Cristina Ballarin

The metabolism and imaging in live cells of the bovine prion protein in its native form or carrying single amino acid substitutions.

Prion diseases are probably caused by an abnormal form of a cellular glycoprotein, the prion protein. Recent evidence suggests that the prion strain causing BSE has been transmitted to humans, thereby provoking a variant form of Creutzfeldt-Jacob disease. In this work, we analyzed the behavior of normal and malformed isoforms of the bovine PrP in transfected mammalian cell lines. Biochemical and immunocytochemical assays were complimented with imaging of live cells expressing fusion constructs between PrP and GFP. Bovine homologues of human E200K and D178N (129M) mutations were used as models of pathogenic isoforms. We show that the GFP does not impair the metabolism of native and mutant bPrPs and is thus a valid marker of PrP cellular distribution. We also show that each amino acid replacement provokes alterations in the cell sorting and processing of bPrP. These are different from those ascribed to both murine mutant homologues. However, human and bovine PrPs carrying the D178N genotype had similar cellular behavior.

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.

Reconstitution of the native mitochondrial outer membrane in planar bilayers. Comparison with the outer membrane in a patch pipette and effect of aluminum compounds

SummaryDetergent-free rat brain outer mitochondrial membranes were incorporated in planar lipid bilayers in the presence of an osmotic gradient, and studied at high (1 m KCl) and low (150 mm KCl) ionic strength solutions. By comparison, the main outer mitochondrial membrane protein, VDAC, extracted from rat liver with Triton X-100, was also studied in 150 mm KCl. In 1 m KCl, brain outer membranes gave rise to electrical patterns which resembled very closely those widely described for detergent-extracted VDAC, with transitions to several subconducting states upon increase of the potential difference, and sensitivity to polyanion. The potential dependence of the conductance of the outer membrane, however, was steeper and the extent of closure higher than that observed previously for rat brain VDAC. In 150 mm KCl, bilayers containing only one channel had a conductance of 700 ± 23 pS for rat brain outer membranes, and 890 ± 29 pS for rat liver VDAC. Use of a fast time resolution setup allowed demonstration of open-close transitions in the millisecond range, which were independent of the salt concentration and of the protein origin. We also found that a potential difference higher than approx. ± 60 mV induced an almost irreversible decrease of the single channel conductance to few percentages of the full open state and a change in the ionic selectivity. These results show that the behavior of the outer mitochondrial membrane in planar bilayers is close to that detected with the patch clamp (Moran et al., 1992, Eur. Biophys. J.20:311–319).The neurotoxicological action of aluminum was studied in single outer membrane channels from rat brain mitochondria. We found that μm concentrations of Al Cl3 and aluminum lactate decreased the conductance by about 50%, when the applied potential difference was positive relative to the side of the metal addition.

Ionic permeability of the mitochondrial outer membrane

The ionic permeability of the outer mitochondrial membrane (OMM) was studied with the patch clamp technique. Electrical recording of intact mitochondria (hence of the outer membrane (OM)), derived from mouse liver, showed the presence of currents corresponding to low conductances (< 50 pS), as well as of four distinct conductances of 99 pS,152 pS, 220 pS and 307 pS (in 150 mM KCl). The latter were voltage gated, being open preferentially at positive (pipette) potentials. Very similar currents were found by patch clamping liposomes containing the isolated OM derived from rat brain mitochondria. Here a conductance of approximately 530 pS, resembling in its electrical characteristics a conductance already attributed to mitochondrial contact sites (Moran et al. 1990), was also detected. Immunoblot assays of mitochondria and of the isolated OM with antibodies against the outer membrane voltage-dependent anion channel (VDAC) (Colombini 1979), showed the presence of the anion channel in each case. However, the typical electrical behaviour displayed by such a channel in planar bilayers could not be detected under our experimental conditions. From this study, the permeability of the OMM appears different from what has been reported hitherto, yet is more in line with that multifarious and dynamic structure which apparently should belong to it, at least within the framework of mitochondrial biogenesis (Pfanner and Neupert 1990).

The brain of the horse: weight and cephalization quotients

The horse is a common domestic animal whose anatomy has been studied since the XVI century. However, a modern neuroanatomy of this species does not exist and most of the data utilized in textbooks and reviews derive from single specimens or relatively old literature. Here, we report information on the brain of Equus caballus obtained by sampling 131 horses, including brain weight (as a whole and subdivided into its constituents), encephalization quotient (EQ), and cerebellar quotient (CQ), and comparisons with what is known about other relevant species. The mean weight of the fresh brains in our experimental series was 598.63 g (SEM ± 7.65), with a mean body weight of 514.12 kg (SEM ± 15.42). The EQ was 0.78 and the CQ was 0.841. The data we obtained indicate that the horse possesses a large, convoluted brain, with a weight similar to that of other hoofed species of like mass. However, the shape of the brain, the noteworthy folding of the neocortex, and the peculiar longitudinal distribution of the gyri suggest an evolutionary specificity at least partially separate from that of the Cetartiodactyla (even-toed mammals and cetaceans) with whom Perissodactyla (odd-toed mammals) are often grouped.

The claustrum of the bottlenose dolphin Tursiops truncatus (Montagu 1821)

The mammalian claustrum is involved in processing sensory information from the environment. The claustrum is reciprocally connected to the visual cortex and these projections, at least in carnivores, display a clear retinotopic distribution. The visual cortex of dolphins occupies a position strikingly different from that of land mammals. Whether the reshaping of the functional areas of the cortex of cetaceans involves also modifications of the claustral projections remains hitherto unanswered. The present topographic and immunohistochemical study is based on the brains of eight bottlenose dolphins and a wide array of antisera against: calcium-binding proteins (CBPs) parvalbumin (PV), calretinin (CR), and calbindin (CB); somatostatin (SOM); neuropeptide Y (NPY); and the potential claustral marker Gng2. Our observations confirmed the general topography of the mammalian claustrum also in the bottlenose dolphin, although (a) the reduction of the piriform lobe modifies the ventral relationships of the claustrum with the cortex, and (b) the rotation of the telencephalon along the transverse axis, accompanied by the reduction of the antero-posterior length of the brain, apparently moves the claustrum more rostrally. We observed a strong presence of CR-immunoreactive (-ir) neurons and fibers, a diffuse but weak expression of CB-ir elements and virtually no PV immunostaining. This latter finding agrees with studies that report that PV-ir elements are rare in the visual cortex of the same species. NPY- and somatostatin-containing neurons were evident, while the potential claustral markers Gng2 was not identified in the sections, but no explanation for its absence is currently available. Although no data are available on the projections to and from the claustrum in cetaceans, our results suggest that its neurochemical organization is compatible with the presence of noteworthy cortical inputs and outputs and a persistent role in the general processing of the relative information.

Primary cell cultures from fetal bovine hypothalamus and cerebral cortex: A reliable model to study P450Arom and α and β estrogen receptors in vitro

Estrogens synthesized by neural P450 aromatase (P450Arom) are implicated in many aspects of mammalian brain development and particularly in sexual differentiation of the central nervous system (CNS). This study analyzes the usefulness of an in vitro model based on bovine primary cell cultures from the hypothalamus and frontal cortex to investigate the role of P450Arom and estrogen receptors (ERs) in the development of fetal neural structures. The mRNA expression of P450Arom, ERalpha and ERbeta was detected using RT-PCR analysis in both hypothalamic and cortical primary cell cultures. P450Arom was identified and localized by immunocytochemistry in both neurons and astrocytes. Our results indicate that, within our experimental settings, astrocytes do not express ERalpha. The experimental model that we propose may represent a standardized dynamic model to study cellular and molecular mechanisms involved in the complex process of brain sexual differentiation.

Ontogenesis of brain aromatase P450 expression in the bovine hypothalamus

Aromatase P450 (P450(AROM)), converting testosterone (T) into estradiol (E), plays an important role in sexual differentiation of neural structures in the developing mammalian brain. The aim of the present study was to characterize the qualitative and quantitative profile of P450(AROM) mRNA expression in the bovine hypothalamus (the region of the central nervous system in which the enzyme is mainly localized) using RT-PCR and quantitative real-time RT-PCR analysis, respectively. P450(AROM) expression was examined in the developing hypothalamus in a series of experimental groups investigated at 10 weeks interval one from the other. Our data indicate that in the bovine fetal hypothalamus P450(AROM) expression peaks at the second quarter of gestation. The presence of neural cells containing P450(AROM) in the bovine fetal hypothalamus was confirmed by immunohistochemistry, and localized in the medial preoptic area. We conclude that second quarter of the gestation is the developmental stage which represents a critical period for hypothalamic differentiation in bovine ontogenesis, an important difference with the rat and mouse, short gestation species in which P450(AROM) activity peaks around delivery.

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 Brain of the Domestic Bos taurus: Weight, Encephalization and Cerebellar Quotients, and Comparison with Other Domestic and Wild Cetartiodactyla.

The domestic bovine Bos taurus is raised worldwide for meat and milk production, or even for field work. However the functional anatomy of its central nervous system has received limited attention and most of the reported data in textbooks and reviews are derived from single specimens or relatively old literature. Here we report information on the brain of Bos taurus obtained by sampling 158 individuals, 150 of which at local abattoirs and 8 in the dissecting room, these latter subsequently formalin-fixed. Using body weight and fresh brain weight we calculated the Encephalization Quotient (EQ), and Cerebellar Quotient (CQ). Formalin-fixed brains sampled in the necropsy room were used to calculate the absolute and relative weight of the major components of the brain. The data that we obtained indicate that the domestic bovine Bos taurus possesses a large, convoluted brain, with a slightly lower weight than expected for an animal of its mass. Comparisons with other terrestrial and marine members of the order Cetartiodactyla suggested close similarity with other species with the same feeding adaptations, and with representative baleen whales. On the other hand differences with fish-hunting toothed whales suggest separate evolutionary pathways in brain evolution. Comparison with the other large domestic herbivore Equus caballus (belonging to the order Perissodactyla) indicates that Bos taurus underwent heavier selection of bodily traits, which is also possibly reflected in a comparatively lower EQ than in the horse. The data analyzed suggest that the brain of domestic bovine is potentially interesting for comparative neuroscience studies and may represents an alternative model to investigate neurodegeneration processes.