bluuebunny/crossref_metadata_2025 huggingface dataset:
Active coatings for SiC particles to reduce the degradation by liquid aluminium during processing of aluminium matrix composites: study of interfacial reactions
Authors: Ureña A. ,   ESCALERA, Rodrigo P. , Baldonedo J. L. , Gil L.
Published: March 2003

The application of a surface coating on SiC particles is studied as an alternative means of solving problems of reactivity between SiC reinforcements and molten aluminium and problems of low wetting which limit the application of casting routes for fabrication of Al–SiCp composites. The selected active barrier was a ceramic composed of SiO2, which was generated by controlled oxidation of the SiC particles. The coating behaves as an active barrier, preventing a direct reaction between molten aluminium and SiC to form Al4C3 as the main degradation product. At the same time, the SiO2 provokes other interfacial reactions, which are responsible for an improvement in wetting behaviour.

Composites were prepared by mixing and compacting SiC particles with Al powders followed by melting in a vacuum furnace, and varying the residence time. Transmission electron microscopy (TEM), high resolution electron microscopy (HREM) and field emission TEM were employed as the main characterization techniques to study the interfacial reactions occurring between the barrier and the molten aluminium. These studies showed that the SiO2 coating behaves as an active barrier which reacts with the molten Al to form a glassy phase Al–Si–O. This compound underwent partial crystallization during the composite manufacture to form mullite. The formation of an outer crystalline layer, composed mainly of Al2O3, was also detected. Participation of other secondary interface reactions inside the active barrier was also identified by HREM techniques.
Control of the vibration damping through surface functionalization of a shear force probe
Authors: Vaccaro L. , Ulrich W.‐P. , Duschl C. , Marquis‐Weible F.
Published: March 2003

Shear force mapping on thiolipid Langmuir–Blodgett monolayers probed with Al‐coated tips leads to a contrast highly dependent on the monolayer molecular organization, which does not correspond to the topographical relief of the sample. The use of functionalized surface probes offers the possibility to better control the probe‐to‐sample interaction. In addition, hydrophilic surface probes are totally insensitive to alkyl chain arrangements in the monolayer. Hydrophobic probes, instead, can be chosen to map shear force on soft samples in liquid environment, since their mechanical properties are not influenced by the surrounding liquid.
Superluminal interactions in near‐field optics
Authors: Broe J. , Keller O.
Published: March 2003

The fact that photons emitted from an electric‐dipole active atom cannot be spatially localized better than to the near‐field zone of the atom is seen as the origin of genuine superluminality. By means of a simple model dipole current density the general theory is used to demonstrate numerically how superluminality enters the near‐field dynamics, and how from a measurement one could be tempted to believe that superluminal propagation effects occur. Furthermore, it is shown how for source‐detector distances larger than a pulse length one should be able to divide the pulse into two separate parts: one purely superluminal part arising solely in the non‐local generation process of the field, and another part seemingly propagating with superluminal speed. We comment on different velocity analyses, and we argue that the only fundamental velocity entering the problem is the vacuum velocity of light, which in a measurement would appear as the velocity of the trailing edge of the pulse.
In situ observation and interferometric characterization of solid–liquid interface morphology in directionally growing transparent model systems
Authors: Jamgotchian H. , Bergeon N. , Benielli D. , Voge P. , Billia B.
Published: March 2003

The performance of a new directional solidification device dedicated to the characterization of solid–liquid interface morphology by means of optical methods is presented in this paper. In contradiction to usual solidification studies on transparent materials carried out on thin films, which eliminates the complex coupling between solidification and convection, this device enables in situ and real time studies on bulk transparent materials. The alloy is contained in a cylindrical crucible and observation is performed in two perpendicular directions: the growth one and the transverse one. In addition to direct observation by light transmission in those directions, an interferometer is also set up in the growth direction to provide information on the shape and the motion of the interface through an analysis of the interferometer fringes. The combined determination of solidification front characteristics by these three observation modes has already given critical information on interface dynamics: front recoil measurements during initial transient, formation of microstructure patterns, and influence of convection on the triggering of instabilities.
A new approach for cryofixation by high‐pressure freezing
Authors: Studer D. , Graber W. , Al‐Amoudi A. , Eggli P.
Published: March 2003

A newly designed high‐pressure freezing machine for cryofixation was established and tested (Leica EMPACT), based on ideas originally proposed by Moor & Riehle in 1968. The new machine, essentially an improved version of our prototype, pressurizes the sample to 2000 bar in a small container (using methylcyclohexane as hydraulic fluid) and at the same time cools the outer surface of the container with a jet of liquid nitrogen. The advantage of this approach is that the machine uses little liquid nitrogen and can be built small and light. The machine is able to vitrify and freeze well a variety of specimens, for example, plant leaves, yeast cells, liver or nerve tissue (more samples are shown at: http://www.ana.unibe.ch/empact). Cooling efficiency is the same as in the traditional machines that use liquid nitrogen to pressurize and simultaneously cool the sample.
Quantitative fractography by digital image processing: NIH Image macro tools for stereo pair analysis and 3‐D reconstruction
Authors: Hein L. R. O.
Published: March 2003

A set of NIH Image macro programs was developed to make qualitative and quantitative analyses from digital stereo pictures produced by scanning electron microscopes. These tools were designed for image alignment, anaglyph representation, animation, reconstruction of true elevation surfaces, reconstruction of elevation profiles, true‐scale elevation mapping and, for the quantitative approach, surface area and roughness calculations. Limitations on time processing, scanning techniques and programming concepts are also discussed.
Thickness measurements of lacey carbon films
Authors: Karlsson G.
Published: March 2003

Lacey carbon filmed grids interact differently with liquid samples during plunging and subsequent studies in cryo‐TEM depending on type and manufacture. One important parameter is the thickness of the film, which is investigated here by ultrathin sectioning. The film thickness of the carbon‐Formvar type is found to be 100–200 nm, with the variation of the thickness occurring in the polymer layer. The pure carbon type is found to be 20–30 nm thick. Knowing the thickness makes it easier to choose the optimal type for each cryo‐TEM sample.
Artificially induced unusual shape of erythrocytes: an atomic force microscopy study
Authors: Girasole M. , Cricenti A. , Generosi R. , Congiu‐Castellano A. , Boumis G. , Amiconi G.
Published: March 2003

We used air operating atomic force microscopy (AFM) to study several morphological modifications of human erythrocytes, artificially produced by addition of exogenous agents including phospholipids, low ionic strength media and drugs. Most experiments were performed on unfixed samples to avoid treating red blood cells (RBCs) with chemical agents that can, in principle, induce morphological alteration.

After detailed quantitative AFM characterization, the artificially produced abnormally shaped RBCs were compared with cells that occur with high incidence in blood pathologies. This morphological approach suggests a new strategy to describe and understand the biochemical and/or mechanical modifications responsible for the underlying pathologically induced changes and prove AFM to be a suitable tool to study erythrocyte deformation.
Confocal imaging, visualization and 3‐D surface measurement of small mammalian teeth
Authors: Evans A. R. , Harper I. S. , Sanson G. D.
Published: March 2003

The difficulties traditionally faced by functional morphologists in representing and interpreting three‐dimensional objects can now be mostly overcome using available laser and computer imaging technologies. A practical method for three‐dimensional imaging of small mammalian teeth using confocal microscopy is reported. Moulding and casting of the teeth were first performed, followed by confocal fluorescence imaging. Accuracy and precision of the scanned structures were tested in morphometric studies by using a new technique to measure the noise in the scan of a three‐dimensional surface, and linear and angular dimensions of the scans were compared with measurements made using traditional morphological tools. It is shown that measurements can be taken with less than 4% difference from the original object. Teeth of the microchiropteran bat Chalinolobus gouldii were scanned and measured to show the potential of the techniques. Methods for visualizing the small teeth in three‐dimensional space, and animating the teeth in occlusion, show the power of this approach in aiding a three‐dimensional understanding of the structure and function of teeth and other three‐dimensional structures.
In‐situ transmission electron microscopy studies of polymer–carbon nanotube composite deformation
Authors: Qian D. , Dickey E. C.
Published: March 2003

This paper demonstrates the viability of in‐situ transmission electron microscopy for studying the deformation mechanisms of polymer nano‐composites. In‐situ straining studies are performed on carbon multiwalled nanotube (MWNT)–polystyrene composite films. The experiments show that load transfer across the nanotube–polystyrene interface is operative well into the plastic deformation regime of the composite film. The MWNTs are observed to bridge cracks propagating through the polystyrene, providing closure stresses across the crack wake. Although some MWNTs fracture by either a sword‐in‐sheath mechanism or transverse shear fracture, most of the MWNTs eventually debond at the MWNT–polymer interface and subsequently pull out of the matrix.
Electron energy loss near edge structure on the nitrogen K‐edge in vanadium nitrides
Authors: Hofer F. , Warbichler P. , Scott A. , Brydson R. , Galesic I. , Kolbesen B.
Published: March 2003

This paper presents electron energy‐loss near‐edge structure (ELNES) data for the N K edges of vanadium nitrides. By rapid thermal processing of vanadium layers in pure nitrogen at high temperatures the two known vanadium nitrides, VN and V2N, have been prepared. The phases have been checked by electron diffraction and quantitative electron energy‐loss spectroscopy (EELS) analysis. Because their crystallographical structures are different, they also exhibit different ELNES features, which can be used as fingerprints for rapidly distinguishing between VN and V2N. The experimental findings are supported by modelling the N K edge using a band structure approach (full linearized augmented plane wave method).
Topographic contrast of partially wetting water droplets in environmental scanning electron microscopy
Authors: Stelmashenko N. A. , Craven J. P. , Donald A. M. , Terentjev E. M. , Thiel B. L.
Published: March 2003

Partially wetting water droplets with sizes smaller than the capillary length acquire a distinct spherical cap shape controlled by the equilibrium contact angle, which is specific for different substrates and conditions. Images of such droplets in an environmental scanning electron microscope (ESEM) show strong topographic contrast. This contrast across the droplets can be analysed within a simple theoretical model, as the droplet sides are inclined smooth surfaces. Very small droplets have ESEM intensity profiles which deviate from this topographic model. Such deviations indicate that other sources of electron signal may be important for such droplets, and also demonstrate the limits of the analytical model. For droplets sufficiently large that they lie within the range of the topographic contrast model, values of contact angles on different substrates can be deduced. These are found to agree with independent direct measurements, as well as the results given in the literature. The possibilities of using this technique to analyse physical properties of different substrates are discussed.
Bias of a length density estimator based on vertical projections
Authors: Hlawiczková M. , Gokhale A. M. , Beneš V.
Published: March 2003

Attention is paid to the stereological estimator of the length density of lineal features in three‐dimensional space. In Gokhale (J. Microsc. (1990) 159, 133–141), the estimator based on measurements performed on a projection of the content of vertical slices with a given thickness Δ was derived. The aim of this paper is to show that using five vertical slices with systematically chosen orientations yields a reliable result, i.e. the bias of the estimator is smaller than 5%. In the case of a choice of the vertical axis such that most lineal features are not perpendicular or nearly perpendicular to the vertical axis, a reliable result can be obtained using only three systematic orientations of vertical slices.
Extracting twins from orientation imaging microscopy scan data
Authors: Wright S. I. , Larsen R. J.
Published: March 2003

Automated electron backscatter diffraction or orientation imaging microscopy (OIM) provides spatially specific measurements of crystallographic orientation. These measurements are typically collected on regular grids. By inspecting the misorientation between neighbouring measurements on the grid, potential twin boundaries can be identified. If the misorientation is within some given tolerance of a specified twin misorientation, the boundary separating the two measurements may be identified as a potential twin boundary. In addition, for a coherent twin, the twinning planes must be coincident with the grain boundary plane. As OIM scans are inherently two‐dimensional, the scan data provide only limited information on the boundary plane. Thus, it is not possible to ascertain definitively whether the twinning planes are coincident with the boundary plane. Nonetheless, the alignment of the surface traces of the twinning planes with the trace of the boundary provides a partial indication of coincidence. An automated approach has been developed that allows data concerning both twin criterion to be extracted from OIM scans. Application of the methodology to deformed zirconium suggests that the twinning planes remain coherent during deformation. The methodology was also used to improve grain size distributions measured by OIM. These results more closely match those obtained by conventional metallography.
Application of electron backscatter diffraction (EBSD) to fracture studies of ferritic steels
Authors: Davies P. A. , Novovic M. , Randle V. , Bowen P.
Published: March 2003

The application of electron backscatter diffraction (EBSD) to fracture studies has provided a new method for investigating the crystallography of fracture surfaces. The crystallographic indices of cleavage planes can be measured both directly from the fracture surface and indirectly from metallographic sections perpendicular to the plane of the adjoining fracture surfaces. The results of direct individual cleavage facet plane orientation measurements are presented for carbon–manganese (C–Mn) and low‐alloy Mn–Mo–Ni (similar to ASTM A553 type‐B). Pressure vessel steel weld metals, obtained from fracture surfaces of Charpy impact test specimens fractured at various test temperatures and for an ultra‐low carbon steel (Fe–0.002C–0.058P) fractured at –196 °C by impact. In addition to the direct measurement from the fracture surface, cleavage facet orientation measurements for the ultra‐low carbon steel were complemented by the results obtained from the metallographic sections. Fractographic observations revealed that cleavage fracture is accommodated by a microvoid coalescence fracture micromechanism, which was induced by decohesion of second phase particles (inclusions). The correlation between the direct and indirect methodologies shows that the cleavage facet planes are dominated by the {001} plane orientations, and indicated that even when information concerning the full five degrees of freedom is inaccessible, the cleavage facet plane could still be determined. Finally, the advantages and disadvantages of direct orientation measurements from the fracture surface and indirectly by a destructive sectioning technique are discussed.
The smooth fractionator
Authors: Gundersen H. J. G.
Published: March 2003

A modification of the general fractionator sampling technique called the smooth fractionator is presented. It may be used in almost every situation in which sampling is performed from distinct items that are uniquely defined, often they are physically separated items or clusters like pieces, blocks, slabs, sections, etc. To each item is associated a ‘guesstimate’ or an associated variable with a more‐or‐less close – and possibly biased − relationship to the content of the item. The smooth fractionator is systematic sampling among the items arranged according to the guesstimates in a unique, symmetric sequence with one peak and minimal jumps. The smooth fractionator is both very simple to implement and so efficient that it should probably always be used unless the natural sequence of the sampling items is equally smooth.

So far, there is no theory for the prediction of the efficiency of smooth fractionator designs in general, and their properties are therefore illustrated with a range of real and simulated examples. At the cost of a slightly more elaborate sampling scheme, it is, however, always possible to obtain an unbiased estimate of the real precision and of some of the variance components.

The only real practical problem for always obtaining a high precision with the smooth fractionator is specimen inhomogeneity, but that is detectable at almost no extra cost.

With careful designs and for sample sizes of about 10, the sampling variation for the primary, smooth fractionator sampling step may in practice often be small enough to be ignored.
The Nonpeptide Growth Hormone Secretagogue, MK‐0677, Activates Hypothalamic Arcuate Nucleus Neurons in vivo
Authors: Bailey  , Smith  , Leng
Published: March 2003

There is accumulating evidence that the hypothalamic arcuate nucleus plays an important role in mediating the effects of growth hormone secretagogues on growth hormone (GH) release from the anterior pituitary gland. One such nonpeptidyl secretagogue, MK‐0677, has been shown to directly stimulate growth hormone release from isolated pituitary cells but its central actions remain to be established. Therefore, in the present study, we have employed both immunocytochemical and in vivo electrophysiological techniques to examine the effects of MK‐0677 within the hypothalamic arcuate nucleus of the male rat. In conscious male rats, both central and systemic injection of MK‐0677 induced fos‐like immunoreactivity specifically within the arcuate nucleus indicating selective neuronal activation of neurons within this region. MK‐0677 induced‐activation was generally confined close to the wall of the third ventricle, whereas systemic injection of the peptide secretagogue, GHRP‐6, also induced fos‐like immunoreactivity in more lateral regions of the nucleus. In urethane anaesthetized rats, intravenous injection of MK‐0677 increased the electrical activity of a population of antidromically identified (i.e. neuroendocrine) arcuate neurons with a similar electrophysiological profile to cells excited by GHRP‐6. The activity of neuroendocrine arcuate neurons excited by MK‐0677 injection could be attenuated by a subsequent systemic injection of somatostatin. However, the activity of neuroendocrine arcuate neurons unaffected by MK‐0677 injection and the activity of non‐neuroendocrine arcuate neurons was unaltered by somatostatin injection. Taken together, the immunocytochemical and electrophysiological results suggest that systemic and central administration of MK‐0677 activates a population of neurons in the arcuate nucleus. Furthermore, the inhibitory effects of somatostatin on MK‐0677‐induced excitation of these neuroendocrine cells is consistent with an action of neurons involved in the regulation of GH release.
Kainate Glutamate Receptors (GluR5–7) in the Rat Arcuate Nucleus: Relationship to Tanycytes, Astrocytes, Neurons and Gonadal Steroid Receptors
Authors: Diano Sabrina , Naftolin Frederick , Horvath Tamas L.
Published: March 2003

Glutamate action, through its ionotropic, kainate receptors, has been implicated in gonadal steroid‐dependent mechanisms of the arcuate nucleus. The objective of the present study was to determine the expression of kainate glutamate receptors in neural and glial elements of this area and their potential relationship to gonadal steroid receptors. Single and double label, light and electron microscopic immunocytochemistry for kainate glutamate receptors and estrogen or androgen receptors revealed the existence of glutamate (GluR) 5–7 kainate receptors in tanycytes, astrocytes and neurons of the arcuate nucleus. In the arcuate nucleus, subsets of GluR5–7‐containing neurons were also immunopositive for estrogen (20%) and/or androgen receptors (23%). Glial elements, however, lacked labeling for gonadal steroid receptors. The coexistence of gonadal and kainate receptors in the same perikarya of arcuate nucleus cell populations suggests hormone regulation of excitatory neurotransmission through ionotropic glutamate receptors in these regions. It is also indicated that a kainate receptor‐mediated glutamate action may participate in neuro–glial interaction in the arcuate nucleus that, in turn, may underlie the morphological synaptic plasticity induced by gonadal steroids.
Indomethacin Attenuates Oxytocin and Hypothalamic‐Pituitary‐Adrenal Axis Responses to Systemic Interleukin‐1β
Authors: Buller  , Xu  , Day
Published: March 2003

Systemic administration of the cytokine IL‐1β produces a significant release of ACTH into the plasma and activation of hypothalamic oxytocin (OT) and corticotropin releasing factor (CRF) cells. However, the mechanism(s) by which systemic IL‐1β induces these responses is not clear. In the present study, we have investigated the proposal that catecholamine cells of the ventrolateral medulla (VLM) and nucleus of the solitary tract (NTS) can relay circulating IL‐1 signals via a prostaglandin‐dependent mechanism to effect the HPA axis responses in the rat. Intra‐arterial administration of IL‐1β (1 pg/kg) to otherwise untreated animals produced a prominent release of ACTH into the plasma, substantial c‐fos expression in paraventricular medial parvocellular (mPVN) corticotropin releasing factor (CRF) cells, supraoptic (SON) and paraventricular nucleus (PVN) OT cells, area postrema cells, NTS and VLM catecholamine cells and cells of the central amygdala. Pretreatment with the prostaglandin synthesis inhibitor, indomethacin (10 mg/kg body weight ia) 15 min before IL‐1β administration (1 pg/kg ia) significantly reduced plasma ACTH release and c‐fos expression in PVN and SON OT cells and MPVN CRF cells. In addition, the area postrema, A1 and C1 catecholamine cell groups of the VLM and A2 and C2 catecholamine cell groups of the NTS, all exhibited concomitant reductions in c‐fos expression. Conversely indomethacin administration did not alter the IL1β‐induced expression of c‐fos in the central amygdala. These data suggest that central pathways involved in the IL‐1β‐induced activation of the HPA axis and OT cells are, at least in part, dependent upon prostaglandin synthesis. It is proposed that neurons in the area postrema, NTS and VLM might mediate this IL‐1β‐induced activation of hypothalamic CRF and OT cells and release of ACTH into the plasma.
Changes in NADPH‐d Staining in the Paraventricular and Supraoptic Nuclei During Pregnancy and Lactation in Rats: Role of Ovarian Steroids and Oxytocin
Authors: Popeski  , Amir  , Woodside
Published: March 2003

Staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH‐d), a histochemical marker for nitric oxide synthase (NOS), is increased in the supraoptic (SON) and paraventricular (PVN) nuclei in late pregnant rats. To determine whether increases in staining were evident at other times during pregnancy and lactation the number of cells that stained for NADPH‐d in the SON and PVN in rats on days 4, 12, 16, and 22 of pregnancy and on days 4, 12, and 20 of lactation was compared to that in virgin females. In a second experiment the influence of ovarian hormones on NADPH‐d staining was assessed by comparing staining in the SON and PVN among ovariectomized animals exposed to either a steroid hormone replacement schedule that mimics late pregnancy (oestrogen and progesterone with progesterone removal), oestrogen alone, oestrogen and progesterone, or cholesterol alone. In the last experiment of this series staining was compared among ovariectomized animals given either oestrogen or cholesterol priming accompanied by oxytocin (OT) or vehicle infusion into the third ventricle for 7 days. The number of cells showing dense staining for NADPH‐d in both the SON and PVN increased on days 12 and 22 of pregnancy and 4 and 12 of lactation compared to that observed in virgins. NADPH‐d staining in these areas was also increased by both the steroid treatment that mimicked late pregnancy and chronic central OT infusion in oestrogen‐primed animals. These data suggest that NADPH‐d staining in the SON and PVN is increased at times when oxytocinergic cells are known to be active and that the hormonal state associated with late pregnancy is sufficient to increase NADPH‐d staining.
Rapid and Sensitive Vasopressin Heteronuclear RNA Responses to Changes in Plasma Osmolality
Authors: Arima  , Kondo  , Kakiya  , Nagasaki  , Yokoi  , Yambe  , Murase  , Iwasaki  , Oiso
Published: March 2003

The plasma concentration of arginine vasopressin (AVP) is closely regulated by plasma osmolality. In this study, we used intronic in‐situ hybridization to investigate the transcriptional activity of the AVP gene in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) after intraperitoneal (i.p.) injection of hypertonic saline inducing small changes in plasma osmolality in rats. After ip injection of 900 mOsm/kg saline (2% BW), plasma [Na] reached the highest level at 10 min (900 mOsm/kg, 146±1 mEq/l; 290 mOsm/kg, 139±1 mEq/l, P<0.01) and maintained that level until 30 min. The expression of AVP heteronuclear (hn) RNA in the SON and PVN increased significantly as early as 10 min and peaked 30 min after ip injection of the hypertonic saline. AVP hnRNA expression showed a significant increase even after a 2 mEq/L elevation in plasma [Na] concentration, and increased dose‐dependently in response to the concentration of saline injection (290–900 mOsm/kg). These results demonstrate that, similar to AVP secretion, AVP gene transcriptional activity in the SON and PVN is closely regulated by plasma osmolality.
Endogenous α‐MSH modulates the Hypothalamic‐Pituitary‐Adrenal Response to the Cytokine Interleukin‐1β
Authors: Papadopoulos  , Wardlaw
Published: March 2003

We and others have previously shown that exogenous α‐MSH antagonizes the stimulatory effects of the cytokine interleukin (IL)‐1 on the hypothalamic‐pituitary‐adrenal (HPA) axis. It is currently unknown, however, if endogenous α‐MSH plays a physiological role in regulating the HPA response to IL‐1. We have therefore examined the HPA response to IL‐1β in rats pretreated with an affinity purified α‐MSH antiserum (AS) infused intracerebroventricularly to neutralize endogenous α‐MSH within the brain. α‐MSH AS or a similarly purified fraction of normal rabbit serum (NRS) was injected intracerebroventricularly at 16 h and at 1 h prior to the i.c.v. injection of IL‐1β (2 ng or 20 ng) and blood samples were collected through an indwelling atrial catheter. After 2 ng IL‐1β, the adrenocorticotropic hormone (ACTH) response was significantly greater in the α‐MSH AS treated rats (n=7) compared to the NRS treated rats (n=7) (P<0.01); the mean ACTH level rose to a peak of 594±208 pg/ml in the α‐MSH AS treated rats vs 274±122 pg/ml in the NRS treated rats. The area under the ACTH response curve in the α‐MSH AS treated animals was 181% of that in the NRS treated animals (P<0.05). A significant effect of α‐MSH AS on the corticosterone response to i.c.v. IL‐1β was also noted during the first 3 h of the study (P<0.05). The mean area under the corticosterone response curve for the first 3 h in the α‐MSH AS treated animals was 144% of that in the NRS treated animals (P<0.05). After 20 ng IL‐1β, the ACTH response over time was again significantly greater in the α‐MSH AS treated rats (n=8) compared to the NRS treated rats (n=9) (P<0.02); the mean ACTH level rose to a peak of 673±190 pg/ml after α‐MSH AS vs 490±115 pg/ml after NRS. Corticosterone levels rose to a peak of 42±3.9 μg/dl in the α‐MSH AS treated rats vs 37±4.6 μg/dl in the NRS treated rats; this difference was not significant. We conclude that the IL‐1β induced stimulation of ACTH is significantly enhanced by antagonizing the activity of α‐MSH. These results support a physiological role for endogenous α‐MSH in limiting the HPA response to this inflammatory cytokine.
Localization of Vasopressin (V1a) Receptor Binding and mRNA in the Rhesus Monkey Brain
Authors: Young  , Toloczko  , Insel
Published: March 2003

Centrally released arginine vasopressin (AVP) has been associated with various behavioural and cognitive effects, such as scent marking, aggression, and memory, which are believed to be mediated by the V1a subtype of the vasopressin receptor. Although the distribution of V1a receptors is conserved in a few brain regions, the pattern of expression of this receptor is, in general, highly species‐specific. We have used receptor autoradiography with the linear V1a receptor ligand (125I‐Phenylacetyl‐D‐Tyr(Me)‐Phe‐Gln‐Asn‐Arg‐Pro‐Arg‐Tyr‐NH2) to characterize the pattern of receptor binding in the rhesus monkey brain. Brain sites of V1a receptor synthesis were defined using in‐situ hybridization. The regions of highest V1a receptor density included the prefrontal, cingulate, pyriform, and entorhinal cortex, as well as the presubiculum and mamillary bodies. In addition, V1a receptor binding and mRNA were detected in several regions reported to have V1a receptor in most rodents, including the amygdala, bed nucleus of the stria terminalis, lateral septum, hypothalamus and the brainstem. The distribution is consistent with a role for vasopressin in higher cognitive functions, especially memory, in primates.
Effects of Sexual Interactions with a Male on Fos‐Like Immunoreactivity in the Female Quail Brain
Authors: Meddle  , Foidart  , Wingfield  , Ramenofskyand  , Balthazart
Published: March 2003

Sexual interactions can cause changes in plasma hormone levels and activate immediate early genes within the mammalian brain. There are marked anatomical differences between the regions activated that relate directly to the sexual specific behaviour and neuroendocrinology of each sex. The aim of this study was to determine if such a sexual dimorphism exists in birds by examining the brain regions stimulated in adult virgin female Japanese quail (Coturnix japonica) during sexual behaviour and comparing this to previously reported data concerning males. Female quail were allowed to freely interact with adult males and both female and male sexual behaviour was recorded. Contrary to previous findings in male quail, no significant induction of Fos‐like immunoreactive (FLI) cells was observed following sexual interactions in the preoptic area of females; this area is fundamentally involved in the control of male‐type copulatory behaviour. Sexual interactions significantly induced FLI cells in the hyperstriatum ventrale, the part of the archistriatum just lateral to the anterior commissure, and the nucleus intercollicularis. Moreover, prominent activation was detected throughout most of the ventromedial nucleus of the hypothalamus, a region reported to be rich in oestrogen receptors. FLI induction was not a consequence of sexual behaviour induced changes in luteinizing hormone (LH) as plasma LH levels were unaltered. Instead, brain activation must be a consequence of copulation‐associated somatosensory inputs or direct stimuli originating from the male. Male quail, like the majority of other birds, lack an intromittant organ (penis) so that the somatosensory inputs to the female are rather different from those in mammals; the precise nature of these inputs is yet to be determined.
Fos Localization in Cytosolic and Nuclear Compartments in Neurones of the Frog, Rana esculenta, Brain: An Analysis Carried Out in Parallel with GnRH Molecular Forms
Authors: Cobellis  , Vallarino  , Meccariello  , Pierantoni  , Masini  , Mathieu  , Pernas‐Alonso  , Chieffi  , Fasano
Published: March 2003

C‐fos activity was determined in the brain of the frog, Rana esculenta, during the annual sexual cycle. The localization of GnRH molecular forms (mammalian‐ and chicken‐GnRHII) was also carried out to determine whether or not the proto‐oncogene and the peptides showed a functional relationship. Northern blot analysis of total RNA revealed the presence of a single strong signal of c‐fos like mRNA of 1.9 Kb during February and April. This was followed by expression of c‐Fos protein (Fos) in several brain areas during March and July shown by immunocytochemistry. In particular, the olfactory region, the lateral and medial pallium, the nucleus lateralis septi, the ventral striatum, the caudal region of the anterior preoptic area, the suprachiasmatic nucleus, the ventral thalamus, tori semicircularis and ependymal layers of the tectum were immunostained. There was no overlap between Fos immunoreactive perikarya and GnRH immunoreactive perikarya (e.g. gonadotrophin‐releasing hormone (GnRH) in the rostral part and Fos in the caudal region of the anterior preoptic area). Interestingly, a cytoplasmic localization of Fos was also observed by immunocytochemistry and gel retardation experiments supported this observation. Cytoplasmic extracts from September–October animals bound the AP1 oligonucleotide. The complex was not available in the nuclear extracts from the same preparation, suggesting that, besides Fos, Jun products were also present. Conversely, nuclear but not cytosolic binding was detected in the brain of animals collected in July. In conclusion, we show that Fos and GnRH activity does not correlate in the frog brain and, for the first time in a vertebrate species, we give evidence of a cytoplasmic AP1 complex in neuronal cells.
Sex‐ and Age‐Specific Differences in Human Brain CYP11A1 mRNA Expression
Authors: Watzka  , Bidlingmaier  , Schramm  , Klingmüller  , Stoffel‐Wagner
Published: March 2003

While the presence of CYP11A1 (P450SCC, cholesterol side‐chain cleavage enzyme) has been well established in the brain of rodents, limited information is available on CYP11A1 expression in human brain. In both species, little is known regarding postnatal changes or sex specific differences in cerebral CYP11A1 expression. In the present study, we used a sensitive competitive reverse transcriptase polymerase chain reaction (RT‐PCR) assay to quantify the amount of CYP11A1 mRNA in a large number of human brain tissue specimens obtained at neurosurgery. CYP11A1 mRNA is expressed ≈200 times lower in the temporal lobe, frontal lobe and hippocampus than in adrenal tissue, known for high CYP11A1 mRNA expression. During childhood CYP11A1 mRNA concentrations in the temporal lobe increase markedly and reach adult levels at puberty. CYP11A1 mRNA is significantly higher in the temporal and frontal lobe cortex of women than in that of men. Our data demonstrate for the first time an age and sex dependent expression of CYP11A1 mRNA in the human brain.
GHRP‐6‐Induced Changes in Electrical Activity of Single Cells in the Arcuate, Ventromedial and periventricular nucleusriventricular Nuclei of a Hypothalamic Slice Preparation in Vitro
Authors: Hewson  , Viltart  , McKenzie  , Dyball  , Dickson
Published: March 2003

Previously, we demonstrated that systemic injection of the growth hormone secretagogue, growth hormone‐releasing peptide (GHRP)‐6, selectively activated cells in the hypothalamic arcuate nucleus, as reflected by increased electrical activity and induction of the immediate early gene c‐fos. The growth hormone secretagogue receptor distribution is not confined to the arcuate nucleus, suggesting that additional sites of action may exist. In the present study we characterized the electrophysiological responses of cells in the arcuate nucleus, ventromedial nucleus and periventricular nucleus in an in‐vitro hypothalamic slice preparation, following bath application of GHRP‐6. Additionally, since central somatostatin administration has been shown to attenuate the induction of the c‐fos gene by GHRP‐6, we sought to determine whether the arcuate cells activated by GHRP‐6 are also somatostatin‐sensitive. Male Wistar rats (100–150 g body weight (BW)) were anaesthetized (urethane; 1.2 g/kg BW) and the brains removed. Coronal sections (400 μm thickness) were cut through a block of hypothalamus and were transferred to a slice chamber perfused with artificial cerebrospinal fluid. Forty‐one arcuate nucleus cells were tested with bath application of 15 μm GHRP‐6 for 10 min, 16 of which were tested subsequently (>30 min later) with application of 10 μM somatostatin. Following GHRP‐6 administration, 19 cells (46.3%) showed a significant increase in firing rate during the 15‐min period after GHRP‐6 application (P<0.001), 17 cells (41.5%) did not respond and the remaining five cells (12.2%) were significantly inhibited. Six of the eight arcuate nucleus cells that were excited by GHRP‐6 were significantly inhibited by somatostatin. By contrast, five of the six arcuate nucleus cells that were unresponsive to GHRP‐6 were also unresponsive to somatostatin. In the ventromedial nucleus, of 19 cells tested, eight cells (42.1%) were excited by GHRP‐6, eight cells (42.1%) were unresponsive and the remaining three cells (15.8%) were significantly inhibited. Of 19 cells recorded in the periventricular nucleus, 13 (68.4%) were unresponsive to GHRP‐6 and six (31.6%) were significantly inhibited. Thus, electrophysiological studies in vitro suggest that: (1) neurones in the hypothalamic arcuate nucleus, ventromedial nucleus and periventricular nucleus show changes in electrical activity in response to GHRP‐6; and (2) the arcuate nucleus cells excited by GHRP‐6 are also subject to inhibitory control by somatostatin.
Transgenic Expression of Green Fluorescent Protein in Mouse Oxytocin Neurones
Authors: Young  , Iacangelo  , Luo  , King  , Duncan  , Ginns
Published: March 2003

Routine targeting of neurones for expression of exogenous genes would facilitate our ability to manipulate their internal milieu or functions, providing insight into physiology of neurones. The magnocellular neurones of the paraventricular and supraoptic nuclei of the hypothalamus have been the objects of limited success by this approach. Here we report on the placement of the enhanced green fluorescent protein (eGFP) coding sequence at various locations within an oxytocin transgene. Placement within the first exon yielded little to no expression, whereas placement in the third exon (as an in‐frame fusion with the carboxyl terminus of the oxytocin preprohormone) resulted in cell‐specific expression of eGFP in oxytocin neurones. Furthermore, placement of the eGFP sequence downstream of a picornavirus internal ribosomal entry site (IRES), also in the third exon, allowed expression of the eGFP as a separate protein. Other coding sequences should now be amenable to expression within oxytocin neurones to study their physiology.
Hypothalamic Vasopressin Gene Expression Increases in Both Males and Females Postpartum in a Biparental Rodent
Authors: Wang Z. X. , Liu Y. , Young L. J. , Insel T. R.
Published: March 2003

In previous studies, the closely related neuropeptide hormones oxytocin and vasopressin have been implicated in the central mediation of parental behaviour. Several studies in rats and sheep have demonstrated a role for oxytocin in the initiation of maternal behaviour. Recently, a few studies in a biparental species, the prairie vole (Microxytocinus ochrogaster) have suggested that vasopressin is important for paternal care. The present study investigated this latter possibility by measuring changes in vasopressin and oxytocin hypothalamic gene expression 1 day and 6 days following parturition in prairie voles which show paternal care and in montane voles (M. montanus) which lack paternal care. In prairie voles, vasopressin gene expression increased in both males and females postpartum, relative to sexually naive controls. In the non‐paternal montane vole, no change in vasopressin gene expression was observed in either sex. In contrast to this species difference in vasopressin gene expression, hypothalamic oxytocin gene expression increased in both prairie and montane vole females, but not in males of either species. To augment measures of gene expression, we assessed vasopressin (V1a) and oxytocin receptor binding in both species. Although forebrain vasopressin V1a receptor binding was not altered following parturition in either species, oxytocin receptor binding increased in the ventromedial nucleus of the hypothalamus in females, but not males, in both prairie and montane voles. In summary, vasopressin gene expression increases in both males and females postpartum in a biparental species and oxytocin gene expression and receptor binding increase selectively in females. These results are consistent with earlier reports of a role for vasopressin in paternal care and for oxytocin in maternal behaviour.
Evidence That Hydrogen Sulphide Can Modulate Hypothalamo‐Pituitary‐Adrenal Axis Function: In Vitro and In Vivo Studies in the Rat
Authors: Dello Russo  , Tringali  , Ragazzoni  , Maggiano  , Menini  , Vairano  , Preziosi  , Navarra
Published: March 2003

The gas hydrogen sulphide (H2S) is normally produced in large amounts in the central nervous system during the metabolism of sulphur‐containing aminoacids. H2S was recently shown to influence long‐term potentiation in the rat hippocampus; this finding suggested that the gas may act as a neuromodulator in the brain. We therefore tested the effect of the gas on the release of corticotropin‐releasing hormone (CRH) from rat hypothalamic explants. CRH immunoreactivity in the incubation media was taken as a marker of peptide release. We found that the addition of NaHS to incubation media was consistently associated with a concentration‐dependent decrease in KCl‐stimulated CRH release, whereas basal secretion was unaffected. Increased endogenous H2S production may be also obtained using an indirect precursor of H2S formation, S‐adenosyl‐
l‐methionine (SAMe). The latter mimicked the effects of NaHS, since it reduced potassium‐stimulated CRH release. In vivo, SAMe showed no effect on hypothalamo‐pituitary‐adrenal (HPA) function under resting conditions, but inhibited stress‐related glucocorticoid increase.
Isolation and Characterization of the Promoter Region of the Rat Vasopressin V1b Receptor Gene
Authors: Rabadan‐Diehl  , Lolait  , Aguilera Greti
Published: March 2003

Regulation of pituitary vasopressin V1b receptors plays a critical role in regulating pituitary adrenocorticotropic hormone (ACTH) secretion during adaptation to stress. The objective of this study was to isolate the promoter regulatory region of the V1b receptor gene to better understand the molecular mechanisms involved in V1b receptor regulation. Screening of a rat genomic library using probes directed to the coding region and to the 5′UTR of the rat V1b receptor resulted in the isolation of several clones containing the 5′upstream regions of the V1b receptor cDNA. Sequencing of an 11.2 Kb fragment revealed 8.2 Kb upsteam of the reported cDNA sequence, which contains a putative promoter regulatory region. The 3′ end of the clone contained 1472 base pairs corresponding to the recognized cDNA sequence, followed by 1506 bp of unknown sequence located at the end of the sixth transmembrane domain, probably corresponding to an intron, characteristic of these family of receptors. An additional 161 bp intron was found in the 5′UTR, similar to that described in the rat oxytocin receptor gene. 5′RACE and RNase protection analysis mapped two major putative transcription start points at −830 and −861 bp from the starting methionine. Analysis of the putative promoter region showed no indication of a proximal TATA box, but the presence of a CACA box, a GAGA box, several AP‐1 and AP‐2 sites and a cluster of Sp1 sites upstream of the AP‐2 sites. A luciferase construct containing a 2.1‐kb of putative promoter, and part of the 5′UTR including the first intron, showed promoter activity when transfected into COS‐7, CHO and PC12 cell lines but not in AtT‐20 cells. A similar construct without the intron and distal 5′UTR sequence has no promoter activity in the same cell lines. In summary, the V1b receptor gene contains at least 3 exons and 2 introns. The 5′flanking sequence contains several potential sites for transcriptional regulation, and induced luciferace activity only in constructs containing intron 1, suggesting that the latter is important for receptor gene activation. The data provide bases for future analysis of the regulatory elements controlling V1b receptor transcription.