Distribution of vasoactive intestinal peptide (VIP) immunoreactivity in the rat pallial and subpallial amygdala and colocalization with γ-aminobutyric acid (GABA).

The amygdaloid complex, also known as the amygdala, is a heterogeneous group of distinct nuclear and cortical pallial and subpallial structures. The amygdala plays an important role in several complex functions including emotional behavior and learning. The expression of calcium-binding proteins and peptides in GABAergic neurons located in the pallial and subpallial amygdala is not uniform and is sometimes restricted to specific groups of cells. Vasoactive intestinal polypeptide (VIP) is present in specific subpopulations of GABAergic cells in the amygdala. VIP immunoreactivity has been observed in somatodendritic and axonal profiles of the rat basolateral and central amygdala. However, a comprehensive analysis of the distribution of VIP immunoreactivity in the various pallial and subpallial structures is currently lacking. The present study used immunohistochemical and morphometric techniques to analyze the distribution and the neuronal localization of VIP immunoreactivity in the rat pallial and subpallial amygdala. In the pallial amygdala, VIP-IR neurons are local inhibitory interneurons that presumably directly and indirectly regulate the activity of excitatory pyramidal neurons. In the subpallial amygdala, VIP immunoreactivity is expressed in several inhibitory cell types, presumably acting as projection or local interneurons. The distribution of VIP immunoreactivity is non-homogeneous throughout the different areas of the amygdaloid complex, suggesting a distinct influence of this neuropeptide on local neuronal circuits and, consequently, on the cognitive, emotional, behavioral and endocrine activities mediated by the amygdala.

Cytoarchitectureal changes in hippocampal subregions of the NZB/W F1 mouse model of lupus.

Over 50% of clinical patients affected by the systemic lupus erythematosus disease display impaired neurological cognitive functions and psychiatric disorders, a form called neuropsychiatric systemic lupus erythematosus. Hippocampus is one of the brain structures most sensitive to the cognitive deficits and psychiatric disorders related to neuropsychiatric lupus. The purpose of this study was to compare, layer by layer, neuron morphology in lupus mice model NZB/W F1 versus Wild Type mice. By a morphometric of cells identified on Nissl-stained sections, we evaluated structural alterations between NZB/W F1 and Wild Type mice in seven hippocampal subregions: Molecular dentate gyrus, Granular dentate gyrus, Polymorph dentate gyrus, Oriens layer, Pyramidal layer, Radiatum layer and Lacunosum molecular layer. By principal component analysis we distinguished healthy Wild Type from NZB/W F1 mice. In NZB/W F1 mice hippocampal cytoarchitecture, the neuronal cells resulted larger in size and more regular than those of Wild Type. In NZB/W F1, neurons were usually denser than in WT. The Pyramidal layer neurons were much denser in Wild Type than in NZB/W F1. Application of principal component analysis, allowed to distinguish NZB/W F1 lupus mice from healthy, showing as NZBW subjects presented a scattered distribution and intrasubject variability. Our results show a hypertrophy of the NZB/W F1 hippocampal neurons associated with an increase in perikaryal size within the CA1, CA2, CA3 region and the DG. These results help advance our understanding on hippocampal organization and structure in the NZB/W F1 lupus model, suggesting the hypothesis that the different subregions could be differentially affected in neuropsychiatric systemic lupus erythematosus disease. Leveraging an in-depth analysis of the morphology of neural cells in the hippocampal subregions and applying dimensionality reduction using PCA, we propose an efficient methodology to distinguish pathological NZBW mice from WT mice."

Connections of the sheep basolateral amygdala: A diffusion tensor imaging study.

BACKGROUND: A large amount of anatomic data published over the past decade has provided novel insight into the connections of the basolateral amygdala (BLA) in the rat, cat, and monkey. The mammalian (rat, cat, and monkey) BLA has strong connections with the cortex (especially piriform, and frontal cortices), the hippocampal region (especially perirhinal cortex, entorhinal cortex, and subiculum), the thalamus (in particular, the posterior internuclear nucleus and medial geniculate nucleus) and, to some extent, the hypothalamus. An important question remains as to how well the data obtained in rodents and primates can be extrapolated to ruminants. NEW METHOD: To address this issue the connections of the sheep BLA has been determined by Magnetic Resonance Imaging (MRI) and Diffusion Tensor Imaging (DTI, Tractography). RESULTS: Tractography showed ipsilateral connections between the BLA and several areas. COMPARISON WITH EXISTING METHOD(S): Reviews based mainly on description of the results obtained using anterograde and retrograde neuronal tracers. In the present research, we prefer to use a non-invasive technique (DTI). CONCLUSIONS: This report shows the existence of specific amygdaloid connections in the sheep.

Multi-aspect testing and ranking inference to quantify dimorphism in the cytoarchitecture of cerebellum of male, female and intersex individuals: a model applied to bovine brains.

The dimorphism among male, female and freemartin intersex bovines, focusing on the vermal lobules VIII and IX, was analyzed using a novel data analytics approach to quantify morphometric differences in the cytoarchitecture of digitalized sections of the cerebellum. This methodology consists of multivariate and multi-aspect testing for cytoarchitecture-ranking, based on neuronal cell complexity among populations defined by factors, such as sex, age or pathology. In this context, we computed a set of shape descriptors of the neural cell morphology, categorized them into three domains named size, regularity and density, respectively. The output and results of our methodology are multivariate in nature, allowing an in-depth analysis of the cytoarchitectonic organization and morphology of cells. Interestingly, the Purkinje neurons and the underlying granule cells revealed the same morphological pattern: female possessed larger, denser and more irregular neurons than males. In the Freemartin, Purkinje neurons showed an intermediate setting between males and females, while the granule cells were the largest, most regular and dense. This methodology could be a powerful instrument to carry out morphometric analysis providing robust bases for objective tissue screening, especially in the field of neurodegenerative pathologies.

Distribution of Calretinin Immunoreactivity in the Lateral Nucleus of the Bottlenose Dolphin (Tursiops truncatus) Amygdala.

The amgdaloid complex consists of different nuclei, each with unique cytoarchitectonic, chemoarchitectonic and connectional characteristics. Most of the inputs coming from cortical and subcortical areas enter the amygdala via the lateral nucleus, which makes it the main receiving structure of the complex. The activity of its neurons is coordinated and modulated by different inhibitory, GABAergic-interneurons, which can be classified for their expression of various calcium-binding proteins, as well as by morphological characteristics. This research based on the analysis of the amygdala of three bottlenose dolphins, provides the first description of the topography, cytoarchitecture and distribution of calretinin immunoreactivity of the lateral nucleus. Our observations on the bottlenose dolphin confirmed the general topography of the mammalian amygdala and of the lateral nucleus. Notably, we identified six subdivision of the nucleus, more than those reported until now in the rat, monkey and human lateral nucleus. This could reveal an outstanding capability of integration and elaboration of external stimuli. In addition, we observed a strong presence of CR-immunoreactive (-ir) neurons and fibres. CR-ir neurons were mainly non-pyramidal inhibitory neurons; in particular, 80% of IR-cells were represented by large and small polygonal neurons. In the lateral nucleus of the human amygdala, CR-ir neurons form inhibitory synapses on calbindin-D28k-IR inhibitory interneurons. Since calbindin-D28k-ir interneurons make inhibitory synapses on the pyramidal cells, the final goal of the CR-ir interneurons could be the synchronization of cells activity, thus playing an important role in the control of information flow in the lateral amygdalar nucleus. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:2008-2016, 2017. © 2017 Wiley Periodicals, Inc.

Gene expression profiles of estrogen receptors α and ß in the fetal bovine hypothalamus and immunohistochemical characterization during development.

Steroid hormones intervene in the structural and functional regulation of neuronal processes during development and thus determine brain differentiation. The effects of estrogens are mediated by two transcription factors, namely estrogen receptor α (ER-α) and estrogen receptor ß (ER-ß), that regulate the expression of target genes through their binding to specific DNA target sequences. We describe the mRNA expression of ER-α and ER-ß in the hypothalamus of developing male and female bovines as revealed by quantitative real-time polymerase chain reaction, and the distribution of the two ERs in hypothalamic sections of all fetal stages as shown by immunohistochemistry. The expression profiles of the mRNAs of both ERs are mutually correlated throughout the gestation period, and their levels increase significantly in the last stages of gestation. No sexual differences in the mRNA expression of either ER-α or ER-ß have been found in our fetal specimens. The use of specific antisera against ER-α and ER-ß has allowed us to characterize and confirm the distribution of these receptors in the hypothalami of all fetal stages considered. Our results offer detailed information concerning the distribution of ER-α and ER-ß in the developing bovine hypothalamus and provide additional insights into the processes involved in the hypothalamic development of a mammal with a long gestation and a highly gyrencephalic brain.

The Inferior Alveolar Nerve of the Horse: Course and Anatomical Relationship with Mandibular Cheek Teeth.

The precise location of neurovascular structures within the relatively long mandibular canal of the horse is of paramount importance in surgical procedures of the area. The inferior alveolar nerve (IAN) enters the mandibular canal on the medial (lingual) surface of the mandible and innervates all the mandibular teeth. During its course, the nerve moves laterally, crossing the roots of the inferior cheek teeth. However, the exact anatomical relationships occurring between the IAN and the roots of the equine mandibular cheek teeth have not been described in detail. In this study, the mandibles of 40 horses were examined with CT scans and then used for bilateral detailed anatomical dissection, to assess the path of the IAN and its relationship to the roots of the lower cheek teeth. The data obtained show that the equine IAN is located ventral to the apices of the molar teeth (311/411, 310/410, 309/409 according to the Triadan numerical system). At the level of PM(4) (308/408), the IAN is located on the lingual side of the roots and coronally to its apices. At the level of PM(3) (307/407), the IAN is then found on the lingual side of the roots but in proximity to the apices. In 2 of 40 horses (=5%), the IAN moves towards the lingual side between the mesial and the distal root of PM(4). Our observations are valuable for planning a surgical approach to the ventral side of the mandible in the horse and to avoid potential post-operative complications.

Expression profile of the pore-forming subunits α1A and α1D in the foetal bovine hypothalamus: a mammal with a long gestation.

This study describes the expression of the voltage operated calcium channels (VOCCs) subunits α1A (typical of the P/Q family) and α1D (of the L family) in the bovine hypothalamus. The expression of both P/Q and L families has been characterized in the brain of adult mammals. However, their distribution and expression during foetal neuronal differentiation have not yet been determined. The expression profile of the α1A and α1D pore-forming subunits was investigated during four embryonic stages in bovine foetuses. Our data suggest that the expressions of α1A and α1D are correlated during development, with an increase only in males that peaks on the last period of gestation. Bovine male hypothalami showed significantly higher α1A and α1D expression values in comparison to female ones during the whole developmental period. In the females, the expression profiles of both genes were constant during all the developmental time. Immunohistochemical studies confirmed the presence of the α1A and α1D protein subunits in foetal hypothalamic neurones starting from the third foetal stage. Our data provide new information on the hypothalamic expression of α1A and α1D subunits during development in a mammal with a long gestation period and a large and convoluted brain.

Characterization of an established endothelial cell line from primary cultures of fetal sheep hypothalamus.

Immortalized cell lines from fetal brain are an experimental model for studying the in vitro molecular pathways regulating neural cell differentiation and the development of neural networks. The procedures are described to obtain an established cell line from the 90-day old fetal sheep hypothalamus. Viral oncogene LT-SV40 transformation was used to isolate a stable cell line (ENOS-01) that was characterized immunocytochemically. Immortalized cells can be classified as an endothelial cell line of hypothalamic microvasculature. Furthermore, mRNA expression and immunocytochemical of estrogen receptors α and ß were also evaluated. Since it is known that cerebral vessels are directly targeted by sex steroids, our established cell line represents an alternative system to study estradiol/receptor interactions during brain development. Our in vitro model can provide a tool to investigate the complex relationships among the cell types forming the blood-brain barrier, which is known to be involved in the pathogenesis of sheep transmissible neurological diseases.

Expression of aromatase P450(AROM) in the human fetal and early postnatal cerebral cortex.

Aromatase (P450(AROM)), the enzyme responsible for the conversion of testosterone (T) into 17-ß estradiol (E(2)), plays a crucial role in the sexual differentiation of specific hypothalamic nuclei. Moreover, recent findings indicate that local E(2) synthesis has an impact on other brain areas including hippocampus, temporal cortex and cerebellum, and may thus influence also cognitive functions. Numerous studies have described the expression and the distribution of P450(AROM) throughout ontogenesis and postnatal development of the central nervous system in several mammals, but data referring to humans are scarce. In the adult human brain, P450(AROM) has been detected in the hypothalamus, limbic areas, and in the basal forebrain, and described in glial cells of the cerebral cortex and hippocampus. In this study we report the expression, distribution and cellular localization of P450(AROM) in the human fetal and early postnatal cerebral cortex. In our series of fetal brains of the second trimester, P450(AROM) expression appeared at gestational week (GW) 17 and resulted limited to groups of cells localized close to the growing neuroepithelium in the ventricular and subventricular zones. At GWs 20-24, scattered P450(AROM) immunoreactive (-ir) neural cells were identified in the intermediate plate and subplate, and in the parietal cortical plate. In perinatal and early postnatal individuals the quantity of P450(AROM)-ir elements increased, and revealed the morphology typical of glial cells. Double labeling immunostaining with anti-GFAP and anti-P450(AROM) antisera, and subsequent confocal analysis, confirmed this observation. Our data show that the expression of P450(AROM) in the fetal cortex starts approx at the end of the fourth gestational month, but increases steadily only in the last trimester or in the early postnatal period. This temporal trend may suggest that P450(AROM) could act as a differentiation-promoting factor, based on timing of the steroid actions.

Stereology of the neocortex in Odontocetes: qualitative, quantitative, and functional implications.

We investigated the quantitative morphology of the neocortex (gray matter) in 2 toothed whale (odontocete) species (harbor porpoise, Phocoena phocoena; bottlenose dolphin, Tursiops truncatus) with stereological methods. The 4 primary projection areas (motor, somatosensory, auditory, and visual fields) are analyzed for their cell densities in layers III and V with standard design-based stereology methods. Along cortical areas M1, S1, A1, and V1 in Tursiops, neuron density is always higher in layer III than in layer V, whereas the data in Phocoena are variable. Moreover, neuron density in layer III is generally around 1.5 times higher in Tursiops than in Phocoena. Maximal density values are seen in layer III of A1 and V1 in Tursiops and the ratio of layer III/layer V density is maximal in A1 of this species. Thus, layer III could have a higher capacity in the bottlenose dolphin with regard to intrinsic connectivity. Extant knowledge on toothed whale neurobiology and behavior suggests that quantitative/stereological differences between the 2 odontocete species regarding the neuron density of standard cortical units may be correlated with specific adaptations to their respective habitats. In contrast to layers V and VI which mainly serve as an executive system, layer III could represent an intermediate level in sensory and premotor processing which works more tangentially in the cortices via horizontal connections with other cortical areas, respectively. The generally higher density of cortical layer III in Tursiops suggests a higher connectivity of this layer in view of the more agile and complicated behavior of these gregarious animals including versatile phonation by complex sound and ultrasound signals.

Expression and localization of aromatase P450AROM, estrogen receptor-α, and estrogen receptor-ß in the developing fetal bovine frontal cortex.

The enzyme aromatase (P450(AROM)) converts testosterone (T) into 17-ß estradiol (E(2)) and is crucial for the control of development of the central nervous system during ontogenesis. The effects of E(2) in various brain areas are mediated by the estrogen receptor alpha (ER-α) and the estrogen receptor beta (ER-ß). During fetal development, steroids are responsible for the sexual differentiation of the hypothalamus. Estrogens are also able to exert effects in other brain areas of the fetus including the frontal cortex, where they act through estrogen receptors (ERs) modulating cognitive function and affective behaviors. In this study we have determined the expression profiles of P450(AROM) and ERs in the fetal bovine frontal cortex by quantitative Real-Time PCR (qRT-PCR) throughout the prenatal development. The data show that the patterns of expression of both ERs are strongly correlated during pregnancy and increase in the last stage of gestation. On the contrary, the expression of P450(AROM) has no correlation with ERs expression and is not developmentally regulated. Moreover, we performed immunochemical studies showing that fetal neurons express P450(AROM) and the ERs. P450(AROM) is localized in the cytoplasm and only seldom present in the fine extensions of the cells; ER-α is detected predominantly in the soma whereas ER-ß is only present in the nucleus of a few cells. This study provides new data on the development of the frontal cortex in a long gestation mammal with a large convoluted brain.

Stimulation of Ca²+ signals in neurons by electrically coupled electrolyte-oxide-semiconductor capacitors.

Electrolyte-oxide-semiconductor capacitors (EOSCs) are a class of microtransducers for extracellular electrical stimulation that have been successfully employed to activate voltage-dependent sodium channels at the neuronal soma to generate action potentials in vitro. In the present work, we report on their use to control Ca²+ signalling in cultured mammalian cells, including neurons. Evidence is provided that EOSC stimulation with voltage waveforms in the microsecond or nanosecond range activates two distinct Ca²+ pathways, either by triggering Ca²+ entry through the plasma membrane or its release from intracellular stores. Ca²+ signals were activated in non-neuronal and neuronal cell lines, CHO-K1 and SH-SY5Y. On this basis, stimulation was tailored to rat and bovine neurons to mimic physiological somatic Ca²+ transients evoked by glutamate. Being minimally invasive and easy to use, the new method represents a versatile complement to standard electrophysiology and imaging techniques for the investigation of Ca²+ signalling in dissociated primary neurons and cell lines.

The upper respiratory tract of dolphins.

The functional anatomy of the respiratory system of dolphins has been scarcely studied. Specifically, the capacity of the system to resist pressure changes during diving has not been fully understood. Here we shortly describe the upper respiratory tract of dolphins based on three common species, the bottlenose dolphin Tursiops truncatus, the Risso's dolphin Grampus griseus, and the striped dolphin Stenella coeruleoalba. We emphasize the keymorphological features that represent evolutionary adaptations to life in the water, and, furthermore, also present a model of the tracheo-bronchial tree based on mechanical characterization and subsequent computational simulation of its biomechanical behaviour. Comparisons with the goat allowed us to determine how different structures may respond to diving-related pressure.

A landmark analysis-based approach to age and sex classification of the skull of the Mediterranean monk seal (Monachus monachus) (Hermann, 1779).

This work aimed at applying geometric morphometric analysis techniques to the skull of the Mediterranean monk seal (Monachus monachus, Hermann, 1779). Inferential analyses were performed using a non-parameteric permutation framework based on a series of skulls of different age classes belonging to individuals of both sexes. Our goal was to establish whether a statistical approach based on osteometric measurements and surface analysis of photographs of the left lateral plane of the skull may lead to a different and scientifically sound method of age and sex classification in this critically endangered marine mammal. Our data indicate that non-parametric combination methodology enables the researcher to give local assessment using a combination with domains. Developing geometric morphometric techniques in a non-parametric permutation framework could be useful in solving high dimensional and small sample size problems as well as classification problems, including zoological classification of specimens within a specific population. The Mediterranean monk seal is believed to be the world's rarest pinniped and one of the most endangered mammals of the world, with fewer than 600 individuals currently surviving. The use of shape analysis would allow new insights into the biological characteristics of the monk seal by simply extracting potentially new information on age and size from museal specimens.

The external and middle ear of the striped dolphin Stenella coeruleoalba (Meyen 1833).

The ear of cetaceans, and especially the middle ear, is very different from that of terrestrial mammals and shows specific adaptations to diving. Our research, performed on six Striped dolphins (Stenella coeruleoalba) stranded along the Italian coasts of the Mediterranean Sea, concentrated on the morphology of the external and middle ear of this species. We report the findings using a proper Veterinary Anatomical Nomenclature and describe the characteristics of the auditory meatus of the external ear and the presence and morphology of the erectile tissue in the middle ear. Our anatomical and histological data highlight the structure and possible functions of the corpus cavernosum located in the middle ear of the Striped dolphin, and suggest a possible role for this structure in relation to pressure regulation during diving. Many of our observations indicate the existence of an internal regulatory system able to prevent barotraumas by regulating pressure and volume inside the middle ear cavity.

Expression of AMH in female fetal intersex gonads in the bovine.

Anti Müllerian Hormone, AMH, is believed to be the main agent in the freemartin syndrome. Supposing an active role of freemartin gonads in AMH secretion, in the present study, we aimed at investigating the presence and the localization of AMH producing cells either in fetal or in adult freemartin gonads. Our finding of positive AMH cells in a 26-week-old freemartin fetus indicates an active role of masculinized freemartin gonads in AMH secretion. However, the positive reaction, limited to few cells grouped in 'nests' in proximity to testis cord-like structures, supports a chimeric origin of such cells, migrated from the male co-twin. No adult freemartin, irrespective from the degree of masculinization, showed any AMH positive cell.

The vasotocinergic system in the hypothalamus and limbic region of the budgerigar (Melopsittacus undulatus).

We report a morphological and biochemical analysis on the presence, distribution and quantification of vasotocin in the hypothalamus and limbic region of the budgerigar Melopsittacus undulatus, using immunohistochemistry on serial sections and competitive enzyme linked immunoadsorbent assay measurements on tissue extracts. Analysis of the sections showed large vasotocin-immunoreactive neurons in three main regions of the diencephalon, of both male and female specimens. Vasotocinergic cell bodies were located in the ventral and lateral areas of the hypothalamus, dorsal to the lateral thalamus and medial to the nucleus geniculatus lateralis. Immunoreactive neurons were placed also periventricularly, close to the walls of the third ventricle, at the level of the magnocellular paraventricular nucleus. Well evident bundles of immunoreactive fibers were placed ventral to the anterior commissure in the same regions of the hypothalamus and thalamus where vasotocinergic perikarya are localized. Fibers were identified close to the third ventricle, and in the lateral hypothalamic area along the lateral forebrain bundle. In contrast to what reported for other oscine and non-oscine avian species, we were not able to identify immunopositive neurons in any region above the anterior commissure, or detect relevant differences on the distribution of the vasotocin immmunoreactivity between sexes. Competitive enzyme linked immunoadsorption assay and image analysis of the extension of immunoreactivity in the tissue sections were consistent with the qualitative observations and indicated that there is no statistically significant dimorphism in the content of vasotocin or in the location and distribution of vasotocinergic elements in the investigated areas of male and female parrot brains.

Sexual differences and effect of photoperiod on melatonin receptor in avian brain.

Several data suggest that melatonin may influence avian reproduction by acting at the level of the hypothalamic-hypophisial-gonadal axis, and/or on neural circuits controlling reproductive behaviours. The action of melatonin is exerted through specific receptors whose distribution and pharmacological properties have been extensively investigated. This review will focus on the distribution, sexual dimorphism, and dependence upon the photoperiod of melatonin binding sites in avian species with a special emphasis on Japanese quail. Melatonin receptors are widely distributed in avian brain. They are mostly present in the visual pathways of all the investigated species and in the song controlling nuclei of oscine birds. Sexual dimorphism of melatonin binding sites (higher density in males than in females) was detected in some telencephalic nuclei of songbirds, in the visual pathways, and in the preoptic area of quail. The last region plays a key role in the activation of male quail copulatory behaviour and it hosts a large population of gonadotropin-releasing hormone-containing neurons. Sexual dimorphism of melatonin-binding sites in the above-mentioned regions suggests a differential role for this hormone in the modulation of visual perception, gonadotropin production, and seasonally activated behaviours in male and female quail. Further studies are necessary to understand interrelationships among photic cues, gonadal steroids, density, and sexually dimorphic distribution of melatonin receptors.

An overview of the central nervous system of the elephant through a critical appraisal of the literature published in the XIX and XX centuries.

The two species of elephants (Indian: Elephas maximus and African: Loxodonta africana) possess the largest brain among land mammals. Due to its size, the elephant brain is discussed in virtually every paper dealing with the evolution of the central nervous system of mammals and comparative brain size. Studies on the social habits of elephants also deal with the skills and the "intelligence" and brain size of these species. Yet most of the descriptions and conclusions reported in comparative studies rely on second-hand data derived from investigations performed several decades before, often dating as far back as the XIX century. Furthermore, many of the original papers actually describing gross and detailed features of the brain of elephants are either no longer available, are written in languages other than English, or are difficult to trace. The present study gives a short description of the anatomy of the central nervous system of elephants, with special attention to its distinctive features, reports all available literature on the subject, and briefly discusses its origins and rationale.