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 and morphology of catecholaminergic neurons and certain pallial terminal networks in the brain of the Nile crocodile, Crocodylus niloticus.

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.

Potential Adult Neurogenesis in the Telencephalon and Cerebellar Cortex of the Nile Crocodile Revealed with Doublecortin Immunohistochemistry.

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.

Comparison of estimates of neuronal number obtained using the isotropic fractionator method and unbiased stereology in day old chicks (Gallus domesticus).

BACKGROUND: The relative size and neuronal density of brain regions are important metrics in both comparative and experimental studies in neuroscience. Consequently, it is imperative to have accurate, reliable and reproducible methods of quantifying cell number. NEW METHOD: The isotropic fractionator (IF) method estimates the number of neurons and non-neurons in the central nervous system by homogenizing tissue into discrete nuclei and determining the proportion of neurons from non-neurons using immunohistochemistry (Herculano- Herculano-Houzel and Lent, 2005). COMPARISON WITH EXISTING METHOD: One of the advantages of IF is that it is considerably faster than stereology. However, as the method is relatively new, concerns about its accuracy remain, particularly whether homogenization results in underestimation of cell number. In this study, we compared estimates of neuronal number in the telencephalon and 'rest of brain' (i.e. the diencephalon and brainstem excluding the optic lobes) of day old chicks using the IF method and stereology. RESULTS: In the telencephalon, there was a significant difference in estimates of neuronal number between the 2 methods, but not estimates of neuronal density (neurons/mg of tissue). Whereas in the 'rest of brain', there was a significant difference in estimates of neuronal density, but not neuronal number. In all cases, stereological estimates were lower than those obtained using the IF method. CONCLUSION: Despite the statistically significant differences, there was considerable overlap (all estimates were within 16% of one another) between estimates obtained using the two methods suggesting that the two methods provide comparable estimates of neuronal number in birds.

Continued Growth of the Central Nervous System without Mandatory Addition of Neurons in the Nile Crocodile (Crocodylus niloticus).

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.

The continuously growing central nervous system of the Nile crocodile (Crocodylus niloticus).

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.

Organisation and chemical neuroanatomy of the African elephant (Loxodonta africana) olfactory bulb.

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.