bluuebunny/crossref_metadata_2025 huggingface dataset:
A comparative study of colloidal particles as imaging standards for microscopy
Authors: Derose  , Revel
Published: March 2003

Colloidal particles have long been used as imaging standards for electron microscopy and, more recently, for scanning probe microscopy. We have analysed gold, polystyrene and silica colloidal particles by both transmission electron microscopy and atomic/scanning force microscopy in an attempt to determine if any can be truly used as ‘standards’ of shape and/or size. From the transmission electron micrographs, we have obtained precise information of the particle circumference and mean diameter. By comparing the ratio of these to the value for π, we obtained a measure of the sphericity of the particles. We have also shadowed the particles with metal at a known angle and have analysed the shadow length to determine the particles' heights and shapes. The height information obtained from the shadow length data collected from the transmission electron micrographs was then compared with that obtained by atomic/scanning force microscopy. Our results show that cleaned (washed) silica or polystyrene particles closely approach true spheres. In the case of gold particles, height data obtained from shadow lengths analysed in transmission electron micrographs show good agreement with that obtained from the atomic/scanning force microscopy images even without washing. However, the gold particles often deviate from sphericity. Based upon both the shape and the physical properties of the colloidal particles, silica would be the best choice as a standard. We also have noticed that metal shadowing of colloidal particle samples used for atomic/scanning force microscopy offers an advantage which we call a ‘nanoscale metric’ visible in the image directly at each particle site. This information can be important if one wishes to use samples prepared from colloidal particles simply and reliably to determine the probe shape for scanning probe microscopy from image deconvolution/restoration methods or as a calibration sample.
Celloidin–wax sandwich microtomy: a novel and rapid method for producing serial semithin sections
Authors: Tyrer
Published: March 2003

A method is described that allows rapid and reliable serial sectioning down to thicknesses of 1 μm. The tissue is first embedded in celloidin and then in wax and trimmed so that the block is sandwiched between two layers of wax. This combines the virtues of both media. The celloidin gives greater support to tissue than wax and enables the cutting of semithin sections. The wax allows ribbons of serial sections to be produced as in conventional wax microtomy. This makes it easy to produce serial semithin sections as a matter of routine.
Image cytometric method for quantifying the relative amount of DNA in bacterial nucleoids using Escherichia coli
Authors: Vischer  , Huls  , Ghauharali  , Brakenhoff  , Nanninga  , Woldringh
Published: March 2003

An image cytometric method for quantifying integrated fluorescence was developed to measure the relative DNA contents of bacterial nucleoids. Image analysis was performed with newly developed macros in combination with the program Object‐Image, all downloadable from . Four aspects of the method were investigated. (i) Good linearity was found over a ten‐fold range of fluorescence intensity in a test with a calibration kit of fluorescent latex spheres. (ii) The accuracy of the method was tested with a narrowly distributed Escherichia coli population, which was obtained by growing cells into stationary phase. The width of the image cytometric distribution was approximately 6%, in good agreement with results obtained by flow cytometry. (iii) The error contribution of manual focusing could be kept below 2%, although a strong dependency between integrated fluorescence and focus position was observed. (iv) The results were verified with a flow cytometer, which gave similar distributions for the DNA contents per cell expressed in chromosome equivalents (4.8 fg of DNA). We used the presented method to evaluate whether the DNA conformation had any effect on the total fluorescence of bacterial nucleoids. Experiments using nucleoids with the same amount of DNA in either a dispersed or a compact conformation showed no significant difference in integrated fluorescence, indicating that it is possible to determine the DNA content per nucleoid independently of the actual organization of the DNA.
Aluminium–aluminium nitride composites fabricated by melt infiltration under pressure
Authors: Chedru  , Vicens  , Chermant  , Mordike
Published: March 2003

Aluminium–matrix composites containing ~55 vol.% AlN particles were fabricated by melt infiltration of aluminium into an AlN preform under a pressure of up to 130 MPa. Two different AlN powders (H.C. Starck, Goslar, Germany, and ESK, Elektroschmelzwerk, Kempten, Germany) and four types of aluminium alloy (2024, 1070, 6060 and 5754) were used. The initial AlN powders were characterized by scanning electron microscopy. The composites were studied by light microscopy, scanning and transmission electron microscopies and energy‐dispersive X‐ray spectroscopy. Particle–matrix interfaces were observed using high‐resolution electron microscopy. As a result of the melt infiltration process, the composites are very dense and the microstructure shows a homogeneous distribution of the reinforcement. The interfaces are clean with very little porosity. Some Al2Cu precipitates were observed in the 2024 matrix.
Collected studies on interfaces and interphases as related to the behaviour of fibre‐reinforced aluminium alloy composites
Authors: Scott  , Chen
Published: March 2003

This paper is an essentially practical treatment of interphases and interfaces and of their influence on the properties of a number of metal matrix composites (MMCs). The illustrations are drawn from the authors' experiences and have been chosen to underline the importance of detailed microstructural analysis for elucidating the fabrication behaviour and the mechanical performance of this group of materials. The work involves a series of MMCs based upon different combinations of aluminium alloy and ceramic/carbon fibre (both continuous and short) and made using the method of low‐pressure liquid metal infiltration (LMI). Detailed analyses of the composite microstructures are given, with particular attention being paid to the interface regions. The data are used to categorize an interface according to the type of bond, that is a mechanical bond resulting from thermal mismatch between the fibre and metal matrix, or a chemical bond, with or without second phase, caused by chemical reaction. The information is then employed to account for aspects of composite fabrication, such as the cast microstructure produced by the LMI method and the effect of heat treatment, and to elucidate composite properties such as stiffness, yield stress and failure strength.
Carbon‐fibre reinforced magnesium alloys: nanostructure and chemistry of interlayers and their effect on mechanical properties
Authors: Feldhoff  , Pippel  , Woltersdorf
Published: March 2003

The micromechanical fracture behaviour of C/Mg–Al composites of varying interface reactivity was investigated by scanning electron microscope bending tests. Structure and chemistry of fibre/matrix interlayers were studied down to the atomic scale by imaging and spectroscopical transmission electron microscope techniques (high‐resolution electron microscopy, energy dispersive X‐ray spectroscopy, parallel‐recording electron energy loss spectroscopy and energy‐filtered transmission electron microscopy). The chemical reactions at the fibre/matrix interfaces of the C/Mg–Al composites were found to form plate‐shaped carbidic precipitates, mainly Al2MgC2, which strongly influence the composite's mechanical properties by changing the fibre/matrix bonding strength.
Image processing experiments for computer‐based three‐dimensional reconstruction of neurones from electron micrographs from serial ultrathin sections
Authors: Shishido  , Yoshida  , Umino
Published: March 2003

This study examined an image processing technique that uses a computer to reconstruct a three‐dimensional image of neurones from electron micrographs of serial ultrathin sections. The major problems involved were: (a) a distortion of features in electron micrographs; (b) a significant change of cross‐section features of neurones in electron micrographs of neighbouring sections; and (c) disagreement between the electron microscopic section face and the coordinate plane desired for the reconstruction. Electron micrographs of a retinal bipolar cell stained with a biotinylated tracer were used. We corrected the distortion of features by means of a warp, a widely used algorithm in morphing image processing. The change of features between neighbouring electron micrographs was minimized by filling the gaps with an interpolated image produced by a dissolve, another algorithm in morphing, as well as the warp. The distortion of the three‐dimensional reconstructed image made by piling up features was corrected by making the image with a wire frame model. Furthermore, in order to estimate a closed contour of features, an active contour model, Snakes, was applied to the electron microscope features. Snakes successfully detected the contour of the target feature, but in some electron microscope images broke into the target feature.
Two‐photon image correlation spectroscopy and image cross‐correlation spectroscopy
Authors: Wiseman P. W. , Squier J. A. , Ellisman M. H. , Wilson K. R.
Published: March 2003

We introduce two‐photon image correlation spectroscopy (ICS) using a video rate capable multiphoton microscope. We demonstrate how video rate two‐photon microscopic imaging and image correlation analysis may be combined to measure molecular transport properties over ranges typical of biomolecules in membrane environments. Using two‐photon ICS, we measured diffusion coefficients as large as 10−8 cm2 s−1 that matched theoretical predictions for samples of fluorescent microspheres suspended in aqueous sucrose solutions. We also show the sensitivity of the method for measuring microscopic flow using analogous test samples. We demonstrate explicitly the advantages of the image correlation approach for measurement of correlation functions with high signal‐to‐noise in relatively short time periods and discuss situations when these methods represent improvements over non‐imaging fluorescence correlation spectroscopy. We present the first demonstration of two‐photon image cross‐correlation spectroscopy where we simultaneously excite (via two‐photon absorption) non‐identical fluorophores with a single pulsed laser. We also demonstrate cellular application of two‐photon ICS for measurements of slow diffusion of green fluorescent protein/adhesion receptor constructs within the basal membrane of live CHO fibroblast cells.
Testing local dependence of spatial structures on images
Authors: Chadœuf J. , Brix A. , Pierret A. , Allard D.
Published: March 2003

Associations between two spatial processes can be due to a real dependence between the two processes or to the dependence on common underlying variables. We propose to test the existence of a real dependence by use of local tests, leading to a global test of real dependence and a map of local interactions. We present first how classical interaction tests based on random rotations between completely observed processes such as those developed by Berman (Berman.Appl. Statist. (1986)35, 54–62), can be integrated in local analyses. For this purpose, tests are first performed locally, and the distribution of theirp‐values is then compared to the corresponding value under the null hypothesis. A similar approach is proposed to test non‐stationarity of a point pattern by using distance statistics popularized by Diggle (Diggle.Statistical Analysis of Spatial Point Patterns. (1983) Academic Press, New York). The problem of testing the interaction between a random field and a censoring area pattern process is discussed and an approach similar to the preceding ones is then proposed. The methods are mainly applied to agricultural examples but they can be applied to any microscopical images for which one wishes to analyse the spatial structure.
Monitoring unidirectional carbon/carbon composite processing by light microscopy
Authors: Casal E. , Granda M. , Bermejo J. , Menéndez R.
Published: March 2003

Unidirectional carbon/carbon composites were prepared using polyacrylonitrile‐based carbon fibres and a commercial coal‐tar pitch. The preparation includes a three‐sequential‐step process: (i) prepreging, (ii) moulding/pressing, and (iii) carbonization to 1000 °C. The effect of the process parameters including pressure, mould characteristics, and temperature and duration of pressure application on the microstructure of composites was monitored by light microscopy. Results show that the application of a pressure of 1 MPa close to the temperature of pitch solidification produces lower porosity composites. This porosity is significantly reduced when the solidification of the pitch is tempered by the introduction of a soak time of 10 h at the temperature at which pressure is applied.
Observation of human dentine by focused ion beam and energy‐filtering transmission electron microscopy
Authors: Hoshi K. , Ejiri S. , Probst W. , Seybold V. , Kamino T. , Yaguchi T. , Yamahira N. , Ozawa H.
Published: March 2003

Molar dentine was sliced into 100 nm ultrathin sections, by means of a focused ion beam, for observation by energy‐filtering transmission electron microscopy (EFTEM). Within the matrix, crystals approximately 10 nm wide and 50–100 nm long were clearly observed. When carbon and calcium were mapped in electron spectroscopic images by EFTEM, carbon failed to localize in crystals. However, it was found in other regions, especially those adjacent to crystals. Because carbon localizations were thought to reflect the presence of organic components, carbon concentration in regions near crystals suggested the interaction of crystals and organics, leading to organic control of apatite formation and growth. Ca was present in almost all regions. The majority of Ca localizing in regions other than crystals may be bound to organic substances present in dentine matrix. These substances are thought to both accumulate Ca and act as reservoirs for crystallization of apatite in dentine.
Techniques for reducing the interfering effects of autofluorescence in fluorescence microscopy: improved detection of sulphorhodamine B‐labelled albumin in arterial tissue
Authors: Staughton T. J. , Mcgillicuddy C. J. , Weinberg P. D.
Published: March 2003

Measurements of the transport of circulating sulphorhodamine B‐labelled albumin into the arterial wall, made by applying digital imaging fluorescence microscopy to sections of arteries fixed in situ, are limited in sensitivity by the low levels of tracer fluorescence and high levels of autofluorescence emitted from the tissue. Three attempts to improve these ratios are described. In the first, spectra of the tracer in solution and of arterial autofluorescence were used to design novel microscope filters for rhodamine‐like dyes. By exciting with the rarely used yellow lines of the mercury arc lamp and detecting a narrow band of emission with Stokes shifts as small as 15 nm, the ratio of tracer fluorescence to autofluorescence was tripled. In the second, effects of different fixatives were investigated. Using a model system, it was confirmed that Karnovsky's fixative gives good tracer immobilization but elevates autofluorescence, whereas fixative‐free buffer solutions give low autofluorescence but do not retain the tracer. It was further found that simple formaldehyde‐based fixatives, hitherto considered to be poor fixatives of albumin, immobilized the tracer as well as the glutaraldehyde‐based fixative, whilst giving autofluorescence levels comparable to those seen with buffer alone; they therefore give excellent tracer fluorescence to autofluorescence ratios. In the third, lowering specimen temperature by 50 °C was found to increase the intensity of tracer fluorescence by 30% whilst autofluorescence was unaffected. These data may have relevance to microscopical studies using other tissues and fluorescent tracers.
State‐selective optical near‐field resonant ionization spectroscopy of atoms near a dielectric surface
Authors: Ohdaira Y. , Kijima K. , Terasawa K. , Kawai M. , Hori H. , Kitahara K.
Published: March 2003

Spin‐sensitive optical near‐field microscopy and spectroscopy are proposed based on the study on the conserved quantities in optical near‐field interactions of atoms with dielectric surfaces. A two‐step photoionization spectra of Cs atoms resolving hyperfine structures are demonstrated near a planar dielectric surface by using evanescent waves. These techniques of state/spin‐selective excitation and highly sensitive detection, combined with the techniques of optical pumping, will open up possibilities of space‐ and polarization‐sensitive detection of optical near‐fields using atomic probes. This novel method provides us with a useful technique for the observation of polarization nature of the optical near‐field and controlling the spin states of mesoscopic electronic systems.
A single gold particle as a probe for apertureless scanning near‐field optical microscopy
Authors: Kalkbrenner T. , Ramstein M. , Mlynek J. , Sandoghdar V.
Published: March 2003

We report on the fabrication, characterization and application of a probe consisting of a single gold nanoparticle for apertureless scanning near‐field optical microscopy. Particles with diameters of 100 nm have been successfully and reproducibly mounted at the end of sharp glass fibre tips. We present the first optical images taken with such a probe. We have also recorded plasmon resonances of gold particles and discuss schemes for exploiting the wavelength dependence of their scattering cross‐section for a novel form of apertureless scanning near‐field optical microscopy.
Light field propagation by metal micro‐ and nanostructures
Authors: Krenn J. R. , Salerno M. , Felidj N. , Lamprecht B. , Schider G. , Leitner A. , Aussenegg F. R. , Weeber J. C. , Dereux A. , Goudonnet J. P.
Published: March 2003

The ability to sustain plasmon oscillations gives rise to unique properties of metal nanostructures, which can be exploited for the controlled manipulation of light fields on the nanoscale. In this context we investigate electromagnetic coupling effects within lithographically produced ensembles of gold nanoparticles with a photon scanning tunnelling microscope. To provide an interface between these nano‐optical devices and classical far‐field optics, we investigate surface plasmon propagation on microstructured metal thin films.
Separating different contributions to the shear force in near‐field microscopy
Authors: Leuschner M. , Schüttler M. , Giessen H.
Published: March 2003

The shear force between a gold and a graphite sample and an approaching near‐field optical probe using tuning fork detection is studied in detail. The adiabatic and dissipative contributions are clearly distinguished by monitoring the amplitude as well as the phase of the tip vibration when approaching the surfaces. Their relative strengths vary differently but characteristically with the distance. The interaction starts in case of graphite at a much larger distance. The adiabatic contribution is larger in the case of gold, whereas graphite shows mostly dissipative interaction. Measurements at various temperatures are performed using a gold sample, showing a dependence of the shear force on the temperature.
Micromanipulation of chloroplasts using optical tweezers
Authors: Bayoudh S. , Mehta M. , Rubinsztein‐Dunlop H. , Heckenberg N. R. , Critchley C.
Published: March 2003

This paper describes experiments using optical tweezers to probe chloroplast arrangement, shape and consistency in cells of living leaf tissue and in suspension. Dual optical tweezers provided two‐point contact on a single chloroplast or two‐point contact on two adhered chloroplasts for manipulation in suspension. Alternatively, a microstirrer consisting of a birefringent particle trapped in an elliptically polarized laser trap was used to induce motion and tumbling of a selected chloroplast suspended in a solution. We demonstrate that displacement of chloroplasts inside the cell is extremely difficult, presumably due to chloroplast adhesion to the cytoskeleton and connections between organelles. The study also confirms that the chloroplasts are very thin and extremely cup‐shaped with a concave inner surface and a convex outer surface.
Peculiarities of imaging one‐ and two‐dimensional structures using an electron microscope in the mirror operation mode
Authors: Nepijko S. A. , Sedov N. N. , Schönhense G.
Published: March 2003

Measurements performed in an electron microscope with the mirror operation mode are most sensitive to local electric fields and geometrical roughness of any kind of the object being studied. The object with a geometrical relief is equivalent to a smooth surface with an effective distribution of microfields. Electrons forming the image interact with the local microfields for an extended time: during approach to the object, deceleration and acceleration away from the object. As a result, the electron trajectories can be strongly distorted, and the contrast changes essentially, leading to image deformation of details of the object under investigation and to lowering of the resolution. These effects are theoretically described and are illustrated by experiments. An analysis of these effects enables the real size and the shape of the object involved to be reconstructed.
Artefacts in restored images due to intensity loss in three‐dimensional fluorescence microscopy
Authors: Markham J. , Conchello J.‐A.
Published: March 2003

Computational algorithms for three‐dimensional deconvolution have proven successful in reducing blurring and improving the resolution of fluorescence microscopic images. However, discrepancies between the imaging conditions and the models on which such deconvolution algorithms are based may lead to artefacts and/or distortions in the images restored by application of the algorithms. In this paper, artefacts associated with a decrease of fluorescence intensity with time or slice in three‐dimensional wide‐field images are demonstrated using simulated images. Loss of intensity, whether due to photobleaching or other factors, leads to artefacts in the form of bands or stripes in the restored images. An empirical method for correcting the intensity losses in wide‐field images has been implemented and used to correct biological images. This method is based on fitting a decreasing function to the slice intensity curve computed by summing all pixel values in each slice. The fitted curve is then used for the calculation of correction factors for each slice.
Characterizing the deformed state in Al‐0.1 Mg alloy using high‐resolution electron backscattered diffraction
Authors: Hurley P. J. , Humphreys F. J.
Published: March 2003

The application of high resolution electron backscatter diffraction (EBSD) in a field emission gun scanning electron microscope to the characterization of a deformed aluminium alloy is discussed and the results are compared with those obtained by transmission electron microscopy. It is shown that the adequate spatial resolution, accompanied by the improvement in angular resolution to ~0.5° that can be achieved by data processing, together with the extensive quantitative data obtainable, make EBSD a suitable method for characterizing the cell or subgrain structures in deformed aluminium. The various methods of analysing EBSD data to obtain subgrain sizes are discussed and it is concluded that absolute subgrain reconstruction is the most accurate.
Light microscopy evaluation of polyurethane vascular grafts porosity by Sudan Black B staining
Authors: Soldani G. , Losi P. , Milioni C. , Raffi A.
Published: March 2003

In small‐diameter vascular grafts, the porosity of the internal surface plays an important role because it affects initial thrombus deposition and therefore the graft’s patency. As well as many other studies reported in the literature we have carried out a study of the relationship between porosity and the manufacturing parameters of polyurethane (PU) grafts by standard scanning electron microscopy (SEM) analysis. However, SEM was not completely satisfactory for evaluating the ‘sponge‐like’ uptake of water by the graft due to the unavoidable water loss and metal coating during preparation. In fact this preparation produces artefacts of the three‐dimensional porous structure. To avoid this problem we investigated the possibility of observing the graft’s internal surface through a stereomicroscope after it had absorbed water. We looked into a simple staining procedure which preferentially colours the PU graft fibres with respect to the void areas. After testing different kinds of stains, we eventually found that Sudan Black B, which usually stains for all kinds of lipid, turned out to be an excellent stain for the water‐loaded PU grafts when diluted with ethanol. This staining procedure, coupled with a computerized image analysis system, allowed us to evaluate the degree of void and average void size of the graft internal surface and to correlate these data with graft density and manufacturing parameters.
Fibre‐optic two‐photon scanning fluorescence microscopy
Authors: Bird D. , Gu M.
Published: March 2003

Two geometries of a novel two‐photon fluorescence microscope incorporating single‐mode fibre optics for the delivery of ultrashort‐pulsed illumination to a remote sample are characterized. First, a 785 nm single‐mode optical fibre is implemented in a scanning microscope, which demonstrates that an improvement in axial resolution is achieved due to the non‐linear response of the fibre to intense ultrashort‐pulsed light. Second, a 785 nm single‐mode optical fibre coupler is adapted, in which case spectral broadening and blue shifting of the ultrashort‐pulsed laser beam caused by the non‐linear response of the fibre to ultrashort‐pulsed illumination are experimentally characterized. An investigation into the impact of temporal broadening of the ultrashort‐pulsed beam on the systems is also considered. The coupling efficiency of both geometries for various illumination wavelengths is also presented. The introduction of the fibre coupler to the system has significant advantages, including an improved optical sectioning effect and a reduction in the number of bulk optical components resulting in a low‐cost, compact instrument. Sets of three‐dimensional images of fluorescent polymer microspheres and biological material confirm these features.
Automatic quantification of viability in epithelial cell cultures by texture analysis
Authors: Malpica N. , Santos A. , Tejedor A. , Torres A. , Castilla M. , García‐Barreno P. , Desco M.
Published: March 2003

Quantification of live cells in phase contrast microscopy images allows in vivo assessment of the viability of cultured cells. An automatic screening procedure seems advisable because of the large number of cells that must be counted to achieve reasonable accuracy. This paper presents a method that quantifies necrosis in cell cultures by texture analysis of microscope images.

The image is divided into regions of equal size that are classified by means of a segmentation algorithm based on texture analysis into three categories: live cells, necrotic cells and background. The classification uses three discriminant functions, built from parameters derived from the histogram and the co‐occurrence matrix and calculated by performing an initial stepwise discriminant analysis on 21 sample images from a training set.

The areas occupied by live and necrotic cells and number of live cells have been obtained for primary cellular cultures in intervals of 48 h during 2 weeks. The results have been compared with those obtained by an experienced observer, showing a very good correlation (Pearson's coefficient 0.95, kappa 0.87, N= 1600).

A method has been developed that provides an accuracy similar to that provided by an expert, while allowing a much higher number of fields to be counted.
Multiple Peptides Infrequently Coexist in Progesterone Receptor‐Containing Neurons in the Ventrolateral Hypothalamic Nucleus of the Guinea‐Pig: An Immunocytochemical Triple‐Label Analysis of Somatostatin, Neurotensin and Substance P
Authors: Dufourny Laurence , Warembourg Maryvonne , Jolivet Andre
Published: March 2003

Progesterone plays an important role in regulating reproductive behaviour in guinea‐pigs through actions exerted at the ventrolateral nucleus (VL), an area of the brain that contains progesterone receptors (PR) and neuroactive peptides, somatostatin (SOM), neurotensin (NT) and substance P (SP). Previous double‐label analyses provided evidence that a substantial proportion of these neuropeptidergic cells contain PR. By means of triple‐label immunofluorescence histochemistry, we examined whether PR are colocalized with two neuropeptides (SOM+NT or SP+SOM or SP+NT) within the same neurons in the VL. Ovariectomized guinea‐pigs were primed with estradiol to induce PR immunoreactivity, and treated with colchicine to visualize immunoreactive (IR) neuropeptidergic cells. Both monoclonal mouse PR and polyclonal rabbit neuropeptide antibodies were used in double staining and in elution‐restaining experiments. In the whole VL, the proportion of each coexisting peptide with PR obtained after double immunofluorescence appeared in decreasing order as: SOM (34%)>NT (25%)>SP (20%). Occasional colocalization was seen between PR and two neuropeptides throughout the rostrocaudal extent of the VL. Combining our various quantitative observations, we found that, of the total population of PR‐IR neurons containing any combination of SOM, NT and SP, only about 1.5% contained SOM and NT, 2% contained SP and SOM and 1.6% contained SP and NT. These results indicate that while many PR‐IR neurons also contain SOM or NT or SP in the guinea‐pig VL, there may be very few PR‐IR neurons that express more than one of these three peptides.
RAPID COMMUNICATION oPer1 is an Early Response Gene Under Photoperiodic Regulation in the Ovine Pars Tuberalis
Authors: Morgan Peter J , Ross Alexander W. , Graham E. Scott , Adam Clare , Messager Sophie , Barrett Perry
Published: March 2003

Mammalian Per1 (or RIGUI) is a recently described putative clock gene that is expressed in the suprachiasmatic nucleus. It is also expressed in the pars tuberalis (PT) of the pituitary, where melatonin appears to drive its expression. This study examines the regulation of Per1 expression. In ovine PT cells, oPer1 is an early response gene transiently expressed after stimulation with forskolin, but melatonin has no independent effect on its expression. In sheep, PT tissue photoperiodic background influences the magnitude or timing of expression of oPer1 2 h after lights‐on. These data demonstrate that oPer1 mRNA is elevated in the PT following the decline in night‐time melatonin, and that the amplitude or timing of this elevation is dependent upon the duration of the nocturnal melatonin signal.
Corticotropin Releasing Factor mRNA Expression in the Hypothalamic Paraventricular Nucleus and the Central Nucleus of the Amygdala is Modulated by Repeated Acute Stress in the Immature Rat
Authors: Hatalski Carolyn G. , Guirguis Christine , Baram Tallie Z.
Published: March 2003

Age‐appropriate acute stress, such as cold exposure, provokes the secretion of corticotropin releasing factor (CRF) from the hypothalamus, leading to a robust increase of plasma corticosterone in the immature rat. This activation of the hypothalamic‐pituitary‐adrenal system is accompanied by a stress‐induced increase of steady‐state CRF‐mRNA expression in the hypothalamic paraventricular nucleus (PVN). In the current study, we analysed changes in CRF‐mRNA expression in the PVN and the central nucleus of the amygdala (ACe) in the immature rat in response to a single episode of cold stress and three repeated exposures to this same stressor. CRF‐mRNA expression in the PVN increased after a single, but not repeated exposures to cold stress, while repeated acute stress increased the content of the CRF peptide in the anterior hypothalamus. In the ACe, repeated episodes of cold stress resulted in increased expression of CRF‐mRNA. These findings indicate a differential regulation of CRF gene expression in the PVN and ACe of the immature rat by single and repeated acute stress.
Excitatory Influence of the Locus Coeruleus in Hypothalamic‐Pituitary‐Adrenocortical Axis Responses to Stress
Authors: Ziegler  , Cass  , Herman
Published: March 2003

The locus coeruleus (LC) is a key brainstem region involved in arousal and is highly responsive to alerting/stressful stimuli, including those that activate the hypothalamic‐pituitary‐adrenocortical (HPA) axis. It is currently unclear whether the LC exerts any regulatory influence on the HPA axis and, consequently, on neuroendocrine responses to stress. The present studies were designed to test the hypothesis that the LC promotes HPA axis responses to acute and chronic stress. Adult male rats received bilateral (6‐hydroxydopamine) lesions of the LC that produced severe cell loss in the LC and 80% depletion of noradrenaline in medial prefrontal cortex. Notably, lesions did not affect dopamine‐β‐hydroxylase protein content in the parvocellular paraventricular nucleus (PVN), indicating a lack of collateral damage to other ascending noradrenergic pathways. LC lesions significantly reduced peak adrenocorticotropic hormone (ACTH) and corticosterone responses to 30 min acute restraint stress. However, LC lesions did not significantly attenuate neuroendocrine or other physiological responses to a 4‐week chronic variable stress regimen. LC lesions did not substantially affect basal concentrations of plasma corticosterone or corticotropin‐releasing hormone mRNA expression in the hypothalamic paraventricular nucleus following chronic stress. We conclude that the LC is a HPA‐excitatory brain region, promoting neuroendocrine and physiological responses primarily to acute stress. However, a potential role for the LC in the induction of HPA axis hyperactivity following chronic stress can not be ruled out.
Evidence that Cholecystokinin Receptors are not Involved in the Hypothalamic‐Pituitary‐Adrenal Response to Intraperitoneal Administration of Interleukin‐1β
Authors: Day  , Akil
Published: March 2003

The purpose of this study was to investigate a putative role for cholecystokinin (CCK) in the activation of the hypothalamic‐pituitary‐adrenal (HPA) axis following intraperitoneal (i.p.) administration of interleukin‐1‐beta (IL‐1β). Previous studies predict that CCKA receptors on vagal sensory afferents may be involved in the initiation of the stress response following an acute i.p. injection of IL‐1β. Adult male rats were given an i.p. injection of a specific CCKA (devazepide, 1 mg/kg) or CCKB (CI‐988, 1 mg/kg) receptor antagonist, 30 min prior to an i.p. injection of rat recombinant IL‐1β (rrIL‐1β), 0.5 μg/kg in 0.9% sterile saline/0.01% rat serum albumin. Blood samples were obtained via an indwelling jugular vein catheter, and the plasma levels of the stress hormones ACTH (adrenocorticotropin hormone) and corticosterone analysed over time as an indicator of HPA axis activation. This dose of rrIL‐1β resulted in a significant release of ACTH and corticosterone, peaking at 30–60 min, and returning to basal levels by 2 h. Pretreatment with either devazepide or CI‐988 had no effect on the rrIL‐1β induced ACTH or corticosterone release. In contrast, the same dose of devazepide completely inhibited the ACTH and corticosterone response to i.p. CCK (octapeptide, sulphated form, CCK‐8S), 5 μg/kg. It is concluded that CCK receptors are not involved in the hormonal stress response to a submaximal i.p. dose of rrIL‐1β.
Serotoninergic and Suprachiasmatic Nucleus Involvement in the Corticotropic Response to Systemic Endotoxin Challenge in Rats
Authors: Givalois  , Becq  , Siaud  , Ixart  , Assenmacher§  , Barbanel¶
Published: March 2003

We have investigated whether the serotonin system participates in the mechanisms underlying the corticotropic response in experimentally infected rats. Intra‐arterial injection of lipopolysaccharide (LPS; 25 μg/kg b.w.) resulted in a slight but significant increase in serotonin (5‐HT) metabolism, detectable 60 min after the stimulus and lasting more than 480 min. Adrenocorticotropin (ACTH) and corticosterone (CORT) responses in intact rats conformed to earlier reports, increasing as early as 30 min after LPS injection and reaching maximal concentrations in the circulation 60 min after the bacterial endotoxin injection. Plasma concentrations of interleukin‐1β (IL‐1β) increased only after 60 min, reaching maximal levels 120 min after LPS. Depletion of hypothalamic 5‐HT (−93%) by pretreatment of the animals with para‐chlorophenylalanine (p‐CPA), resulted in a halved ACTH response to LPS, despite an overall unchanged secretory pattern. Neither CORT nor IL‐1β secretory patterns were affected in these rats pretreated with p‐CPA. Complete bilateral electrochemical lesions of the suprachiasmatic nucleus (SCN), which is innervated by mesencephalic 5‐HT, impaired the early phase of the ACTH (−75% at 30 min) and CORT (−40% at 30 min) responses but did not affect the later increases of the corticotropic and the plasma IL‐1β responses following the LPS injection. These results indicate that serotonin pathways and SCN are involved in the earlier mechanisms of corticotropic axis recruitment following systemic LPS endotoxemia.
Differential Regulation of Neuropeptide Y mRNA Expression in the Arcuate Nucleus and Locus Coeruleus by Stress and Antidepressants
Authors: Makino  , Baker  , Smith  , Gold
Published: March 2003

In rats, circulating corticosterone and insulin are involved in regulation of the hypothalamic neuropeptide Y (NPY) system, which in turn, is involved in regulation of the hypothalamic‐pituitary‐adrenal (HPA) axis. Since the HPA axis and stress responsivity is altered in diseases such as depression, we investigated interactions between the effects of stress and antidepressant drug treatment on arcuate nucleus and locus coeruleus NPY mRNA expressions using in‐situ hybridization histochemistry. After acute (2 h) and repeated immobilization (2 h daily, for 14 days), plasma concentrations of corticosterone increased, and those of insulin decreased. The expression of NPY mRNA was significantly increased in the arcuate nucleus, but was unchanged in the locus coeruleus following acute and repeated immobilization. Adrenalectomized rats with systemic corticosterone replacement (ADX+CORT), whose corticosterone concentration was maintained at ≈50–100 ng/ml during repeated stress, showed a decrease in plasma insulin and an increase in arcuate nucleus NPY mRNA similar to that observed in sham rats, suggesting that changes in NPY mRNA levels are more closely tied to circulating insulin than to circulating corticosterone. In contrast, locus coeruleus NPY mRNA expressions in ADX+CORT rats were significantly higher than those in sham rats after repeated stress. Desmethylimipramine (DMI) treatment for 24 days did not affect basal plasma concentrations of corticosterone or insulin, or arcuate nucleus NPY mRNA expressions, but significantly decreased basal levels of locus coeruleus NPY mRNA compared to saline‐treated rats. After repeated immobilization (2 h daily, for 4 days), DMI significantly reduced the stress‐induced rise in locus coeruleus NPY mRNA levels, but potentiated the stress‐induced rise in arcuate nucleus NPY mRNA expression. These results demonstrate that: (1) the increase in arcuate nucleus NPY mRNA expressions in stressed rats closely follows the decrease in plasma concentrations of insulin; (2) increases in NPY mRNA expressions occur in the absence of changes in plasma corticosterone; and (3) desipramine treatment potentiated the effect of stress on arcuate nucleus NPY mRNA expressions, but blocked the repeated stress‐induced increase in locus coeruleus NPY mRNA expressions. Thus, NPY mRNA expression in the arcuate nucleus and the locus coeruleus is sensitive to the effects of stress and to the antidepressant drug desipramine, but the arcuate nucleus NPY system is regulated by different mechanisms than the locus coeruleus NPY system. The results provide further evidence for the importance of circulating insulin in the regulation of the arcuate nucleus NPY system.
Chronic Corticosterone Administration Dose‐Dependently Modulates Aβ(1–42)− and NMDA‐Induced Neurodegeneration in Rat Magnocellular Nucleus Basalis
Authors: Abrahám I. , Harkany T. , Horvath K. M. , Veenema A. H. , Penke B. , Nyakas C. , Luiten P. G. M.
Published: March 2003

The impact of glucocorticoids on β‐amyloid(1–42) (Aβ(1–42)) and NMDA‐induced neurodegeneration was investigated in vivo. Aβ(1–42) or NMDA was injected into the cholinergic magnocellular nucleus basalis in adrenalectomized (ADX) rats, ADX rats supplemented with 25%, 100%, 2×100% corticosterone pellets, or sham‐ADX controls. Aβ(1–42)‐ or NMDA‐induced damage of cholinergic nucleus basalis neurones was assessed by quantitative acetylcholinesterase histochemistry. Plasma concentrations of corticosterone and cholinergic fibre loss after Aβ(1–42) or NMDA injection showed a clear U‐shaped dose–response relationship. ADX and subsequent loss of serum corticosterone potentiated both the Aβ(1–42) and NMDA‐induced neurodegeneration. ADX+25% corticosterone resulted in a 10–90 nM plasma corticosterone concentration, which significantly attenuated the Aβ(1–42) and NMDA neurotoxicity. ADX+100% corticosterone (corticosterone concentrations of 110–270 nM) potently decreased both Aβ(1–42)‐ and NMDA‐induced neurotoxic brain damage. In contrast, high corticosterone concentrations of 310–650 nM potentiated Aβ(1–42)‐ and NMDA‐triggered neurodegeneration. In conclusion, chronic low or high corticosterone concentrations increase the vulnerability of cholinergic cells to neurotoxic insult, while slightly elevated corticosterone levels protect against neurotoxic injury. Enhanced neurotoxicity of NMDA in the presence of high concentrations of specific glucocorticoid receptor agonists suggests that the corticosterone effects are mediated by glucocorticoid receptors.
Vasopressin (V1a) Receptor Binding, mRNA Expression and Transcriptional Regulation by Androgen in the Syrian Hamster Brain
Authors: Young L. J. , Wang Z. , Cooper T. T. , Albers H. Elliott
Published: March 2003

Arginine vasopressin plays an important role in the regulation of social behaviours in rodents. In the Syrian hamster, vasopressin injected directly into the brain stimulates scent marking and aggressive behaviour in a steroid dependent manner and is therefore a useful model for investigating steroid‐peptide–behaviour interactions. In this study, we used in situ hybridization and radioligand binding assays on adjacent sections of hamster brains to compare the relative distribution of vasopressin (V1a) receptor mRNA and V1a receptor binding. V1a receptor mRNA and binding are abundant in the lateral septum, bed nucleus of the stria terminalis, medial preoptic nucleus, anterodorsal thalamus and suprachiasmatic nucleus. Moderate receptor binding and low levels of receptor mRNA are present in the central nucleus of the amygdala and a lateral zone from the medial preoptic area through the anterior hypothalamus. V1a receptor mRNA is anatomically more restricted in several areas compared to the ligand binding pattern, which is consistent with significant spread of receptor protein along neuronal processes. Comparison of V1a receptor ligand binding and mRNA in intact, castrated, and castrated‐testosterone treated animals reveals that V1a receptors in the medial preoptic nucleus are regulated by androgen, most likely by an upregulation of V1a receptor gene expression in a cluster of neurones concentrated in the ventromedial part of this nucleus. This study confirms the presence of the V1a subtype of vasopressin receptors in behaviourally important regions of the hamster brain and suggests that transcriptional regulation by gonadal steroids may play a role in modulating behavioural sensitivity to vasopressin.
Effect of Superior Cervical Ganglionectomy on 24‐h Variations in Hormone Secretion from the Anterior Hypophysis and in Hypothalamic Monoamine Turnover During the Preclinical Phase of Freund's Adjuvant Arthritis in Rats
Authors: Castrillón P. O. , Cardinali D. P. , Pazo D. , Cutrera R. A. , Esquifino A. I.
Published: March 2003

The effect of superior cervical ganglionectomy (SCGx) on 24‐h rhythms of circulating adrenocorticotropic hormone (ACTH), growth hormone (GH), prolactin and luteinizing hormone (LH) and of hypothalamic noradrenaline content and dopamine and serotonin turnover, was assessed in rats 3 days after administering Freund's complete adjuvant. In sham‐operated rats, Freund's adjuvant injection increased serum ACTH without affecting its diurnal rhythmicity. SCGx, performed 10 days earlier, suppressed 24‐h rhythmicity and augmented mean values of circulating ACTH. A depressive effect of immunization on GH release was found in both sham‐operated and SCGx rats. GH concentrations did not exhibit diurnal rhythmicity and decreased after immunization. Time‐of‐day‐related changes in serum prolactin were significant for all examined groups, except for SCGx‐immunized rats. Freund's adjuvant administration augmented prolactin secretion. Daily changes in serum LH concentration and a decrease after immunization were found in both sham‐operated and SCGx rats. SCGx: (i) counteracted inhibition of daily variations of noradrenaline content in medial hypothalamus of Freund's adjuvant‐injected rats; (ii) decreased anterior hypothalamic dopamine turnover and augmented it in the medial hypothalamus; (iii) lowered amplitude of serotonin turnover rhythm in medial hypothalamus. The data indicate that several early changes in levels and 24‐h rhythms of circulating ACTH and prolactin, and in hypothalamic noradrenaline content and dopamine and serotonin turnover, were modified by a previous SCGx in Freund's adjuvant‐injected rats.
Agonist‐Mediated Endocytosis of Rat Somatostatin Receptor Subtype 3 Involves β‐Arrestin and Clathrin Coated Vesicles
Authors: Kreuzer O. J. , Krisch B. , Déry O. , Bunnett N. W. , Meyerhof W.
Published: March 2003

Agonist‐induced endocytosis of somatostatin receptors determines subsequent cellular responsiveness to peptide agonist and influences somatostatin receptor scintigraphy, a technique to image various tumours. We examined the internalization of sst3HSV, an epitope‐tagged type 3 somatostatin receptor, in transfected rat neuroendocrine insulinoma cells. Stimulation of these cells with somatostatin induced trafficking of coexpressed enhanced green fluorescence protein/β‐arrestin1 fusion protein and sst3HSV to colocalize in the same endocytic vesicles. Coexpression of a dominant negative mutant of the arrestin fusion protein with the receptor blocked the internalization of sst3HSV. Stimulation with somatostatin also induced the transient translocation of α‐adaptin, a component of the adaptor protein complex 2, to the plasma membrane. α‐adaptin and clathrin colocalized with the receptor. By electron microscopy, we observed internalized sst3 in clathrin coated pits, endosomes and at the limiting membrane of multivesicular bodies, a location typical for receptors being recycled. Concordantly, we observed sst3HSV colocalized with Rab11 in a perinuclear compartment which is likely to correspond to the pericentriolar recycling endosome. Thus, agonist‐induced endocytosis of sst3 depends on its interaction with β‐arrestin, involves the adaptor protein complex 2 and proceeds via clathrin coated vesicles to the recycling compartment.