Bruno Cozzi

Expression of calcium-binding proteins and selected neuropeptides in the human, chimpanzee, and crab-eating macaque claustrum

The claustrum is present in all mammalian species examined so far and its morphology, chemoarchitecture, physiology, phylogenesis and ontogenesis are still a matter of debate. Several morphologically distinct types of immunostained cells were described in different mammalian species. To date, a comparative study on the neurochemical organization of the human and non-human primates claustrum has not been fully described yet, partially due to technical reasons linked to the postmortem sampling interval. The present study analyze the localization and morphology of neurons expressing parvalbumin (PV), calretinin (CR), NPY, and somatostatin (SOM) in the claustrum of man (# 5), chimpanzee (# 1) and crab-eating monkey (# 3). Immunoreactivity for the used markers was observed in neuronal cell bodies and processes distributed throughout the anterior-posterior extent of human, chimpanzee and macaque claustrum. Both CR- and PV-immunoreactive (ir) neurons were mostly localized in the central and ventral region of the claustrum of the three species while SOM- and NPY-ir neurons seemed to be equally distributed throughout the ventral-dorsal extent. In the chimpanzee claustrum SOM-ir elements were not observed. No co-localization of PV with CR was found, thus suggesting the existence of two non-overlapping populations of PV and CR-ir interneurons. The expression of most proteins (CR, PV, NPY), was similar in all species. The only exception was the absence of SOM-ir elements in the claustrum of the chimpanzee, likely due to species specific variability. Our data suggest a possible common structural organization shared with the adjacent insular region, a further element that emphasizes a possible common ontogeny of the claustrum and the neocortex.

Characterization and Mapping of Melatonin Receptors in the Brain of Three Mammalian Species: Rabbit, Horse and Sheep

Melatonin receptors were characterized in the brains of three mammals (rabbit, horse and sheep) by an in vitro binding technique, using 2-[125I]iodomelatonin as labelled ligand. Although binding sites for melatonin have been described recently in several vertebrate species (including the sheep), the rabbit and the horse have not been the subject of investigation so far. Apart from characterization, the present report describes receptor distribution in a number of brain regions, thus allowing for direct interspecies comparison under the same methodological conditions. 2-[125I]iodomelatonin labelled high-affinity binding sites in crude membrane preparations from these species. A series of kinetic and saturation experiments revealed that the binding was rapid, stable, saturable, reversible, of high affinity (Kd in the low picomolar range) and low capacity (Bmax between 1 and 20 fmol/mg protein). The competition studies showed that the relative order of potency of a variety of indoles for inhibition of 2-[125I]iodomelatonin binding was as follows: 2-iodomelatonin greater than 6-chloromelatonin greater than melatonin much much greater than 5-methoxytryptophol greater than 5-methoxytryptamine, and that it was similar in the different brain regions. Prazosin, which has been reported as an extremely potent melatonin analog in the hamster brain, possessed no potency in all preparations from different regions in the three species under investigation. The regional distribution of the receptor showed insignificant species differences. Highest density was always recorded in the median eminence/pars tuberalis (ME/PT) area. Other regions (SCN, POA and certain cortical areas), showed lower, but significant, receptor content. Saturation and competition studies revealed that these binding sites were also of high affinity, low capacity and high specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

The serotoninergic system in the brain of the Japanese quail

SummaryThe presence and topographical localization of the serotoninergic system in the brain of the Japanese quail (Coturnix coturnix japonica) have been studied by means of peroxidase-anti-peroxidase immunocytochemistry. The perimeter, diameter, area, and shape factor of immunoreactive cells have been recorded and analyzed morphometrically for intra- and interspecies comparison. The data reported here confirm and extend results previously obtained in the brain of other avian species. Serotonin-immunoreactive neurons of the quail are mainly located in the hypothalamic paraventricular organ and adjacent areas, and in the brainstem where they form three separate groups. The first of these groups consists of small-sized neurons located in the ventro-rostral mesencephalon. The second group is composed of medium-sized neurons located in the dorsal mesencephalo-pontine region. The third group is also formed by medium-sized neurons, and is located ventrally in the ponto-medullary region. In the quail brain, serotoninergic neurons are not restricted to nuclei located in the vicinity of the midsagittal plane, but show some lateralization, especially in the brainstem. The organization of the different groups of immunoreactive neurons based on this topographical distribution and morphometric analysis has been compared with descriptions of the serotoninergic system in other birds. Serotonin-immunoreactive nerve fibers are widely distributed throughout the brain, but appear to be particularly abundant in regions involved in the control of reproductive activities, such as the septal region, the medial preoptic nucleus, the nucleus intercollicularis, and the external zone of the median eminence. The data reported here have allowed the drawing of a map of serotoninimmunoreactive structure.

The chemical neuroanatomy of the mammalian pineal gland: Neuropeptides

The mammalian pineal gland contains multiple afferent peptidergic nerve fibres. Sympathetic nerve fibres, with their origin in the superior cervical ganglia, contain neuropeptide Y colocalized with norepinephrine. Other pinealopetal nerve fibres, probably originating in the pterygopalatine ganglion, contain vasoactive intestinal peptide and peptide histidine isoleucine. Fibres containing substance P and calcitonin gene-related peptide have also been demonstrated in pinealopetal nerve fibres. These fibres might originate in the trigeminal ganglion. The neurotransmitter content of the fibres of the central innervation, innervating the gland from the brain via the pineal stalk, has not been elucidated. However, strong indications for the presence of neuropeptide Y, substance P, somatostatin, and vasopressin in these fibres have been presented. Recent immunohistochemical studies have further shown the presence of subtypes of pinealocytes containing neuropeptides. Thus, pinealocytes containing beta-endorphin, leu-enkephalin, and somatostatin have been demonstrated in the gland. Immunohistochemistry at the electron microscopical level has shown, that in some species, leu-enkephalin containing pinealocytes make synaptic contacts with other pinealocytes indicating of paracrine regulation of the pineal gland. It must however be emphasized that large interspecies variations exist with regard to the peptidergic pineal innervation and its content of peptidergic cells.

Distribution and characterization of melatonin receptors in the brain of the Japanese quail, Coturnix japonica

2-[125I]iodomelatonin was used to study the distribution and properties of the melatonin receptor in the Japanese quail brain. High receptor density was detected in the major targets of direct retinal input (optic tectum, nucleus of the optic basal rout, ventrolateral geniculate nucleus), as well as areas representing terminals in the visual pathways (nucleus rotundus, ectostriatum, thalamo-hyperstriatal pathway). Binding was also found in the piriform cortex, the hypophyseal pars tuberalis, the oculomotorius nucleus and the associated Edinger-Westphal nucleus, and in the nuclei of the third, fourth and sixth cranial nerves. A comparison of the receptor pharmacological profile to that of the mammalian brain demonstrated pharmacological identity of the two binding sites. In the saturation experiments, GPT gamma S decreased the binding affinity, numerical Kd values increasing from approximately 35 pM to approximately 150 pM.

Age dependent changes in plasma anti-Müllerian hormone concentrations in the bovine male, female, and freemartin from birth to puberty: relationship between testosterone production and influence on sex differentiation.

To understand the behaviour of the gonads, in terms of hormonal secretion, in a model of intersexual development naturally occurring in mammals, we determined plasma concentrations of testosterone, progesterone, and anti-Müllerian hormone (AMH) in bovine freemartins, and compared them to normal levels measured in males and females from birth to puberty. We found that newborn males and freemartins have very high concentrations of AMH (over 700ng/ml). Conversely, plasma AMH concentration is always below 120ng/ml in females. While values remain stable in males for the first five months of life, they sharply decrease in the freemartins within the first fortnight, and reach female levels, which demonstrates that AMH is essentially originated in the male twin. In young bulls the trend of plasma testosterone concentrations is opposite to that of the AMH. The rise in testosterone production at puberty corresponds to a sharp decline in AMH concentrations. Bovine plasma concentrations of AMH are surprisingly higher than those measured in other mammals, including man and mouse. The results obtained are discussed in reference to comparative aspects of endocrine functions.

Accumulation of copper and other metal ions, and metallothionein I/II expression in the bovine brain as a function of aging

Accumulation of metal ions in the brain contributes to heighten oxidative stress and neuronal damage as evidenced in aging and neurodegenerative diseases, both in humans and in animals. In the present paper we report the analysis of Cu, Zn and Mn in the brain of two series of respectively young (8-16 months) and adult (9-12 years) bovines. Our data indicate that the concentrations of Cu varied of one order of magnitude between 1.67 and 15.7microg/g wet tissue; the levels of Zn varied between 6.13 and 17.07microg/g wet tissue and the values of Mn resulted between 0.19 and 1.24microg/g wet tissue. We found relevant age-dependent differences in the distribution of Cu and Zn, whose concentrations were markedly higher in older animals. By contrast, Mn seemed to redistribute in the different cerebral areas rather than drastically change with age. Tissues from bovine brain were also analysed immunohistochemically for the presence and distribution of metallothionein I/II and also for the expression of glial fibrillary acidic protein. Metallothionein I/II immunoreactive elements included ependymal cells lining the lateral ventricles and neural cells in middle layer of the cerebellar cortex. No age differences were evident between calves and adult. The presence of liquor-contacting metallothionein I/II in cells confirms that their functions in the central nervous system are not yet completely established.

Localization and characterization of melatonin binding sites in the brain of the rabbit (Oryctolagus cuniculus) by autoradiography and in vitro ligand-receptor binding

The distribution and the properties of the melatonin binding sites were characterized in the brain of the rabbit by combined use of autoradiography and in vitro ligand-receptor binding. Autoradiography revealed widespread specific binding in the brain. The pars tuberalis of the pituitary gland, suprachiasmatic nuclei, ventromedial hypothalamic nuclei, tapetum, hippocampus, indusium griseum, cingulate gyrus, cortex and the choroid plexus were intensely labelled. Diffuse specific binding was recorded in the olfactory bulb and the anterior hypothalamus. Series of in vitro ligand-receptor binding experiments, using the anterior hypothalamus, confirmed that the binding was of high affinity and specificity. Coincubation with a non-hydrolyzable GTP analogue provoked a shift in the binding affinity, the numerical values of the Kd increasing from 20-30 pM to 280-300 pM. Apparently the melatonin receptor in the rabbit brain is linked to its second messenger via a G protein, similarly to what has been described for the brain of other vertebrates.

Demonstration of an orexinergic central innervation of the pineal gland of the pig.

Orexins/hypocretins, two isoforms of the same prepropeptide, are widely distributed throughout the brain and are involved in several physiological and neuroendocrine regulatory patterns, mostly related to feeding, sleep, arousal, and cyclic sleep‐wake behaviors. Orexin‐A and orexin‐B bind with different affinities to two G‐protein‐coupled transmembrane receptors, orexin‐1 and orexin‐2 receptors (OR‐R1 and OR‐R2, respectively). Because of the similarities between the human and the swine brain, we have studied the pig to investigate the orexinergic system in the diencephalon, with special emphasis on the neuroanatomical projections to the epithalamic region. By using antibodies against orexin‐A and orexin‐B, immunoreactive large multipolar perikarya were detected in the hypothalamic periventricular and perifornical areas at the light and electron microscopic levels. In the region of the paraventricular nucleus, the orexinergic neurons extended all the way to the lateral hypothalamic area. Immunoreactive nerve fibers, often endowed with large varicosities, were found throughout the hypothalamus and the epithalamus. Some periventricular immunoreactive nerve fibers entered the epithalamic region and continued into the pineal stalk and parenchyma to disperse among the pinealocytes. Immunoelectron microscopy confirmed the presence of orexinergic nerve fibers in the pig pineal gland. After extraction of total mRNA from the hypothalamus and pineal gland, we performed RT‐PCR and nested PCR using primers specific for porcine orexin receptors. PCR products were sequenced, verifying the presence of both OR‐R1 and OR‐R2 in the tissues investigated. These findings, supported by previous studies on rodents, suggest a hypothalamic regulation of the pineal gland via central orexinergic nervous inputs. J. Comp. Neurol. 471:113–127, 2004.

The NADPH-diaphorase-containing system in the brain of the budgerigar (Melopsittacus undulatus)

Abstract.In the present investigation we studied the presence and distribution of histochemically detected neuronal NADPH-diaphorase (ND) in the brain of the budgerigar, Melopsittacus undulatus. Positive neurons are widely distributed throughout the central nervous system. ND-containing neurons are present in the telencephalon and the paleostriatal-parolfactory lobe complex. Positive cells were observed also in the neostriatum, including the main auditory area (field L), in several nuclei of the archistriatum and in the hyperstriatum (accessory, dorsal, and ventral). In the diencephalon, positive neurons were present both in the lateral hypothalamic and periventricular areas, and in a segregate area at the confluence of the anterior commissure and the lateral prosencephalic bundle. A group of positive perikarya was located lateral to the dorsal part of the IIIrd ventricle, and continued laterally into the thalamus. Weakly stained neurons were observed in the thalamic dorsomedial posterior nucleus. In the mesencephalon, ND-containing neurons were scattered in the reticular formation (pars lateralis and pars medialis) and in the optic tecta. A large population of positive neurons was observed in the substantia nigra, the ventral area of Tsai and the nucleus interpeduncularis. Positive neurons extended through the tegmental nuclei to the locus coeruleus. In the cerebellum, the granular neurons were weakly stained and the internal cerebellar nuclei were surrounded by a wide network of positive fibers. In the medulla the number of positive cells was highly reduced, but stained neurons were observed in the cochlear as well in the vestibular nuclei. The data here presented suggest that the distribution of ND-containing neurons in the brain of the budgerigar is different from those of the chicken and quail. The locations of positive neurons suggest also a possible involvement in sound perception and production pathways, and visual perception.