Nuclear parcellation and numbers of orexinergic neurons in five species of larger brained birds.

The orexinergic/hypocretinergic system, while having several roles, appears to be a key link in the balance between arousal and food intake. In birds, to date, this system has only been examined anatomically in four species, all with brains smaller than 3.5 g and of limited phylogenetic range. Here, using orexin-A immunohistochemistry, we describe the distribution, morphology, and nuclear parcellation of orexinergic neurons within the hypothalami of a Congo gray and a Timneh gray parrot, a pied crow, an emu, and a common ostrich. These birds represent a broad phylogeny, with brains ranging in size from 7.85 to 26.5 g. Within the hypothalami of the species studied, the orexinergic neurons were organized in two clusters, and a densely packed paraventricular hypothalamic nucleus cluster located within the medial hypothalamus (Hyp), but not contacting the ventricle, and a more loosely packed lateral hypothalamic cluster in the lateral Hyp. Stereological analysis revealed a strong correlation, using phylogenetic generalized least squares regression analyses, between brain mass and the total number of orexinergic neurons, as well as soma parameters such as volume and area. Orexinergic axonal terminals evinced two types of boutons, larger and the smaller en passant boutons. Unlike the orexinergic system in mammals, which has several variances in cluster organization, that of the birds studied, in the present and previous studies, currently shows organizational invariance, despite the differences in brain and body mass, phylogenetic relationships, and life-histories of the species studied.

Apparent absence of hypothalamic cholinergic neurons in the common ostrich and emu: Implications for global brain states during sleep.

We examined the presence/absence and parcellation of cholinergic neurons in the hypothalami of five birds: a Congo grey parrot (Psittacus erithacus), a Timneh grey parrot (P. timneh), a pied crow (Corvus albus), a common ostrich (Struthio camelus), and an emu (Dromaius novaehollandiae). Using immunohistochemistry to an antibody raised against the enzyme choline acetyltransferase, hypothalamic cholinergic neurons were observed in six distinct clusters in the medial, lateral, and ventral hypothalamus in the parrots and crow, similar to prior observations made in the pigeon. The expression of cholinergic nuclei was most prominent in the Congo grey parrot, both in the medial and lateral hypothalamus. In contrast, no evidence of cholinergic neurons in the hypothalami of either the ostrich or emu was found. It is known that the expression of sleep states in the ostrich is unusual and resembles that observed in the monotremes that also lack hypothalamic cholinergic neurons. It has been proposed that the cholinergic system acts globally to produce and maintain brain states, such as those of arousal and rapid-eye-movement sleep. The hiatus in the cholinergic system of the ostrich, due to the lack of hypothalamic cholinergic neurons, may explain, in part, the unusual expression of sleep states in this species. These comparative anatomical and sleep studies provide supportive evidence for global cholinergic actions and may provide an important framework for our understanding of one broad function of the cholinergic system and possible dysfunctions associated with global cholinergic neural activity.

Orexinergic neurons in the hypothalami of an Asiatic lion, an African lion, and a Southeast African cheetah.

Employing orexin-A immunohistochemistry, we describe the distribution, morphology, and nuclear parcellation of orexinergic neurons within the hypothalami of an Asiatic lion (Panthera leo subsp. persica), an African lion (Panthera leo subsp. melanochaita), and a Southeast African cheetah (Acinonyx jubatus subsp. jubatus). In all three felids, the clustering of large, bipolar, and multipolar hypothalamic orexinergic neurons primarily follows the pattern observed in other mammals. The orexinergic neurons were found, primarily, to form three distinct clusters-the main, zona incerta, and optic tract clusters. In addition, large orexinergic neurons were observed in the ventromedial supraoptic region of the hypothalamus, where they are not typically observed in other species. As has been observed in cetartiodactyls and the African elephant, a cluster of small, multipolar orexinergic neurons, the parvocellular cluster, was observed in the medial zone of the hypothalamus in all three felids, although this parvocellular cluster has not been reported in other carnivores. In both subspecies of lions, but not the cheetah, potential orexin-immunopositive neurons were observed in the paraventricular hypothalamic nucleus, supraoptic nucleus, the lateral part of the retrochiasmatic area, and the inner layer of the median eminence. The distribution and parcellation of orexinergic neurons in the hypothalami of the three felids studied appear to be more complex than observed in many other mammals and for the two subspecies of lion may be even more complex. These findings are discussed in terms of potential technical concerns, phylogenetic variations of this system, and potentially associated functional aspects of the orexinergic system.

Nuclear organization of orexinergic neurons in the hypothalamus of a lar gibbon and a chimpanzee.

Employing orexin-A immunohistochemical staining we describe the nuclear parcellation of orexinergic neurons in the hypothalami of a lar gibbon and a chimpanzee. The clustering of orexinergic neurons within the hypothalamus and the terminal networks follow the patterns generally observed in other mammals, including laboratory rodents, strepsirrhine primates and humans. The orexinergic neurons were found within three distinct clusters in the ape hypothalamus, which include the main cluster, zona incerta cluster and optic tract cluster. In addition, the orexinergic neurons of the optic tract cluster appear to extend to a more rostral and medial location than observed in other species, being observed in the tuberal region in the anterior ventromedial aspect of the hypothalamus. While orexinergic terminal networks were observed throughout the brain, high density terminal networks were observed within the hypothalamus, medial and intralaminar nuclei of the dorsal thalamus, and within the serotonergic and noradrenergic regions of the midbrain and pons, which is typical for mammals. The expanded distribution of orexinergic neurons into the tuberal region of the ape hypothalamus, is a feature that needs to be investigated in other primate species, but appears to correlate with orexin gene expression in the same region of the human hypothalamus, but these neurons are not revealed with immunohistochemical staining in humans. Thus, it appears that apes have a broader distribution of orexinergic neurons compared to other primate species, but that the neurons within this extension of the optic tract cluster in humans, while expressing the orexin gene, do not produce the neuropeptide.

Cortical and thalamic connectivity of occipital visual cortical areas 17, 18, 19, and 21 of the domestic ferret (Mustela putorius furo).

The present study describes the ipsilateral and contralateral corticocortical and corticothalamic connectivity of the occipital visual areas 17, 18, 19, and 21 in the ferret using standard anatomical tract-tracing methods. In line with previous studies of mammalian visual cortex connectivity, substantially more anterograde and retrograde label was present in the hemisphere ipsilateral to the injection site compared to the contralateral hemisphere. Ipsilateral reciprocal connectivity was the strongest within the occipital visual areas, while weaker connectivity strength was observed in the temporal, suprasylvian, and parietal visual areas. Callosal connectivity tended to be strongest in the homotopic cortical areas, and revealed a similar areal distribution to that observed in the ipsilateral hemisphere, although often less widespread across cortical areas. Ipsilateral reciprocal connectivity was observed throughout the visual nuclei of the dorsal thalamus, with no contralateral connections to the visual thalamus being observed. The current study, along with previous studies of connectivity in the cat, identified the posteromedial lateral suprasylvian visual area (PMLS) as a distinct network hub external to the occipital visual areas in carnivores, implicating PMLS as a potential gateway to the parietal cortex for dorsal stream processing. These data will also contribute to a macro connectome database of the ferret brain, providing essential data for connectomics analyses and cross-species analyses of connectomes and brain connectivity matrices, as well as providing data relevant to additional studies of cortical connectivity across mammals and the evolution of cortical connectivity variation.

Cortical and thalamic connectivity of temporal visual cortical areas 20a and 20b of the domestic ferret (Mustela putorius furo).

The present study describes the ipsilateral and contralateral corticocortical and corticothalamic connectivity of the temporal visual areas 20a and 20b in the ferret using standard anatomical tract-tracing methods. The two temporal visual areas are strongly interconnected, but area 20a is primarily connected to the occipital visual areas, whereas area 20b maintains more widespread connections with the occipital, parietal and suprasylvian visual areas and the secondary auditory cortex. The callosal connectivity, although homotopic, consists mainly of very weak anterograde labeling which was more widespread in area 20a than area 20b. Although areas 20a and 20b are well connected with the visual dorsal thalamus, the injection into area 20a resulted in more anterograde label, whereas more retrograde label was observed in the visual thalamus following the injection into area 20b. Most interestingly, comparisons to previous connectional studies of cat areas 20a and 20b reveal a common pattern of connectivity of the temporal visual cortex in carnivores, where the posterior parietal cortex and the central temporal region (PMLS) provide network points required for dorsal and ventral stream interaction enroute to integration in the prefrontal cortex. This pattern of network connectivity is not dissimilar to that observed in primates, which highlights the ferret as a useful animal model to understand visual sensory integration between the dorsal and ventral streams. The data generated will also contribute to a connectomics database, to facilitate cross species analysis of brain connectomes and wiring principles of the brain.

Cortical and thalamic connectivity of posterior parietal visual cortical areas PPc and PPr of the domestic ferret (Mustela putorius furo).

The present study describes the ipsilateral and contralateral cortico-cortical and cortico-thalamic connectivity of the parietal visual areas, posterior parietal caudal cortical area (PPc) and posterior parietal rostral cortical area (PPr), in the ferret using standard anatomical tract-tracing methods. The two divisions of posterior parietal cortex of the ferret are strongly interconnected, however area PPc shows stronger connectivity with the occipital and suprasylvian visual cortex, while area PPr shows stronger connectivity with the somatomotor cortex, reflecting the functional specificity of these two areas. This pattern of connectivity is mirrored in the contralateral callosal connections. In addition, PPc and PPr are connected with the visual and somatomotor nuclei of the dorsal thalamus. Numerous connectional similarities exist between the posterior parietal cortex of the ferret (PPc and PPr) and the cat (area 7 and 5), indicative of the homology of these areas within the Carnivora. These findings highlight the existence of a frontoparietal network as a shared feature of the organization of parietal cortex across Euarchontoglires and Laurasiatherians, with the degree of expression varying in relation to the expansion and areal complexity of the posterior parietal cortex. This observation indicates that the ferret is a potentially valuable experimental model animal for understanding the evolution and function of the posterior parietal cortex and the frontoparietal network across mammals. The data generated will also contribute to a connectomics database, to further cross-species analyses of connectomes and illuminate wiring principles of cortical connectivity across mammals.

The brain of the tree pangolin (Manis tricuspis). V. The diencephalon and hypothalamus.

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.

Organization and number of orexinergic neurons in the hypothalamus of two species of Cetartiodactyla: a comparison of giraffe (Giraffa camelopardalis) and harbour porpoise (Phocoena phocoena).

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.

Distribution of orexinergic neurons and their terminal networks in the brains of two species of African mole rats.

The distribution of orexinergic cell bodies and terminal networks within the brains of two species of African mole rat (Cape-dune mole rat--Bathyergus suillus and highveld mole rat--Cryptomys hottentotus) were identified using immunohistochemistry for orexin-A. The aim of the study was to investigate possible differences in the nuclear complement and terminal distribution of this system by comparing those of the mole rats to published studies of other rodents and mammals. The wild-caught mole rats used in this study live a subterranean lifestyle and are well known for their regressed visual system, which may lead to the prediction of differences in the distribution of the cell bodies and the terminal networks; however, we found that both species of mole rat displayed orexinergic nuclei limited to the hypothalamus in regions similar to those previously reported for other rodent and mammalian species. No immunoreactive neurons could be identified, in either species of mole rat within the anterior hypothalamic paraventricular nucleus, as has been reported for Murid rodents. The terminal networks, while remaining similar between the species, are more strongly expressed in the Cape-dune mole rat than in the highveld mole rat.

The superior colliculus of the ferret: cortical afferents and efferent connections to dorsal thalamus.

By injecting both biotinylated and fluorescent dextran amines into the superior colliculus of the ferret we traced its afferent connections from the cerebral cortex and its projections to nuclei of the dorsal thalamus. All visual and auditory cortical areas had retrogradely labelled layer 5 pyramidal neurons projecting to the superior colliculus, with the highest density in areas 18 and 21. Secondary somatosensory and motor cortical regions also projected to the superior colliculus. No retrograde labelling was observed in primary somatosensory, primary motor or prefrontal cortex. All visual dorsal thalamic nuclei received connections from the superior colliculus. Within the LGN, lamina C and the medial interlaminar nucleus (MIN) received dense afferents terminating with large labelled boutons. Within the lateral posterior nucleus (LP) a distinct band of moderately dense small, labelled boutons was observed, and within the pulvinar a broader and less dense region of small bouton labelling was observed. For the most part these connections are similar to those seen in the cat and other mammals, however, specific comparisons appear to delineate potential evolutionary trends related to complexity of the visual system.

The thalamus of the monotremes: cyto- and myeloarchitecture and chemical neuroanatomy.

Echidna and platypus brains were sectioned and stained by Nissl or myelin stains or immunocytochemically for calcium-binding proteins, gamma aminobutyric acid (GABA) or other antigens. Cyto- and myeloarchitecture revealed thalami that are fundamentally mammalian in organization, with the three principal divisions of the thalamus (epithalamus, dorsal thalamus and ventral thalamus) identifiable as in marsupials and eutherian mammals. The dorsal thalamus exhibits more nuclear parcellation than hitherto described, but lack of an internal medullary lamina, caused by splaying out of afferent fibre tracts that contribute to it in other mammals, makes identification of anterior, medial and intralaminar nuclear groups difficult. Differentiation of the ventral nuclei is evident with the ventral posterior nucleus of the platypus enormously expanded into the interior of the cerebral hemisphere, where it adopts a relationship to the striatum not seen in other mammals. Other nuclei such as the lateral dorsal become identifiable by expression of patterns of calcium-binding proteins identical to those found in other mammals. GABA cells are present in the ventral and dorsal thalamic nuclei, and in the ventral thalamus form a remarkable continuum with GABA cells of the two segments of the globus pallidus and pars reticulata of the substantia nigra.

Areal organization of the posterior parietal cortex of the ferret (Mustela putorius).

On grounds of electrophysiological mapping, cytoarchitecture, myeloarchitecture and callosal and thalamic connectivity, we have identified two cortical areas in the posterior parietal cortex of the ferret: posterior parietal caudal and rostral (PPc and PPr). These areas occupy the lateral and suprasylvian gyri, from the cingulate sulcus (medially) to the suprasylvian sulcus (laterally) and lie between visual areas 18 and 21 (posteriorly) and the somatosensory areas (anteriorly). Within both areas a coarse representation of the visual field was found and within PPr there was also a representation of the body. Each representation mirrors those within neighboring areas. Cytoarchitectonic and myeloarchitectonic fields within this cortical region did not correspond in any simple way to the physiological representations. The architectonic differences correlate to differential callosal connectivity, with predominant connectivity corresponding to the upper hemifield/head representations. PPr and PPc receive thalamic projections from a different, but overlapping, complement of thalamic nuclei. The superimposition of somatic and visual maps in PPr might relate to the probable role of this area in transforming retinal-centered to body-centered spatial coordinates. The organization of the parietal areas in the ferret resembles that of the flying fox and might unveil a common organizational plan from which the primate posterior parietal cortex evolved.