bluuebunny/crossref_metadata_2025 huggingface dataset:
Mortality amongst participants in Vasaloppet: a classical long‐distance ski race in Sweden
Authors: Farahmand B. Y. , Ahlbom A. , Ekblom Ö. , Ekblom B. , Hållmarker U. , Aronson D. , Brobert G. Persson
Published: March 2003

Abstract. Farahmand BY, Ahlbom A, Ekblom Ö, Ekblom B, Hållmarker U, Aronson D, Persson Brobert G (Stockholm Center of Public Health, and Institute of Environmental Medicine; Institute of Physiology and Pharmacology, Karolinska Institutet, Stockholm; Mora Hospital; and Astrazeneca R&D, Södertälje; Sweden). Mortality amongst participants in Vasaloppet: a classical long‐distance ski race in Sweden.

J Intern Med 2003; 253: 276–283.

Objective. The aim of this study was to assess mortality amongst participants in long‐distance ski races during the Vasaloppet week. We considered the 90 km races for men and 90 or 30 km for women. The vast majority of the participants in these races are not competing on the elite level. It is assumed, however, that they have to undergo regular physical training during a long period of time in order to successfully finish the race.

Design. The cohort study consisted of 49 219 men and 24 403 women, who participated in any of the races during 1989–1998. All subjects were followed up in the National‐Cause‐of‐Death‐Register until 31 December 1999. We computed the standardized mortality ratios (SMRs) adjusting for age and calendar year.

Results. Overall, 410 deaths occurred, compared with 850.6 expected, yielding an SMR of 0.48 [95% confidence interval (CI) 0.44–0.53]. Low SMRs were found in all age groups in both men and women and in all groups after categorization by finishing time and number of races. The lowest SMRs were found amongst older participants and in those who participated in several races. A decreased mortality was observed in all major diagnostic groups, namely cancers (SMR = 0.61; 95% CI 0.52–0.71), diseases of the circulatory system (SMR = 0.43; 95% CI 0.35–0.51), and injuries and poisoning (SMR =0.73; 95% CI 0.60–0.89). For lung cancer the SMR was 0.22, but even after exclusion of lung cancer the all‐cancer mortality was low (SMR = 0.72; 95% CI 0.59–0.86).

Conclusions. We conclude that participants in long‐distance skiing races, which demand prolonged regular physical training, have low mortality. The extent to which this is due to physical activity, related lifestyle factors, genetics or selection bias is yet to be assessed.
Electron energy‐loss spectroscopy (EELS) of iodine in thyroxine: parameters of radiation‐induced loss of iodine
Authors: Haking  , Richter  , TRÖster  , Spring  , Spiess  , Trendelenburg
Published: March 2003

The loss of iodine during irradiation with electrons was measured in order to determine whether it can be detected by electron spectroscopic imaging (ESI). Since iodine can be bound to organic molecules, it could be a candidate for tracer studies by ESI in biomedical research. A solution of thyroxine spread over a carbon film was used as a test specimen. Thyroxine contains four iodine atoms covalently bound to aromatic rings. For ESI, higher doses have to be used than in conventional electron microscopy. The iodine content was therefore measured after irradiation with doses of up to 3 × 107 el nm−2. The measurements were carried out for different specimen thicknesses, temperatures and dose rates, and it was found that iodine can be detected with ESI if moderate dose rates are used at a temperature of −160 °C.
Observation of vortices in metal‐ and oxide‐superconductors using electron waves
Authors: Tonomura
Published: March 2003

Magnetic vortices in superconductors were directly and dynamically observed using electron waves; magnetic lines of force of vortices were measured by holographic interference microscopy and the dynamics of vortices were observed by Lorentz microscopy. The interaction between vortices and defects in superconductors was observed.
Surface profiling of combustion chamber deposits using aperture correlation confocal microscopy
Authors: Reed  , Wilson  , Juškaitis  , Neil
Published: March 2003

The depth discrimination capability of a confocal microscope can be used to generate height‐coded maps of surface topography from reflective surfaces. However, this surface profiling ability is severely limited when black surfaces are examined. In this paper we describe how a new form of confocal microscopy, known as self‐correlating aperture microscopy, can be used to obtain surface topographies from the black carbonaceous deposits found in the combustion chambers of internal combustion engines. The technique is nondestructive and requires no sample preparation. The stereo pair images presented show the range of different morphologies found in combustion deposits generated by different fuel chemistries.
Autofluorescence of living cells
Authors: Andersson  , Baechi  , Hoechl  , Richter
Published: March 2003

We have investigated the autofluorescence of viable mammalian cells (DU‐145 and V79) with a confocal laser scanning microscope equipped with a UV laser. Our aim was to investigate the autofluorescence dependence on different treatments in mitochondria and lysosomes by using different reagents and to improve the confocal laser scanning microscope image quality by deconvolution. The following conclusions were drawn from the results: (1) not all of the autofluorescence comes from mitochondria; (2) one can significantly affect the signal which comes from the mitochondria; (3) the other organelles involved are probably lysosomes; (4) it is harder to affect the autofluorescence signal from the lysosomes than that from the mitochondria, and (5) deconvoluted autofluorescence images provide better information than undeconvoluted ones.
En bloc optical sectioning of resin‐embedded specimens using a confocal laser scanning microscope
Authors: PRIOR D. A. M. , OPARKA K. J. , Roberts I. M.
Published: March 2003

Reconstruction of 3D structures of specimens embedded for light or electron microscopy is usually achieved by cutting serial sections through the tissues, then assembling the images from each section to reconstruct the original structure or feature. This is both time‐consuming and destructive, and may lead to areas of particular interest being missed. This paper describes a method of examining specimens which have been fixed in glutaraldehyde and embedded in epoxy resin, by utilising the autofluorescence preserved or enhanced by aldehyde fixation, and by using a confocal laser scanning microscope to section optically such specimens in the block down to a depth of about 200 μm. In this way, the accurate estimation of the depth of particular features could be used to facilitate subsequent sectioning at the light microscope or electron microscope level for more detailed studies, and 3D images of tissues/structures within the block could be easily prepared if required.
Imaging streptavidin 2D crystals on biotinylated lipid monolayers at high resolution with the atomic force microscope
Authors: SCHEURING S. , MÜLLER D. J. , RINGLER P. , HEYMANN J. B. , ENGEL A.
Published: March 2003

Streptavidin crystals were grown on biotinylated lipid monolayers at an air/water interface and transferred onto highly oriented pyrolytic graphite (HOPG). These arrays could be imaged to a resolution below 1 nm using the atomic force microscope. The surface topographs obtained were compared with negative‐stain electron microscopy images and the atomic model as determined by X‐ray crystallography. The streptavidin tetramer (60 kDa) exposes two free biotin‐binding sites to the buffer solution, while two are occupied by linkage to the lipid monolayer. Therefore, the streptavidin 2D crystals can be used as nanoscale matrices for binding biotinylated compounds. Furthermore, this HOPG‐based preparation method provides a general novel approach to study the structure of protein arrays assembled on lipid monolayers with the AFM.
Ultrastructural characterization of isolated rat Kupffer cells by transmission X‐ray microscopy
Authors: SCHARF J.‐G. , SCHNEIDER G.
Published: March 2003

The ultrastructure of primary cultured rat Kupffer cells was studied using transmission X‐ray microscopy as well as transmission electron microscopy. X‐ray microscopical images of intact, hydrated Kupffer cells demonstrated structures such as cell nucleus separated by a nuclear membrane and filaments concentrated in the perinuclear area. Within the cytoplasm, a number of vacuoles were visible; some of these were crescent‐shaped vacuoles that were half X‐ray lucent, half X‐ray dense; others were uniformly dense. The number of crescent‐shaped vacuoles was predominant. After phagocytosis of haematite particles, enlarged vacuoles containing the ingested material were visible within the cytoplasm of Kupffer cells while crescent‐shaped vacuoles were no longer detectable. Densitometric analysis of the two types of vacuole revealed that the X‐ray absorption of the uniform vacuole was approximately half that of the dense part of the crescent‐shaped vacuoles. This observation led to speculation on the existence of only one type of vacuole in the cytoplasm of Kupffer cells. The different morphological aspects — crescent‐shaped versus uniform vacuoles — might be due to different three‐dimensional orientation with respect to the image plane. Using transmission electron microscopy, the morphology of vacuoles differed more widely in diameter, density and shape. Two main types of vacuole were identified: electron‐lucent and electron‐dense. Based on the observation of only one type of vacuole by transmission X‐ray microscopy, the different morphological aspects of vacuoles obtained by transmission electron microscopy could be explained by imaging several different sections of a crescent‐shaped vacuole. From the present data it can be concluded that transmission X‐ray microscopy is a versatile technique that reveals the ultrastructure of intact, unsectioned biological specimens in their aqueous environment, thereby allowing a more comprehensive interpretation of data obtained by transmission electron microscopy.
Polarization effects in near‐field excitation — collection probe optical microscopy of a single quantum dot
Authors: Chavez‐Pirson A. , Chu S. T.
Published: March 2003

We solve numerically the three‐dimensional vector form of Maxwell's equation for the situation of near‐field excitation and collection of luminescence from a single quantum dot, using a scanning near‐field optical fibre probe with sub‐wavelength resolution. We highlight the importance of polarization‐dependent effects in both the near‐field excitation and collection processes. Applying a finite‐difference time domain method, we calculate the complete vector fields emerging from a realistic probe structure which is in close proximity to a semiconductor surface. We model the photoluminescence from the quantum dot in terms of electric dipoles of different polarization directions, and determine the near‐field luminescence images of the dot captured by the same probe. We show that a collimating effect in the high index semiconductor significantly improves the spatial resolution in the excitation–collection mode. We find that the spatial resolution, image shape and collection efficiency of near‐field luminescence imaging strongly depend on the polarization direction as represented by the orientation of the radiating electric dipoles inside the quantum dot.
Modified fabrication process for aperture probe cantilevers
Authors: Vollkopf A. , Rudow O. , Leinhos T. , Mihalcea C. , Oesterschulze E.
Published: March 2003

We report the development of cantilever‐ and fibre‐based probes for scanning near‐field optical microscopy. Both probe concepts rely on the integration of a microfabricated aperture tip with reproducible optical and mechanical properties. Numerical calculations were carried out using a finite integration code to investigate the polarization‐sensitive transmission behaviour of aperture tips. In order to establish technological guidelines for the optimization of the properties of the optical tip the distinct influence of the tip geometry on the intensity distribution in the vicinity of the aperture is studied in detail.
Characterization of reflection scanning near‐field optical microscopy and scanning tunnelling optical microscopy/photon scanning tunnelling microscopy working in preliminary approach constant height scanning mode
Authors: Barchiesi D.
Published: March 2003

The resolution in near‐field images is currently determined by the visual inspection of recorded images. One of the major questions in near‐field optical microscopy is ‘what resolution can be reached, the tip‐to‐sample distance being known?’ This knowledge is critical when choosing the scanning step and the distance between the tip and the sample, in a preliminary scan. This preliminary scan is often the only way to detect the interesting parts of the sample, with limited risk of tip crash and topographical artefacts. The method proposed here needs two scans of the same area, of the same sample, in constant height mode, recorded at two tip‐to‐sample distances. The pseudo‐transfer function is the ratio of the Fourier transform of these two data maps. This function enables the evaluation of the limit of resolution. Theoretical considerations are introduced to assess the method.
The nucleus: a highly organized but dynamic structure
Authors: Gonzalez‐Melendi P. , Beven A. , Boudonck K. , Abranches R. , Wells B. , Dolan L. , Shaw P.
Published: March 2003

The nucleus in plants and animals is a highly structured organelle containing several well‐defined subregions or suborganelles. These include the nucleolus, interphase chromosome territories and coiled bodies. We have visualized transcription sites in plants at both light‐ and electron‐microscopy level by the incorporation of BrUTP. In the nucleolus many dispersed foci are revealed within the dense fibrillar component, each of which probably corresponds to a single gene copy. In the nucleoplasm there are also many dispersed foci of transcription, but not enough to correspond to one site per transcribed gene. We have shown that in wheat, and probably many other plant species, interphase chromosome territories are organized in a very regular way, with all the chromosomes in the Rabl configuration, all the centromeres clustered at the nuclear membrane and all the telomeres located at the nuclear membrane on the opposite side of the nucleus. However, despite this regular, polarized structure, there is no sign of polarization of transcription sites, or of any preferred location for them with respect to chromosome territorial boundaries. The nucleus is also highly dynamic. As an example, we have shown by the use of a green fluorescent protein fusion to the spliceosomal protein U2B′′ that coiled bodies move and coalesce within the nucleus, and may act as transport structures within the nucleus and nucleolus.
Is it reasonable to assume a uniformly distributed cooling‐rate along the microslide of a directional solidification stage?
Authors: Rabin Y.
Published: March 2003

It is commonly assumed that the cooling‐rate along the microslide of a directional solidification stage is uniformly distributed, an assumption which is typically applied in low cooling‐rates studies. A new directional solidification stage has recently been presented, which is specified to achieve high cooling‐rates of up to 1.8 × 104 °C min−1, where cooling‐rates are still assumed to be uniformly distributed. The current study presents a closed‐form solution to the temperature distribution and to the cooling‐rate in the microslide. Thermal analysis shows that the cooling‐rate is by no means uniformly distributed and can vary by several hundred percent along the microslide in some cases. Therefore, the mathematical solution presented in this study is essential for experimental planning of high cooling‐rate experiments.
Characterization of the cutting edge of glass knives for ultramicrotomy by scanning force microscopy using cantilevers with a defined tip geometry
Authors: Matzelle T. R. , Kruse N. , Reichelt R.
Published: March 2003

The geometry of glass knife edges for ultramicrotomy was studied with nanoscale resolution using scanning force microscopy (SFM) in the contact mode. The local shape of the cutting edge was estimated from single line profiles of the SFM topographic images by taking into account the exact radius of the ultrasharp silicon tip. The tip radius was estimated from secondary electron micrographs recorded at low voltage by field emission scanning electron microscopy (FESEM). The radius of the investigated cutting edges was found to be in range 5–20 nm. The results obtained illustrate that the combination of SFM and high resolution FESEM provides a unique means to determine precisely the radius of glass knives.
Detection of shape changes in biological features *
Authors: Le H. , Kume A.
Published: March 2003

The question of analysing shape changes over time, such as during growth and during the progress of disease, is an important fundamental issue for many applications. Recent mathematical developments in the understanding of the detailed structure of shape spaces have made possible the quantitative study of shape variation. In this paper, we combine the classical multidimensional scaling technique with knowledge of the geometry of shape spaces to examine the role played by the geodesics in shape spaces. We present some promising early results answering questions about shape changes over time.
Spatial distribution and polarization dependence of the optical near‐field in a silicon microfabricated probe
Authors: Minh P. N. , Ono T. , Tanaka S. , Esashi M.
Published: March 2003

This paper reports on the spatial distribution and polarization behaviour of the optical near‐field at the aperture of a Si micromachined probe. A sub‐100 nm aperture at the apex of a SiO2 tip on a Si cantilever was successfully fabricated by selective etching of the SiO2 tip in a buffered‐HF solution using a thin Cr film as a mask. The aperture, 10–100 nm in size, can be reproducibly fabricated by optimizing the etching time. The optical throughput of several apertures was measured. For a 100 nm aperture, a throughput of 1% was approved. The probe shows a very high optical throughput owing to the geometrical structure of the tip. The spatial distribution of the near‐field light is measured and simulated using a finite difference‐time domain method. The polarization behaviour of apertures with different shapes was analysed using a photon counting camera system.
Microstructure, toughness and flexural strength of self‐reinforced silicon nitride ceramics doped with yttrium oxide and ytterbium oxide
Authors: Zheng Y. S. , Knowles K. M. , Vieira J. M. , Lopes A. B. , Oliveira F. J.
Published: March 2003

Self‐reinforced silicon nitride ceramics with additions of either yttrium oxide or ytterbium oxide have been investigated at room temperature after various processing heat treatments. Devitrification of the intergranular phase in these materials is very sensitive to the heat treatment used during processing and does not necessarily improve their strength and toughness. Hot‐pressed ceramics without a subsequent devitrification heat treatment were the strongest. The ytterbium oxide‐doped silicon nitride ceramics were consistently tougher, but less strong, than the yttrium oxide‐doped silicon nitride ceramics. In all the ceramics examined, the fracture toughness showed evidence for R‐curve behaviour. This was most significant in pressureless sintered ytterbium oxide‐doped silicon nitride ceramics. A number of toughening mechanisms, including crack deflection, bridging, and fibre‐like grain pull‐out, were observed during microstructural analysis of the ceramics. In common with other silicon nitride‐based ceramics, thin amorphous films were found at the grain boundaries in each of the ceramics examined. Arrays of dislocations left in the elongated silicon nitride grains after processing were found to belong to the {101¯0}<0001> primary slip system.
Centrifuge polarizing microscope. I. Rationale, design and instrument performance
Authors: Inoué S. , Knudson R. A. , Goda M. , Suzuki K. , Nagano C. , Okada N. , Takahashi H. , Ichie K. , Iida M. , Yamanaka K.
Published: March 2003

We first describe early uses of the centrifuge for deciphering physical properties and molecular organization within living cells, as well as the development and use of centrifuge microscopes for such studies. The rationale for developing a centrifuge microscope that allows high‐extinction polarized light microscopy to observe dynamic fine structures in living cells is next discussed. We then describe a centrifuge polarizing microscope (CPM) that we developed for observing fine structural changes in living cells which are being exposed to up to ≈ 11 500 times earth's gravitational field (g).

With the specimen housed in a rotor supported on an air spindle motor, and imaged through an external microscope illuminated by a precisely synchronized flash of less than 10 ns duration from a Nd:YAG laser, the image of the spinning object remains steady up to the maximum speed of 11 700 rev min−1, or up to ≈ 11 500 × g. The image is captured, at up to 25 frames s−1, by an interference‐fringe‐free CCD camera that is synchronized to the centrifuge rotor. At all speeds (in 100 rev min−1 increments), the image is resolved to better than 1 µm, while birefringence of the specimen, housed in a specially designed specimen chamber that suffers low‐stress birefringence and prevents leakage of the physiological solutions, is detected with a retardance sensitivity of better than 1 nm. Differential interference contrast and fluorescence images (532 nm excitation) of the spinning specimen can also be generated with the CPM.

The second part of this study (Inouéet al., J. Microsc. 201 (2001) 357–367, describes several biological applications of the CPM that we have explored. Individual live cells, such as oocytes and blood cells, are supported on a sucrose or Percoll density gradient while other cells, such as cultured fibroblasts and Dictyostelium amoebae, are observed crawling on glass surfaces. Observations of these cells exposed to the high G fields (centripetal acceleration/g) in the CPM are yielding many new results that lead to intriguing questions regarding the organization and function of fine structures in living cells and related quasi‐fluid systems.
Photocatalytic deposition of a gold nanoparticle onto the top of a SiN cantilever tip
Authors: Okamoto T. , Yamaguchi I.
Published: March 2003

We propose a new technique for depositing a gold nanoparticle onto the tip of a dielectric support. We employed the photocatalytic effect of titanium dioxide for the deposition. When the titanium dioxide immersed in a solution including gold ions is subject to optical exposure, the excited electrons in the conduction band reduce gold ions into gold metal. Illumination by an evanescent wave generated with a total reflection configuration limits the deposition region to the very tip. In experiments we obtained 100–300 nm gold particles on SiN cantilever tips for atomic force microscopes. The contrast of evanescent interference fringes measured by a near‐field scanning optical microscope with this gold nanoparticle probe has proved to be higher than that with a non‐deposited SiN probe by a factor of 1.5.
Theoretical study of transient phenomena in near‐field optics
Authors: Parent G. , Van Labeke D. , Baida F. I.
Published: March 2003

A time‐domain study of the propagation of a light pulse is made by the finite‐difference time‐domain method. This method is described briefly and then two applications are presented: creation and diffraction of surface plasmons in the time‐domain, and propagation of a light pulse through two tip models, a dielectric one and a metal‐coated one. Results on propagation speed of surface plasmons, spatio‐temporal shape and spectral study of the field emitted by a tip are presented.
Preparation of transmission electron microscopy cross‐section specimens of crack tips using focused ion beam milling
Authors: Huang Y. Z. , Lozano‐Perez S. , Langford R. M. , Titchmarsh J. M. , Jenkins M. L.
Published: March 2003

The preparation of transmission electron microscope (TEM) thin foil specimens from metal alloys containing cracks is usually thwarted by the difficulty in preventing preferential erosion of material along the flanks and at the tips of cracks. Recent developments in focused ion beam (FIB) micromachining methods have the potential to overcome this inherent problem. In this article we describe the development of new procedures, one using FIB alone and the other using a combination of FIB with more conventional ion milling to generate TEM specimens that largely retain the microstructural information at stress corrosion cracks in austentic alloys. Examples of corrosion product phase identification and interfacial segregation are included to verify that detailed information is not destroyed by ion bombardment during specimen preparation.
The application of a hot deformation SEM stage, backscattered electron imaging and EBSD to the study of thermomechanical processing
Authors: Huang Y. , Humphreys F. J. , Brough I.
Published: March 2003

The technique of combining in situ hot‐deformation and high resolution electron backscattered diffraction (EBSD) has been applied to study the mechanisms operating during the thermomechanical processing of metals. A simple hot tensile‐straining stage is installed in a field emission gun scanning electron microscope equipped with an EBSD system and has been used successfully for a number of preliminary investigations. These investigations include substructure formation, dynamic subgrain and grain growth, superplastic deformation in aluminium alloys, and dynamic recrystallization in copper. Despite the surface topography, which inevitably increases during plastic deformation, channelling contrast backscattered electron micrographs have been successfully obtained after strains of up to ∼50%. Good quality EBSD maps have been obtained after strains of up to 100%. Most observations and measurements from the in situ experiments are consistent with what is known about the mechanisms occurring in the bulk. The microstructures revealed in the centre of the in situ samples after later repolishing are generally similar to those at the surface.
Estimation of tissue layer level by sequential morphological reconstruction
Authors: Landini G. , Othman I. E.
Published: March 2003

An automated method for labelling layers of clustered elements in two dimensional images based on morphological operations is presented here. Unlike the various distance transforms for regular lattices, the procedure deals successfully with clusters composed of different shaped and sized elements and provides a distance measure, in number of layers, from each element in the cluster to any other reference element (or elements). The method was applied to histological sections of two different types of polystratified epithelia. Local clusters in the different cell profile layers can subsequently be used to define the orientation of the tissue layers relative to each element in it.
Immunological Localization and Ontogenetic Development of Inhibin α Subunit in Rat Brain
Authors: Fujimura  , Ohsawa  , Funaba  , Murata  , Murata  , Takahashi  , Abe  , Torii
Published: March 2003

This study examined the immunolocalization and ontogeny of the inhibin‐specific α subunit in the brain of male rats. Immunohistochemistry using antiserum directed against the mature region of porcine inhibin α (1–19, Tyr20) revealed positive reactions in process‐bearing cells resembling astroglia in several regions, especially in the dorsal region of the third ventricle, medial and ventral arcuate nucleus, hippocampal dentate gyrus, and layers 1–3 of the cerebral cortex. Generally, inhibin α‐positive cells in the limbic cortex had larger cell bodies and longer processes than those in the hypothalamus. These inhibin α‐positive cells were verified to be positive for glial fibrillary acidic protein (GFAP), a differentiated astroglial marker, by double immunolabelling. The expression of inhibin α mRNA was higher in the brains of neonatal rats than in those of adult rats, as revealed by reverse transcription‐competitive polymerase chain reaction, although the similar changes of immunoreactive inhibin α subunit in the brain was not observed. Orchiectomy did not affect expression of inhibin α mRNA in the hypothalamic area. This study suggests that inhibin‐related peptide is produced by differentiated astrocytes, especially in the hypothalamic arcuate nucleus, the hippocampal dentate gyrus, and the cerebral cortex, and that the expression of inhibin α is regulated during brain development.
The Effect of Growth Hormone Secretagogues and Neuropeptide Y on Hypothalamic Hormone Release from Acute Rat Hypothalamic Explants
Authors: Korbonits  , Little  , Forsling  , Tringali  , Costa  , Navarra  , Trainer  , Grossman
Published: March 2003

Growth hormone (GH) secretagogues (GH‐releasing peptides and their non‐peptide analogues) stimulate growth hormone release via specific G‐protein coupled receptors both directly from the pituitary gland and through stimulation of the hypothalamus. The exact mechanism of action in the hypothalamus is not known. The presence of endogenous GH releasing hormone (GHRH) seems to be necessary for the in‐vivo actions of growth hormone secretagogues (GHSs), but data suggest that further factors must be involved as well. The effect of GHSs is not entirely specific for the GH axis; they release prolactin and stimulate the hypothalamo‐pituitary‐adrenal axis causing elevations in circulating ACTH and cortisol levels in both animal and human studies. Recently, it has also been suggested that GHSs stimulate hypothalamic neuropeptide Y (NPY) neurones. In the present study, we have therefore investigated the direct effect of several GHSs (GHRP‐6, hexarelin and the non‐peptide analogues L‐692, 429 and L‐692, 585) on GHRH, somatostatin (SS), corticotrophin‐releasing hormone (CRH) and arginine vasopressin (AVP) release in vitro in an acute rat hypothalamic incubation system. We also assessed the effect of NPY on GHRH, SS and AVP release. Freshly removed hypothalami were incubated in control media for 20 min and then in 1–4 consecutive 20‐min periods in each of the test substances at different concentrations. There was no significant change in either the basal or potassium‐stimulated release of GHRH or SS at low concentrations of any of the secretagogues; however, at millimolar doses a paradoxical inhibition of GHRH was observed with GHRP‐6, hexarelin and L‐692 585 (data are expressed as the ratio of treated to preceding basal release; at 20 min control group: 0.97±0.02, GHRP‐6: 0.55±0.04, P<0.001 compared to control group; hexarelin: 0.56±0.06, P<0.001, L‐692,585: 0.70±0.03, P<0.001), while SS was stimulated after 60 or 80 min (at 80 min control: 0.80±0.03, hexarelin: 1.23±0.07, P<0.05 and L‐692,585: 1.37±0.11, P<0.05). GHSs stimulated hypothalamic AVP release (at 20 min control: 0.99±0.06 ratio to basal release, 10−4 M concentration of GHRP‐6: 6.31±1, P<0.001, hexarelin: 1.88±0.4, P<0.01, L‐692,429: 1.90±0.5, P<0.05 and L‐692,585: 2.34±0.96, P<0.01), while no stimulatory effect was found on CRH release. NPY significantly stimulated SS and inhibited basal and potassium‐stimulated GHRH release, while potentiating potassium‐evoked AVP secretion. The Y1 receptor antagonist BIBP 3226 did not inhibit the effects of NPY on SS, GHRH or AVP release. We therefore conclude that, in this in‐vitro rat hypothalamic incubation model, growth hormone secretagogues stimulate the release of AVP but have no effect on either GHRH, SS or CRH at low doses; at high doses paradoxically they inhibit the hypothalamic GH axis similar to in‐vivo data in the rat. We speculate that these effects might be mediated by NPY.
Reduction of Luteinzing Hormone Secretion Induced by Long‐Term Feed Restriction in Male Rats Is Associated with Increased Expression of GABA‐Synthesizing Enzymes Without Alterations of GnRH Gene Expression
Authors: Leonhardt  , Shahab  , Luft  , Wuttke  , Jarry
Published: March 2003

In rats, fasting or restriction of feed intake impairs the activity of the hypothalamic gonadotropin‐releasing hormone (GnRH) pulse generator which results in reduced luteinizing hormone (LH) secretion. It is still unknown which neurotransmitters are involved in this phenomenon. However, it is known that increased GABA concentrations in the hypothalamus reduce GnRH biosynthesis and release. Therefore, we examined whether 17 days of feed restriction in male rats affected the hypothalamic gene expression of GnRH and the GABA‐synthesizing enzymes glutaminase (GLS) and glutamic acid decarboxylase–which exists in two forms, GAD67 and GAD65–in the mammalian brain. Furthermore, the expression of the GnRH receptor (GnRH‐R) and the GABA transporter 1 (GAT‐1) were investigated. Feed restriction resulted in a 75% reduction in body weight (b.w.) compared to rats fed ad libitum. Serum concentrations of LH and testosterone in the feed restricted group were significantly reduced to ≈15% of that of rats fed ad libitum, while the FSH concentration remained unchanged. In the mediobasal hypothalamus (MBH) where GnRH is released into the portal vessels, mRNA levels of GAD67 and GLS were increased twofold compared to rats fed ad libitum while no changes were observed in the preoptic area of the hypothalamus (POA) where GnRH is biosynthesised. Neither the expression of preoptic GnRH mRNA nor the expression of GAD65 and of GnRH‐R mRNA in both hypothalamic structures was affected by feed restriction. In the anterior pituitary, a significant reduction of the expression of GnRH‐R, LH‐β and the α subunit was observed in the feed restricted rats, whereas FSH‐β mRNA levels remained constant. Thus, feed restriction selectively increased the expression of GABA‐synthesizing enzymes in the MBH but did not modify GnRH expression in the POA. However, the reduced expression of the LH‐β‐ and α‐subunit and of the GnRH‐R in the anterior pituitary indicates that pulsatile GnRH release may have been attenuated or even abolished. We suggest, that enhanced expression of GABA‐synthesizing enzymes reflects increased GABAergic neurotransmission and thereby reducing GnRH release from the MBH.
Regulation of Corticotropin‐Releasing Factor‐Binding Protein Expression in Amygdalar Neuronal Cultures
Authors: Kasckow  , Regmi  , Seasholtz  , Mulchahey
Published: March 2003

Corticotropin‐releasing factor‐binding protein (CRF‐BP) is known to regulate the bioavailability of CRF and may also play a role in stress behaviours. CRF‐BP has been localized in the pituitary as well as central nervous system (CNS) limbic and cortical areas, including the amygdala. The signal transduction pathways which regulate amygdalar CRF‐BP are not well understood. In this report, we have examined the effect of protein kinase A and C activators, CRF, dexamethasone and interleukin‐6 (IL6) on CRF‐BP mRNA and protein expression in dissociated fetal amygdalar cultures. CRF‐BP mRNA levels were determined by Northern analysis following 12 h treatment with the following agents: forskolin (1–30 μM), CRF (1–1000 nM), phorbol‐12‐myristate‐13‐acetate (TPA; 1–50 nM), dexamethasone (1–100 nM) and IL6 (10–500 pM). Significant increases in CRF‐BP mRNA were observed in response to forskolin (30 mM), CRF (100, 1000 nM), IL6 (100, 500 pM), TPA (50 nM) and dexamethasone (100 nM; P<0.05 for all; n=3–6 for all). We extended our observations of CRF‐BP expression to the protein level by performing semiquantitative Western analysis of total cellular protein after treatment with the same agents. Twenty‐four hour treatment with 30 μM forskolin, 1000 nM CRF, 50 nM TPA, 100 pM IL6 or 100 nM dexamethasone significantly increased CRF‐BP expression (P<0.05, n=3 for each treatment). The primary cultures were then transfected with a rat CRF‐BP‐reporter construct containing 3500 base pairs of CRF‐BP 5′ flanking DNA. Treatment with all five agents produced statistically significant increases above control (P<0.05; n=3 for each). The results suggest that CRF‐BP in the amygdala is stimulated by numerous pathways which may play a significant role in promoting behavioural changes.
The Influence of Gonadal Steroids on Pre‐Pro Melanin‐Concentrating Hormone mRNA in Female Rats
Authors: Murray  , Baker  , Levy  , Wilson
Published: March 2003

Melanin‐concentrating hormone (MCH) may have a regulatory role in the control of luteinizing hormone (LH) release. We have investigated if gonadal steroids induce changes in the expression of pre‐pro MCH (ppMCH) that are associated with changes in the pattern of LH release. Using quantitative in‐situ hybridization histochemistry we have determined the effect of administration of either oestradiol benzoate (5 μg/rat) or oestradiol benzoate followed 44 or 48 h later by progesterone (0.5 mg/rat) to ovariectomized rats on the expression of ppMCH in the medial and lateral zona incerta and the lateral hypothalamus. The prevalence of ppMCH transcripts in the intact female rat at 12.00 and 19.00 h on proestrus and the first day of dioestrus was also examined. Oestrogen reduced the intensity of hybridization signal for ppMCH mRNA and this was associated with both a decrease in the number of cells in which the message was detected in the medial zona incerta and a negative feedback effect on LH release in ovariectomized rats. Progesterone administration to oestradiol benzoate‐primed rats did not alter the reduced expression in the medial zona incerta in spite of its positive feedback effect on LH release. We suggest that progesterone may act only on post‐translational events. Expression in the MCH cell bodies of the lateral zona incerta were not affected but there was a transient decrease 4 h after progesterone treatment in the oestradiol benzoate‐primed rats in expression in the lateral hypothalamus. No changes in ppMCH mRNA were seen in intact animals on proestrus or the first day of dioestrus indicating that gonadal steroids are not important in the modulation of ppMCH gene expression during the oestrous cycle. In other steroid‐dependent physiological situations, however, oestrogen may influence the expression of ppMCH in a subpopulation of cell bodies in the medial zona incerta.
Glucocorticoids Regulate Pituitary Growth Hormone Secretagogue Receptor Gene Expression
Authors: Tamura H. , Kamegai J. , Sugihara H. , Kineman R. D. , Frohman L. A. , Wakabayashi I.
Published: March 2003

Glucocorticoids regulate growth hormone (GH) secretion by modulating both hypothalamic and pituitary function. At the level of the pituitary, glucocorticoids increase GH and GH‐releasing hormone receptor (GHRH‐R) gene expression. To test if glucocorticoids might also regulate the pituitary expression of the recently identified GH secretagogue (GHS) receptor, GHS‐R; adult male rats were adrenalectomized or sham operated, and treated with the synthetic glucocorticoid (dexamethasone, 200 µg/day) or vehicle for 8 days. Pituitary GHS‐R mRNA levels were assessed by reverse transcriptase polymerase chain reaction (RT‐PCR). Adrenalectomy decreased pituitary GHS‐R mRNA to 45% of vehicle‐treated, sham‐operated rats (P < 0.05). Administration of dexamethasone increased GHS‐R mRNA levels in sham‐operated as well as in adrenalectomized rats (199 ± 24% (P < 0.05) and 369 ± 48% (P < 0.01) of vehicle‐treated controls). Addition of dexamethasone to primary rat pituitary cell cultures increased GHS‐R mRNA levels in a dose‐ and time‐dependent manner while the transcriptional inhibitor, actinomycin D, completely blocked the stimulatory action of dexamethasone. Taken together, these results suggest glucocorticoids directly increase pituitary GHS‐R mRNA levels by stimulating GHS‐R gene transcription.
Peripheral or Central Administration of Motilin Suppresses LH Release in Female Rats: A Novel Role for Motilin
Authors: Tsukamura  , Tsukahara  , Maekawa  , Moriyama  , Reyes  , Sakai  , Niwa  , Foster
Published: March 2003

Motilin is secreted in a clear episodic pattern during fasting or during the interdigestive phase, but feeding promptly stops this secretory pattern, and plasma concentrations of motilin decrease. We have previously determined that fasting markedly suppresses pulsatile luteinizing hormone (LH) secretion in female rats in the presence of oestrogen. In the present study, we wished to learn if motilin may mediate the fasting‐induced suppression of LH secretion by determining the effects of motilin administration on LH release and on food intake. Intravenous (i.v.) injection of motilin (37 nmol/rat) suppressed LH release and significantly decreased mean LH concentrations both in ovariectomized (OVX) and oestradiol‐implanted ovariectomized (OVX+E2) rats. Food intake was significantly increased by i.v. motilin injection in OVX rats, but not in OVX+E2rats. It is likely that motilin inhibits LH release via inhibition of the gonadotrophin‐releasing hormone (GnRH)‐releasing mechanism at the hypothalamic level, because motilin (3.7 nmol/rat) also suppressed LH secretion when centrally administered, and because LH release in i.v. motilin‐treated rats increased in response to exogenous GnRH. These results suggest that motilin may be a peripheral signal for the suppression of LH secretion through central sensors.
Adrenalectomy Dramatically Modifies the Dynamics of Neuropeptide and c‐fos Gene Responses to Stress in the Hypothalamic Paraventricular Nucleus
Authors: Tanimura S. M. , Watts A. G.
Published: March 2003

We have used in situ hybridization and radio‐immunoassay to compare temporal dynamics of components in the hypothalamo‐pituitary limb of the hypothalamo‐pituitary‐adrenal axis during sustained hypovolemic stress in adrenalectomized (ADX) rats to those previously reported in intact animals. We asked three questions: first, does corticotropin‐releasing hormone (CRH) gene transcription occur in neuroendocrine neurones of the hypothalamic paraventricular nucleus (PVH) of ADX rats, and if so, how is it temporally organized; second, what is the expression pattern of the vasopressin and other genes known to be colocalized in these neuroendocrine neurones; third, if adrenocorticotropin hormone (ACTH) secretion occurs, what is its temporal profile? We found that sustained hypovolemia evoked a brief episode of CRH gene transcription in ADX rats that occurred earlier than in intact rats. However, in contrast to saline‐injected controls, this activation was not maintained because declines in CRH hnRNA and mRNA were seen as the stress continued. Although increased vasopressin gene transcription was not seen in intact hypovolemic rats, robust increases were measured throughout in ADX rats, suggesting that in the absence of corticosterone the vasopressin gene is transcribed preferentially to the CRH gene during sustained hypovolemia. c‐fos and preproenkephalin mRNA profiles also exhibited earlier onsets compared to intact rats. Finally, the onset and duration of ACTH secretion was the same in ADX rats as previously reported in intact rats. Collectively, these data support two hypotheses regarding the actions of corticosterone. First, that it provides some form of facilitatory signal allowing neuroendocrine CRH transcriptional mechanisms to remain active during sustained hypovolemia. Second, that it strongly inhibits the response of the vasopressin gene to hypovolemic stress.
Steroid Implants in the Medial Preoptic Area or Ventromedial Nucleus of the Hypothalamus Activate Female Sexual Behaviour in the Musk Shrew
Authors: Veney S. L. , Rissman E. F.
Published: March 2003

Female musk shrews are induced ovulators that do not exhibit a spontaneous behavioural oestrous cycle. Testosterone produced by the ovaries and adrenal glands, is the major steroid hormone in circulation at times of mating, and as such, regulates sexual behaviour. In the first experiment, we identified the neural site(s) of action for testosterone. Hormone implants were placed in one of three targeted brain regions. The neural sites selected were the medial anterior division of the bed nucleus of the stria terminalis (BNSTMA), medial preoptic area (mPOA) and the ventromedial nucleus of the hypothalamus (VMN). Ovariectomized females who received a unilateral testosterone propionate implant in either the mPOA or VMN, were significantly more likely to display sexual behaviour as compared to females who received an implant in the BNSTMA or any other hypothalamic nucleus. In experiments 2 and 3, we investigated whether the behavioural effects of testosterone propionate were mediated by an oestrogen receptor or the androgen receptor. Ovariectomized females that received oestradiol (E2) implants in either the mPOA or VMN were more likely to display receptivity, and had significantly shorter behavioural latencies, as compared to females implanted with either dihydrotestosterone or cholesterol. These data show that neural aromatization of testosterone to E2 in the mPOA or VMN is necessary for optimal activation of female musk shrew sexual behaviour. This finding implies a degree of neural redundancy in the networks that control the expression of sexual receptivity.
Differences in Arginine Vasotocin Gene Transcripts and Cortisol Secretion in Trout with High or Low Endogenous Melanin‐Concentrating Hormone Secretion
Authors: Gilchriest B. J. , Tipping D. J. , Hake L. , Levy A. , Baker B. I.
Published: March 2003

Previous studies on trout suggest that melanin‐concentrating hormone (MCH) acts at both hypothalamic and pituitary levels to restrain the release of adrenocorticotropic hormone and hence cortisol during stress. Using in situ hybridization, the present work examined whether high rates of MCH secretion were associated with changes in the synthesis of arginine vasotocin (AVT), one of the corticotropin secretogogues. It also examined whether high endogenous MCH secretion restrains cortisol secretion during intense as well as mild stress, and how exogenous MCH affects the rise in plasma cortisol following injection stress. Trout were reared in black‐ or white‐coloured tanks for 1 year or more to achieve maximal differences in MCH secretion. Following a mild stress, cortisol secretion was greater in black‐reared fish with low MCH secretion which is in line with previous findings but, following a more severe stress, plasma cortisol concentrations were similar in the two groups. Injection of MCH into black‐adapted fish restrained the stress‐induced rise in plasma cortisol concentration during the first hour but did not affect final cortisol values. In two separate experiments, AVT mRNA levels were significantly lower in the hypothalamus of black‐reared fish. Possible explanations for this include a greater negative‐feedback restraint by cortisol, which is likely to rise higher in black‐adapted fish during the moderate, daily stresses of aquarium life; or the possibility that exposure to a white background may be psychologically stressful, stimulating AVT transcription. The possibility that MCH directly stimulates AVT transcription cannot be excluded but seems less likely. The results suggest that while MCH may restrain the release of hypothalamo‐pituitary stress hormones under moderately stressful conditions, it does not restrain AVT synthesis.