, cerebellum and telencephalon, with exponents of between 0.08 and 0.20, but not in the brainstem and diencephalon, the brain structures that receive inputs and send outputs to the growing body. Densities of both neurons and non-neurons decrease with increasing body mass. These results indicate that increasing body mass with growth in the Nile crocodile is associated with a general addition of non-neurons and increasing cell size throughout CNS structures, but is only associated with an addition of neurons in some structures (and at very small rates) and not in those brain structures directly connected to the body. Larger bodies thus do not imperatively require more neurons to operate them., (© 2016 S. Karger AG, Basel.)

The nuclear organization of the cholinergic, catecholaminergic, serotonergic and orexinergic systems in the brains of three species of strepsirrhine primates is presented. We aimed to investigate the nuclear complement of these neural systems in comparison to those of simian primates, megachiropterans and other mammalian species. The brains were coronally sectioned and immunohistochemically stained with antibodies against choline acetyltransferase, tyrosine hydroxylase, serotonin and orexin-A. The nuclei identified were identical among the strepsirrhine species investigated and identical to previous reports in simian primates. Moreover, a general similarity to other mammals was found, but specific differences in the nuclear complement highlighted potential phylogenetic interrelationships. The central feature of interest was the structure of the locus coeruleus complex in the primates, where a central compactly packed core (A6c) of tyrosine hydroxylase immunopositive neurons was surrounded by a shell of less densely packed (A6d) tyrosine hydroxylase immunopositive neurons. This combination of compact and diffuse divisions of the locus coeruleus complex is only found in primates and megachiropterans of all the mammalian species studied to date. This neural character, along with variances in a range of other neural characters, supports the phylogenetic grouping of primates with megachiropterans as a sister group., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The hippocampus is essential for the formation and retrieval of memories and is a crucial neural structure sub-serving complex cognition. Adult hippocampal neurogenesis, the birth, migration and integration of new neurons, is thought to contribute to hippocampal circuit plasticity to augment function. We evaluated hippocampal volume in relation to brain volume in 375 mammal species and examined 71 mammal species for the presence of adult hippocampal neurogenesis using immunohistochemistry for doublecortin, an endogenous marker of immature neurons that can be used as a proxy marker for the presence of adult neurogenesis. We identified that the hippocampus in cetaceans (whales, dolphins and porpoises) is both absolutely and relatively small for their overall brain size, and found that the mammalian hippocampus scaled as an exponential function in relation to brain volume. In contrast, the amygdala was found to scale as a linear function of brain volume, but again, the relative size of the amygdala in cetaceans was small. The cetacean hippocampus lacks staining for doublecortin in the dentate gyrus and thus shows no clear signs of adult hippocampal neurogenesis. This lack of evidence of adult hippocampal neurogenesis, along with the small hippocampus, questions current assumptions regarding cognitive abilities associated with hippocampal function in the cetaceans. These anatomical features of the cetacean hippocampus may be related to the lack of postnatal sleep, causing a postnatal cessation of hippocampal neurogenesis.

This study investigated the nuclear organization of four immunohistochemically identifiable neural systems (cholinergic, catecholaminergic, serotonergic and orexinergic) within the brains of three male Tasmanian devils (Sarcophilus harrisii), which had a mean brain mass of 11.6g. We found that the nuclei generally observed for these systems in other mammalian brains were present in the brain of the Tasmanian devil. Despite this, specific differences in the nuclear organization of the cholinergic, catecholaminergic and serotonergic systems appear to carry a phylogenetic signal. In the cholinergic system, only the dorsal hypothalamic cholinergic nucleus could be observed, while an extra dorsal subdivision of the laterodorsal tegmental nucleus and cholinergic neurons within the gelatinous layer of the caudal spinal trigeminal nucleus were observed. Within the catecholaminergic system the A4 nucleus of the locus coeruleus complex was absent, as was the caudal ventrolateral serotonergic group of the serotonergic system. The organization of the orexinergic system was similar to that seen in many mammals previously studied. Overall, while showing strong similarities to the organization of these systems in other mammals, the specific differences observed in the Tasmanian devil reveal either order specific, or class specific, features of these systems. Further studies will reveal the extent of change in the nuclear organization of these systems in marsupials and how these potential changes may affect functionality., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Elephants are thought to possess excellent long-term spatial-temporal and social memory, both memory types being at least in part hippocampus dependent. Although the hippocampus has been extensively studied in common laboratory mammalian species and humans, much less is known about comparative hippocampal neuroanatomy, and specifically that of the elephant. Moreover, the data available regarding hippocampal size of the elephant are inconsistent. The aim of the current study was to re-examine hippocampal size and provide a detailed neuroanatomical description of the hippocampus in the African elephant. In order to examine the hippocampal size the perfusion-fixed brains of three wild-caught adult male African elephants, aged 20-30 years, underwent MRI scanning. For the neuroanatomical description brain sections containing the hippocampus were stained for Nissl, myelin, calbindin, calretinin, parvalbumin and doublecortin. This study demonstrates that the elephant hippocampus is not unduly enlarged, nor specifically unusual in its internal morphology. The elephant hippocampus has a volume of 10.84 ± 0.33 cm³ and is slightly larger than the human hippocampus (10.23 cm(3)). Histological analysis revealed the typical trilaminated architecture of the dentate gyrus (DG) and the cornu ammonis (CA), although the molecular layer of the dentate gyrus appears to have supernumerary sublaminae compared to other mammals. The three main architectonic fields of the cornu ammonis (CA1, CA2, and CA3) could be clearly distinguished. Doublecortin immunostaining revealed the presence of adult neurogenesis in the elephant hippocampus. Thus, the elephant exhibits, for the most part, what might be considered a typically mammalian hippocampus in terms of both size and architecture.

Cerebral asymmetries result from hemispheric specialization and interhemispheric communication pattern that develop in close gene-environment interactions. To gain a deeper understanding of developmental and functional interrelations, we investigated interhemispheric information exchange in pigeons, which possess a lateralized visual system that develops in response to asymmetrical ontogenetic light stimulation. We monocularly trained pigeons with or without embryonic light experience in color discriminations whereby they learned another pair of colors with each eye. Thereby, information from the ipsilateral eye had to be transferred. Monocular tests confronting the animals with trained and transferred color pairs demonstrated that embryonic light stimulation modulates the balance of asymmetrical handling of transfer information. Stronger embryonic stimulation of the left hemisphere significantly enhanced access to interhemispheric visual information, thereby reversing the right-hemispheric advantage that develops in the absence of embryonic light experience. These data support the critical role of environmental factors in molding a functionally lateralized brain.

The claustrum, a subcortical telencephalic structure, is known to be reciprocally interconnected to almost all cortical regions; however, a systematic analysis of claustrocortical connectivity with physiologically identified lower and higher order visual cortical areas has not been undertaken. In the current study we used biotinylated dextran amine to trace the connections of the ferret claustrum with lower (occipital areas 17, 18, 19 and 21) and higher (parietal and temporal areas posterior parietal caudal visual area (PPc), posterior parietal rostral visual area (PPr), 20a, 20b, anterior ectosylvian visual area (AEV)) order visual cortical areas. No connections between the claustrum and area 17 were observed. Occipital visual areas 18, 19 and 21 revealed a reciprocal connectivity mainly to the caudal part of the claustrum. After injection into parietal areas PPc and PPr labeled neurons and terminals were found throughout almost the entire rostrocaudal extent of the dorsal claustrum. Area 20b revealed reciprocal connections mainly to the caudal-ventral claustrum, although some labeled neurons and terminals were observed in the dorso-central claustrum. No projection from the claustrum to areas AEV and 20a could be observed, though projections from AEV and 20a to the claustrum were found. Only injections placed in areas PPr and AEV resulted in anterogradely labeled terminals in the contralateral claustrum. Our results suggest that lower order visual areas have clearly defined connectivity zones located in the caudal claustrum, whereas higher order visual areas, even if not sending and/or receiving projections from the entire claustrum, show a more widespread connectivity.

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., (© 2014 S. Karger AG, Basel.)

It is a central assumption that larger bodies require larger brains, across species but also possibly within species with continuous growth throughout the lifetime, such as the crocodile. The current study investigates the relationships between body growth (length and mass) and the rates of growth of various subdivisions of the central nervous system (CNS) (brain, spinal cord, eyes) in Nile crocodiles weighing between 90 g and 90 kg. Although the brain appears to grow in two phases in relation to body mass, initially very rapidly then very slowly, it turns out that brain mass increases continuously as a power function of body mass with a small exponent of 0.256, such that a 10-fold increase in body mass is accompanied by a 1.8-fold in brain mass. Eye volume increases slowly with increasing body mass, as a power function of the latter with an exponent of 0.37. The spinal cord, however, grows more rapidly in mass, accompanying body mass raised to an exponent of 0.54, and increasing in length as predicted, with body mass raised to an exponent of 0.32 (close to the predicted 1/3). While supporting the expectation formulated by Jerison that larger bodies require larger brains to operate them, our findings show that: (1) the rate of increase in brain size is very small compared to body growth; and (2) different parts of the CNS grow at different rates accompanying continuous body growth, with a faster increase in spinal cord mass and eye volume, than in brain mass., (Copyright © 2013 Wiley Periodicals, Inc.)

The present study describes the organisation of the cholinergic, catecholaminergic, and serotonergic neurons in the brains of the giant otter shrew, the Hottentot golden mole and the four-toed sengi, and the orexinergic (hypocretinergic) system in the giant otter shrew and four-toed sengi. The aim of the present study was to investigate the possible differences in the nuclear complement of these neural systems in comparison to previous studies on other Afrotherian species and mammalian species in general. Brains of the golden mole, sengi and giant otter shrew were coronally sectioned and immunohistochemically stained with antibodies against cholineacetyl-transferase, tyrosine hydroxylase, serotonin and orexin-A. The majority of nuclei revealed in the current study were similar among the species investigated, to other Afrotherian species, and to mammals generally, but certain differences in the nuclear complement highlighted phylogenetic interrelationships. The golden mole was observed to have cholinergic interneurons in the cerebral cortex, hippocampus, olfactory bulb and amygdala. The four-toed sengi had cholinergic neurons in both colliculi and in the cochlear nucleus, but lacked the catecholaminergic A15d group in the hypothalamus. In both the golden mole and the four-toed sengi, the locus coeruleus (A6d group) was made up of few neurons. The golden mole also exhibited an unusual foreshortening of the brain, such that a major (mesencephalic?) flexure in the brainstem was evident., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The current study examined the organization of the diencephalon and brainstem of the African elephant (Loxodonta africana) - a region of the elephant brain that has not been examined for at least 50 years. The current description, employing material amenable for use with modern neuroanatomical methods, shows that, for the most part, the elephant diencephalon and brainstem are what could be considered typically mammalian, with subtle differences in proportions and topology. The variations from these previous descriptions, where they occurred, were related to four specific aspects of neural information processing: (1) the motor systems, (2) the auditory and vocalization systems, (3) the orexinergic satiety/wakefulness centre of the hypothalamus and the locus coeruleus, and (4) the potential neurogenic lining of the brainstem. For the motor systems, three specific structures exhibited interesting differences in organization - the pars compacta of the substantia nigra, the facial motor nerve nucleus, and the inferior olivary nuclear complex, all related to the timing and learning of movements and likely related to the control of the trunk. The dopaminergic neurons of the substantia nigra appear to form distinct islands separated from each other by large fibre pathways, an appearance unique to the elephant. Each island may send topologically organized projections to the striatum forming a dopaminergic innervation mosaic that may relate to trunk movements. At least five regions of the combined vocalization production and auditory/seismic reception system were specialized, including the large and distinct nucleus ellipticus of the periaqueductal grey matter, the enlarged lateral superior olivary nucleus, the novel transverse infrageniculate nucleus of the dorsal thalamus, the enlarged dorsal column nuclei and the ventral posterior inferior nucleus of the dorsal thalamus. These specializations, related to production and reception of infrasound, allow the proposal of a novel concept regarding the reception and localization of infrasonic sources. The orexinergic system of the hypothalamus displayed a medial hypothalamic parvocellular cluster of neurons in addition to the magnocellular clusters typical of mammals located in the lateral hypothalamus, and a novel medial division of the locus coeruleus was observed in the pons. These systems are related to appetitive drive and promotion of wakefulness, two aspects of elephant behaviour that appear to be inextricably linked. Lastly, we observed an extensive potential neurogenic lining of the ventricles throughout the brainstem that is present in even quite old elephants, although the function of these cells remains elusive. These observations combined demonstrate that, while much of the elephant brainstem is typically mammalian, certain aspects of the anatomy related to specialized behaviour of elephants are present and instructive in understanding elephant behaviour.

The current paper details our developing understanding of the evolution of large brains in mammals. In order to do this, we first define brains that we consider to be large--those that have passed the apparent 700-gram ceiling on brain mass evolution in the class Mammalia. The over-700-gram club includes certain species within the genus Homo, order Cetacea, order Proboscidea, and suborder Pinnipedia. Our analysis suggests that selection for body size appears to be the most important factor in the evolution of large brain size, but there also appear to be internal morphophysiological constraints on large brain size evolution that need to be overcome in order for brains to break the 700-gram barrier. These two aspects appear to be common themes in the evolution of large brains. This significantly diminishes the explanatory value of selection for greater cognitive capacities as a principal factor in the evolution of enlarged brain sizes above the 700-gram threshold., (© 2013 S. Karger AG, Basel.)

The present study describes the organization of the orexinergic (hypocretinergic) neurons in the hypothalamus of the giraffe and harbour porpoise--two members of the mammalian Order Cetartiodactyla which is comprised of the even-toed ungulates and the cetaceans as they share a monophyletic ancestry. Diencephalons from two sub-adult male giraffes and two adult male harbour porpoises were coronally sectioned and immunohistochemically stained for orexin-A. The staining revealed that the orexinergic neurons could be readily divided into two distinct neuronal types based on somal volume, area and length, these being the parvocellular and magnocellular orexin-A immunopositive (OxA+) groups. The magnocellular group could be further subdivided, on topological grounds, into three distinct clusters--a main cluster in the perifornical and lateral hypothalamus, a cluster associated with the zona incerta and a cluster associated with the optic tract. The parvocellular neurons were found in the medial hypothalamus, but could not be subdivided, rather they form a topologically amorphous cluster. The parvocellular cluster appears to be unique to the Cetartiodactyla as these neurons have not been described in other mammals to date, while the magnocellular nuclei appear to be homologous to similar nuclei described in other mammals. The overall size of both the parvocellular and magnocellular neurons (based on somal volume, area and length) were larger in the giraffe than the harbour porpoise, but the harbour porpoise had a higher number of both parvocellular and magnocellular orexinergic neurons than the giraffe despite both having a similar brain mass. The higher number of both parvocellular and magnocellular orexinergic neurons in the harbour porpoise may relate to the unusual sleep mechanisms in the cetaceans., (Copyright © 2012 Elsevier B.V. All rights reserved.)

This study investigated the nuclear organization of four immunohistochemically identifiable neural systems (cholinergic, catecholaminergic, serotonergic and orexinergic) within the brain of the African pygmy mouse (Mus minutoides). The African pygmy mice studied had a brain mass of around 275 mg, making these the smallest rodent brains to date in which these neural systems have been investigated. In contrast to the assumption that in this small brain there would be fewer subdivisions of these neural systems, we found that all nuclei generally observed for these systems in other rodent brains were also present in the brain of the African pygmy mouse. As with other rodents previously studied in the subfamily Murinae, we observed the presence of cortical cholinergic neurons and a compactly organized locus coeruleus. These two features of these systems have not been observed in the non-Murinae rodents studied to date. Thus, the African pygmy mouse displays what might be considered a typical Murinae brain organization, and despite its small size, the brain does not appear to be any less complexly organized than other rodent brains, even those that are over 100 times larger such as the Cape porcupine brain. The results are consistent with the notion that changes in brain size do not affect the evolution of nuclear organization of complex neural systems. Thus, species belonging to the same order generally have the same number and complement of the subdivisions, or nuclei, of specific neural systems despite differences in brain size, phenotype or time since evolutionary divergence., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The olfactory system of mammals can be divided into a main and accessory olfactory system with initial processing for each system occurring in the olfactory bulb. The main and accessory olfactory bulbs have similar structural features, even though they appear to be functionally independent. In mammals the main olfactory bulb (MOB) is also one of two established sites of lifelong generation of new cells. The present study describes the histological and immunohistochemical neuroanatomy of the olfactory bulb of the African elephant (Loxodonta africana). The morphology of MOB of the elephant does not differ significantly from that described in other mammals; however, it lacks the internal plexiform layer. In addition, the glomeruli of the glomerular layer are organised in 2-4 "honey-combed" layers, a feature not commonly observed. The cell types and structures revealed with immunohistochemical stains (parvalbumin, calbindin, calretinin, tyrosine hydroxylase, orexin-A, glial fibrillary acidic protein) were similar to other mammals. Neurogenesis was examined using the neurogenic marker doublecortin. Migration of newly generated cells was observed in most layers of the MOB. No accessory olfactory bulb (AOB) was observed. Based on the general anatomy and the immunohistochemical observations, it is evident that the morphology of the African elephant MOB is, for the most part, similar to that of all mammals, although very large in absolute size., (© Springer-Verlag 2011)

A large body of evidence indicates that pigeons use olfactory cues to navigate over unfamiliar areas with a differential contribution of the left and right hemispheres. In particular, the right nostril/olfactory bulb (OB) and left piriform cortex (Cpi) have been demonstrated to be crucially involved in navigation. In this study we analysed behaviour-induced activation of the olfactory system, indicated by the expression of the immediate early gene ZENK, under different homing conditions. One experimental group was released from an unfamiliar site, the second group was transported to the unfamiliar site and back to the loft, and the third group was released in front of the loft. To evaluate the differential contribution of the left and/or right olfactory input, the nostrils of the pigeons were either occluded unilaterally or not. Released pigeons revealed the highest ZENK cell density in the OB and Cpi, indicating that the olfactory system is activated during navigation from an unfamiliar site. The groups with no plug showed the highest ZENK cell density, supporting the activation of the olfactory system probably being due to sensory input. Moreover, both Cpis seem to contribute differently to the navigation process. Only occlusion of the right OB resulted in a decreased ZENK cell expression in the Cpi, whereas occlusion of the left nostril had no effect. This is the first study to reveal neuronal activation patterns in the olfactory system during homing. Our data show that lateralized processing of olfactory cues is indeed involved in navigation over unfamiliar areas.