The African wild dog is endemic to sub-Saharan Africa and belongs to the family Canidae which includes domestic dogs and their closest relatives (i.e., wolves, coyotes, jackals, dingoes, and foxes). The African wild dog is known for its highly social behavior, co-ordinated pack predation, and striking vocal repertoire, but little is known about its brain and whether it differs in any significant way from that of other canids. We employed gross anatomical observation, magnetic resonance imaging, and classical neuroanatomical staining to provide a broad overview of the structure of the African wild dog brain. Our results reveal a mean brain mass of 154.08 g, with an encephalization quotient of 1.73, indicating that the African wild dog has a relatively large brain size. Analysis of the various structures that comprise their brains and their topological inter-relationships, as well as the areas and volumes of the corpus callosum, ventricular system, hippocampus, amygdala, cerebellum and the gyrification index, all reveal that the African wild dog brain is, in general, similar to that of other mammals, and very similar to that of other carnivorans. While at this level of analysis we do not find any striking specializations within the brain of the African wild dog, apart from a relatively large brain size, the observations made indicate that more detailed analyses of specific neural systems, particularly those involved in sensorimotor processing, sociality or cognition, may reveal features that are either unique to this species or shared among the Canidae to the exclusion of other Carnivora.

The large external pinnae and extensive vocal repertoire of the African wild dog (Lycaon pictus) has led to the assumption that the auditory system of this unique canid may be specialized. Here, using cytoarchitecture, myeloarchitecture, and a range of immunohistochemical stains, we describe the systems-level anatomy of the auditory system of the African wild dog. We observed the cochlear nuclear complex, superior olivary nuclear complex, lateral lemniscus, inferior colliculus, medial geniculate body, and auditory cortex all being in their expected locations, and exhibiting the standard subdivisions of this system. While located in the ectosylvian gyri, the auditory cortex includes several areas, resembling the parcellation observed in cats and ferrets, although not all of the auditory areas known from these species could be identified in the African wild dog. These observations suggest that, broadly speaking, the systems-level anatomy of the auditory system, and by extension the processing of auditory information, within the brain of the African wild dog closely resembles that observed in other carnivores. Our findings indicate that it is likely that the extraction of the semantic content of the vocalizations of African wild dogs, and the behaviors generated, occurs beyond the classically defined auditory system, in limbic or association neocortical regions involved in cognitive functions. Thus, to obtain a deeper understanding of how auditory stimuli are processed, and how communication is achieved, in the African wild dog compared to other canids, cortical regions beyond the primary sensory areas will need to be examined in detail.

The current study provides a detailed architectural analysis of the subpallial telencephalon of the tree pangolin. In the tree pangolin, the subpallial telencephalon was divided into septal and striatopallidal regions. The septal region contained the septal nuclear complex, diagonal band of Broca, and the bed nuclei of the stria terminalis. The striatopallidal region comprised of the dorsal (caudate, putamen, internal and external globus pallidus) and ventral (nucleus accumbens, olfactory tubercle, ventral pallidum, nucleus basalis, basal part of the substantia innominata, lateral stripe of the striatum, navicular nucleus, and the major island of Calleja) striatopallidal complexes. In the tree pangolin, the organization and numbers of nuclei forming these regions and complexes, their topographical relationships to each other, and the cyto-, myelo-, and chemoarchitecture, were found to be very similar to that observed in commonly studied mammals. Minor variations, such as less nuclear parcellation in the bed nuclei of the stria terminalis, may represent species-specific variations, or may be the result of the limited range of stains used. Given the overall similarity across mammalian species, it appears that the subpallial telencephalon of the mammalian brain is highly conserved in terms of evolutionary changes detectable with the methods used. It is also likely that the functions associated with these nuclei in other mammals can be translated directly to the tree pangolin, albeit with the understanding that the stimuli that produce activity within these regions may be specific to the life history requirements of the tree pangolin.

A large population of infracortical white matter neurons, or white matter interstitial cells (WMICs), are found within the subcortical white matter of the mammalian telencephalon. We examined WMICs in three species of megachiropterans, Megaloglossus woermanni, Casinycteris argynnis, and Rousettus aegyptiacus, using immunohistochemical and stereological techniques. Immunostaining for neuronal nuclear marker (NeuN) revealed substantial numbers of WMICs in each species-M. woermanni 124,496 WMICs, C. argynnis 138,458 WMICs, and the larger brained R. aegyptiacus having an estimated WMIC population of 360,503. To examine the range of inhibitory neurochemical types we used antibodies against parvalbumin, calbindin, calretinin, and neural nitric oxide synthase (nNOS). The calbindin and nNOS immunostained neurons were the most commonly observed, while those immunoreactive for calretinin and parvalbumin were sparse. The proportion of WMICs exhibiting inhibitory neurochemical profiles was ~26%, similar to that observed in previously studied primates. While for the most part the WMIC population in the megachiropterans studied was similar to that observed in other mammals, the one feature that differed was the high proportion of WMICs immunoreactive to calbindin, whereas in primates (macaque monkey, lar gibbon and human) the highest proportion of inhibitory WMICs contain calretinin. Interestingly, there appears to be an allometric scaling of WMIC numbers with brain mass. Further quantitative comparative work across more mammalian species will reveal the developmental and evolutionary trends associated with this infrequently studied neuronal population.

We examined the number, distribution, and immunoreactivity of the infracortical white matter neuronal population, also termed white matter interstitial cells (WMICs), in the brain of a lesser ape, the lar gibbon. Staining for neuronal nuclear marker (NeuN) revealed WMICs throughout the infracortical white matter, these cells being most numerous and dense close to cortical layer VI, decreasing significantly in density with depth in the white matter. Stereological analysis of NeuN-immunopositive cells revealed a global estimate of ~67.5 million WMICs within the infracortical white matter of the gibbon brain, indicating that the WMICs are a numerically significant population, ~2.5% of the total cortical gray matter neurons that would be estimated for a primate brain the mass of that of the lar gibbon. Immunostaining revealed subpopulations of WMICs containing neuronal nitric oxide synthase (nNOS, ~7 million in number, with both small and large soma volumes), calretinin (~8.6 million in number, all of similar soma volume), very few WMICs containing parvalbumin, and no calbindin-immunopositive neurons. These nNOS, calretinin, and parvalbumin immunopositive WMICs, presumably all inhibitory neurons, represent ~23.1% of the total WMIC population. As the white matter is affected in many cognitive conditions, such as schizophrenia, autism and also in neurodegenerative diseases, understanding these neurons across species is important for the translation of findings of neural dysfunction in animal models to humans. Furthermore, studies of WMICs in species such as apes provide a crucial phylogenetic context for understanding the evolution of these cell types in the human brain.

We examined the number, distribution, and immunoreactivity of the infracortical white matter neuronal population, also termed white matter interstitial cells (WMICs), throughout the telencephalic white matter of an adult female chimpanzee. Staining for neuronal nuclear marker (NeuN) revealed WMICs throughout the infracortical white matter, these cells being most numerous and dense close to the inner border of cortical layer VI, decreasing significantly in density with depth in the white matter. Stereological analysis of NeuN-immunopositive cells revealed an estimate of approximately 137.2 million WMICs within the infracortical white matter of the chimpanzee brain studied. Immunostaining revealed subpopulations of WMICs containing neuronal nitric oxide synthase (nNOS, approximately 14.4 million in number), calretinin (CR, approximately 16.7 million), very few WMICs containing parvalbumin (PV), and no calbindin-immunopositive neurons. The nNOS, CR, and PV immunopositive WMICs, possibly all inhibitory neurons, represent approximately 22.6% of the total WMIC population. As the white matter is affected in many cognitive conditions, such as schizophrenia, autism, epilepsy, and also in neurodegenerative diseases, understanding these neurons across species is important for the translation of findings of neural dysfunction in animal models to humans. Furthermore, studies of WMICs in species such as apes provide a crucial phylogenetic context for understanding the evolution of these cell types in the human brain.

The spinal cord of the tree pangolin is known to be very short compared to the overall length of the body and tail. Here, we provide a description of the tree pangolin spinal cord to determine whether the short length contributes to specific structural, and potentially functional, differences. The short spinal cord of the adult tree pangolin, at around 13 cm, terminates at the midthoracic level. Within this shortened spinal cord, we could identify six regions, which from rostral to caudal include the prebrachial, brachial, interramal, crural, postcrural, and caudal regions, with both the brachial and crural regions showing distinct swellings. The chemoarchitecture of coronal sections through these regions confirmed regional assignation, being most readily delineated by the presence of cholinergic neurons forming the intermediolateral column in the interramal region and the sacral parasympathetic nucleus in the postcrural region. The 10 laminae of Rexed were observed throughout the spinal cord and presented with an anatomical organization similar to that observed in other mammals. Despite the shortened length of the tree pangolin spinal cord, the regional and laminar anatomical organization is very similar to that observed in other mammals. This indicates that the functional aspects of the short tree pangolin spinal cord can be inferred from what is known in other mammals.

The diencephalon (dorsal thalamus, ventral thalamus, and epithalamus) and the hypothalamus, play central roles in the processing of the majority of neural information within the central nervous system. Given the interactions of the diencephalon and hypothalamus with virtually all portions of the central nervous system, the comparative analysis of these regions lend key insights into potential neural, evolutionary, and behavioral specializations in different species. Here, we continue our analysis of the brain of the tree pangolin by providing a comprehensive description of the organization of the diencephalon and hypothalamus using a range of standard and immunohistochemical staining methods. In general, the diencephalon and hypothalamus of the tree pangolin follow the organization typically observed across mammals. No unusual structural configurations of the ventral thalamus, epithalamus, or hypothalamus were noted. Within the dorsal thalamus, the vast majority of typically identified nuclear groups and component nuclei were observed. The visual portion of the tree pangolin dorsal thalamus appears to be organized in a manner not dissimilar to that seen in most nonprimate and noncarnivore mammals, and lacks certain features that are present in the closely related carnivores. Within the ventral medial geniculate nucleus, a modular organization, revealed with parvalbumin neuropil immunostaining, is suggestive of specialized auditory processing in the tree pangolin. In addition, a potential absence of hypothalamic cholinergic neurons is suggestive of unusual patterns of sleep. These observations are discussed in an evolutionary and functional framework regarding the phylogeny and life history of the pangolins.

A cyto-, myelo-, and chemoarchitectonic analysis of the pallial telencephalon of the tree pangolin is provided. As certain portions of the pallial telencephalon have been described previously (olfactory pallium, hippocampal formation, and amygdaloid complex), we focus on the claustrum and endopiriform nuclear complex, the white matter and white matter interstitial cells, and the areal organization of the cerebral cortex. Our analysis indicates that the organization of the pallial telencephalon of the tree pangolin is similar to that observed in many other mammals, and specifically quite similar to the closely related carnivores. The claustrum of the tree pangolin exhibits a combination of insular and laminar architecture, while the endopiriform nuclear complex contains three nuclei, both reminiscent of observations made in other mammals. The population of white matter interstitial cells resembles that observed in other mammals, while a distinct laminated organization of the intracortical white matter was revealed with parvalbumin immunostaining. The cerebral cortex of the tree pangolin presented with indistinct laminar boundaries as well as pyramidalization of the neurons in both layers 2 and 4. All cortical regions typically found in mammals were present, with the cortical areas within these regions often corresponding to what has been reported in carnivores. Given the similarity of the organization of the pallial telencephalon of the tree pangolin to that observed in other mammals, especially carnivores, it would be reasonable to assume that the neural processing afforded the tree pangolin by these structures does not differ dramatically to that of other mammals.

Here, we describe the cytoarchitecture and chemoarchitecture of the amygdaloid body of the tree pangolin. Our definition of the amygdaloid body includes the pallial portions of the amygdala, and the centromedial group that is a derivative of the subpallium and part of the extended amygdala. The remainder of the extended amygdala is not described herein. Within the amygdaloid body of the tree pangolin, we identified the basolateral group (composed of the lateral, basal, and accessory basal amygdaloid nuclei), the superficial, or cortical nuclei (the anterior and posterior cortical nuclei, the periamygdaloid cortex, and nuclei of the olfactory tract), the centromedial group (the central amygdaloid nucleus and the medial nuclear cluster), and other amygdaloid nuclei (the anterior amygdaloid area, the amygdalohippocampal area, the intramedullary group, and intercalated islands). The location within and relative to each other within the amygdaloid body and the internal subdivisions of these groups were very similar to that reported in other mammalian species, with no clearly derived features specific to the tree pangolin. The only variation was the lack of an insular appearance of the intercalated islands, which in the tree pangolin were observed as a continuous band of neurons located dorsomedial to the basolateral group similar in appearance to and almost continuous with the intramedullary group. In carnivores, the closest relatives of the pangolins, and laboratory rats, a similar appearance of portions of the intercalated islands has been noted.

The brainstem (midbrain, pons, and medulla oblongata) and cerebellum (diencephalic prosomere 1 through to rhombomere 11) play central roles in the processing of sensorimotor information, autonomic activity, levels of awareness and the control of functions external to the conscious cognitive world of mammals. As such, comparative analyses of these structures, especially the understanding of specializations or reductions of structures with functions that have been elucidated in commonly studied mammalian species, can provide crucial information for our understanding of the behavior of less commonly studied species, like pangolins. In the broadest sense, the nuclear complexes and subdivisions of nuclear complexes, the topographical arrangement, the neuronal chemistry, and fiber pathways of the tree pangolin conform to that typically observed across more commonly studied mammalian species. Despite this, variations in regions associated with the locus coeruleus complex, auditory system, and motor, neuromodulatory and autonomic systems involved in feeding, were observed in the current study. While we have previously detailed the unusual locus coeruleus complex of the tree pangolin, the superior olivary nuclear complex of the auditory system, while not exhibiting additional nuclei or having an altered organization, this nuclear complex, particularly the lateral superior olivary nucleus and nucleus of the trapezoid body, shows architectonic refinement. The cephalic decussation of the pyramidal tract, an enlarged hypoglossal nucleus, an additional subdivision of the serotonergic raphe obscurus nucleus, and the expansion of the superior salivatory nucleus, all indicate neuronal specializations related to the myrmecophagous diet of the pangolins.

Employing a range of standard and immunohistochemical stains we provide a description of the hippocampal formation in the brain of the tree pangolin. For the most part, the architecture, chemical neuroanatomy, and topological relationships of the component parts of the hippocampal formation of the tree pangolin were consistent with that observed in other mammalian species. Within the hippocampus proper fields CA1, 3, and 4 could be identified with certainty, while CA2 was tentatively identified as a small transitional zone between the CA1 and CA3 fields. Within the dentate gyrus evidence for adult hippocampal neurogenesis at a rate comparable to other mammals was observed. The subicular complex and entorhinal cortex also exhibited divisions typically observed in other mammalian species. In contrast to many other mammals, an architecturally and neurochemically distinct CA4 field was observed, supporting Lorente de Nó's proposed CA4 field, at least in some mammalian species. In addition, up to seven laminae were evident in the dentate gyrus. Calretinin immunostaining revealed the three sublamina of the molecular layer, while immunostaining for vesicular glutamate transporter 2 and neurofilament H indicate that the granule cell layer was composed of two sublamina. The similarities and differences observed in the tree pangolin indicate that the hippocampal formation is an anatomically and neurochemically conserved neural unit in mammalian evolution, but minor changes may relate to specific life history features and habits of species.

This study employed a range of neuroanatomical stains to determine the organization of the main and accessory olfactory systems within the brain of the tree pangolin. The tree pangolin has a typically mammalian olfactory system, but minor variations were observed. The main olfactory system is comprised of the layered main olfactory bulb (MOB), the anterior olfactory nucleus (AON), the rostral olfactory cortex (including the taenia tecta, anterior hippocampal continuation and induseum griseum), the olfactory tubercle (Tu), the lateral olfactory tract (lot) and the olfactory limb of the anterior commissure, the nucleus of the lateral olfactory tract (NLOT), the piriform cortex (PIR) and a typically mammalian rostral migratory stream (RMS). The accessory olfactory system included the layered accessory olfactory bulb (AOB) and the nucleus of the accessory olfactory tract (NAOT). Volumetric analysis of the relative size of the MOB and PIR indicate that the tree pangolin has an olfactory system that occupies a proportion of the brain typical for the majority of mammals. Within the MOB, the glomeruli of the tree pangolin, at 200 μm diameter, are larger than observed in most other mammalian species, and the MOB lacks a distinct internal plexiform layer. In addition, the laminate appearance of the NLOT was not observed in the tree pangolin. The accessory olfactory system appears to lack the posterior compartment of the accessory olfactory bulb. These observations are contextualized in relation to olfactory-mediated behaviors in pangolins.

Here, we used a range of immunohistochemical stains, focussing on tyrosine hydroxylase and dopamine-β-hydroxylase, to show that within the pons of tree pangolins clusters of noradrenergic neurons are present. No noradrenergic neurons were observed in the pontine periventricular gray matter (A6 and A4 groups missing), with all noradrenergic neurons being found within the pontine tegmentum (A7 and A5 groups). The tree pangolin is unique in lacking the locus coeruleus (A6) cell group observed in all vertebrates previously studied; however, noradrenergic axons and terminal networks were found throughout the cerebral cortex. We propose this is achieved through a unique structural reorganization of this system. First, the number of noradrenergic neurons in the compact portion of the subcoeruleus (A7sc) of the tree pangolin is increased, providing a total number of noradrenergic neurons in the pontine tegmentum (A7diffuse, A7sc, A5) that is equivalent to the entire locus coeruleus complex in related species of similar brain mass. Second, the most medially located noradrenergic neurons of the A7sc have dendrites that extend into the ventrolateral periventricular gray matter, in the location where the A6 neurons should have been located, forming a "pseudo A6" region. Third, the topological relationships of this "pseudo A6" region to other neurochemical systems that interact with the A6 neurons, such as the orexinergic, cholinergic, and serotonergic systems, appear to be maintained. Thus, a unique structural plasticity of this region appears to maintain the standard functions of the locus coeruleus complex in this unusual mammalian species.

In the current study, we examined the number, distribution, and aspects of the neurochemical identities of infracortical white matter neurons, also termed white matter interstitial cells (WMICs), in the brains of a southern lesser galago (Galago moholi), a black-capped squirrel monkey (Saimiri boliviensis boliviensis), and a crested macaque (Macaca nigra). Staining for neuronal nuclear marker (NeuN) revealed WMICs throughout the infracortical white matter, these cells being most dense close to inner cortical border, decreasing in density with depth in the white matter. Stereological analysis of NeuN-immunopositive cells revealed estimates of approximately 1.1, 10.8, and 37.7 million WMICs within the infracortical white matter of the galago, squirrel monkey, and crested macaque, respectively. The total numbers of WMICs form a distinct negative allometric relationship with brain mass and white matter volume when examined in a larger sample of primates where similar measures have been obtained. In all three primates studied, the highest densities of WMICs were in the white matter of the frontal lobe, with the occipital lobe having the lowest. Immunostaining revealed significant subpopulations of WMICs containing neuronal nitric oxide synthase (nNOS) and calretinin, with very few WMICs containing parvalbumin, and none containing calbindin. The nNOS and calretinin immunopositive WMICs represent approximately 21% of the total WMIC population; however, variances in the proportions of these neurochemical phenotypes were noted. Our results indicate that both the squirrel monkey and crested macaque might be informative animal models for the study of WMICs in neurodegenerative and psychiatric disorders in humans.