bluuebunny/crossref_metadata_2025 huggingface dataset:
Polarization properties of bent‐type optical fibre probe for magneto‐optical imaging
Authors: Ishibashi T. , Yoshida T. , Iijima A. , Sato K. , Mitsuoka Y. , Nakajima K.
Published: March 2003

Quantitative evaluation of magneto‐optical parameters is necessary in order to apply scanning near‐field optical microscope (SNOM) technology to the study of magnetism on the mesoscopic scale. For this purpose, quantitative knowledge of polarization transmission properties through an optical fibre probe is required. We therefore determined the Stokes parameters of the bent‐type optical fibre probe that is used as a cantilever for atomic force microscope operation in our SNOM system. As a result, it is found that the degree of polarization is maintained in the light emitted from the probe, although the probe acts as if it were a wave plate. This anisotropic polarization state of the light emitted from the probe was compensated for by using a Berek compensator placed in front of the fibre coupler.
Domain formation in thin lipid films probed with near‐field scanning optical microscopy
Authors: Shiku H. , Dunn R. C.
Published: March 2003

High‐resolution near‐field scanning optical microscopy (NSOM) fluorescence and topographic images of l‐α‐dipalmitoylphosphatidylcholine (DPPC) monolayers doped with a fluorescent dye are presented. DPPC monolayers are deposited onto mica substrates from the air–water interface at several surface pressures using the Langmuir–Blodgett technique. Sub‐diffraction limit phase domain structures are observed in both fluorescence and topographic NSOM images of the lipid films. The morphology of the resulting monolayers depends strongly on the surface pressure and composition of the subphase used in the film transfer. Mechanisms for lipid domain formation and growth are discussed.
The depolarization near‐field scanning optical microscope: comparison of experiment and theory
Authors: Von Freymann G. , Adelmann CH. , Scheiber G. , Schimmel TH. , Wegener M.
Published: March 2003

The depolarization near‐field scanning optical microscope allows for sub‐wavelength optical resolution with uncoated optical fibre tips. We show by a simple thought experiment, by numerical solution of the Maxwell equations for a realistic geometry, and by direct experiments, that this novel apparatus images gradients of the dielectric function of a sample in the direction of the incident electric field vector. The near‐field images obtained from experiment and theory agree well.
Near‐field optical studies of local photomodification in nanostructured materials
Authors: Bragg W. D. , Safonov V. P. , Kim W. , Banerjee K. , Young M. R. , Zhu J. G. , Ying Z. C. , Armstrong R. L. , Shalaev V. M.
Published: March 2003

Fractal aggregates of silver nanoparticles are studied experimentally using atomic force microscopy and photon scanning tunnelling microscopy. Large changes in the near‐field optical response of fractal aggregates are observed after the irradiation of samples with nanosecond laser pulses. The threshold energy density for photomodification using a 532 nm laser is measured to be 9 mJ cm−2. It is shown that photomodification‐induced changes in the local optical response can be two orders of magnitude larger than changes in far‐field absorption.
Near‐field optical photomask repair with a femtosecond laser
Authors: Lieberman K. , Shani Y. , Melnik I. , Yoffe S. , Sharon Y.
Published: March 2003

We present a high‐resolution near‐field optical tool designed for repair of opaque defects in binary photomasks. Both instrument design and near‐field imaging and patterning results will be presented. Designed for ablative processing of thin metal films, the MR‐100 incorporates an industrial amplified femtosecond laser, third harmonic generator and built‐in autocorrelator. The ultrashort duration of the femtosecond pulses enables the tool to remove chrome layers with negligible damage to the surrounding metal or the underlying quartz substrate. The micropipette based near‐field writing head can deliver power densities of hundreds of GW/cm2 to spots of several hundred nanometres and below. Repairs on sample masks will be presented and the repair quality will be discussed.
Microstructure and properties of pitch‐based carbon composites
Authors: Blanco  , Santamaría  , Bermejo  , Bonhomme  , Menéndez
Published: March 2003

Pitches prepared in the laboratory by thermal treatment and air‐blowing of a commercial coal‐tar pitch were used as matrix precursors of carbon composites using granular petroleum coke, foundry coke, amorphous graphite and anthracite. Pitches were characterized by standard procedures (elemental analysis, softening point, solubility tests and carbon yield) and light microscopy (mesophase content). Pitch pyrolysis behaviour was monitored by thermogravimetric analysis and from the optical texture of cokes. Pitch wettability to the different carbons, at different temperatures, was also studied. Experimental conditions selected for the preparation of composites were based on pitch composition and properties. The main microstructural features of composites were determined by light microscopy and scanning electron microscopy. Composite properties were described in terms of their density, porosity and compressive strength, and related to composite microstructure and the characteristics of the precursors. Thermal treatment and air‐blowing of pitch improved carbon composite structure and properties. The lowest porosities and best mechanical properties were observed in those composites obtained with the thermally treated pitches combined with foundry coke and anthracite.
Post failure analysis of 0°/90° ultra high molecular weight polyethylene composite after ballistic testing
Authors: Taylor  , Carr
Published: March 2003

The ballistic performance of a 0°/90° ultra high molecular weight polyethylene (UHMWPE) composite was investigated. Two areal densities were considered: one for fragmentation protection and one for high velocity bullet protection.

The role of microscopy in the post‐failure analysis of the UHMWPE was explored. Stereo, optical and environmental scanning electron microscopy were utilized and attempts were made to correlate the results obtained with other characterization techniques available. These included high speed photography, visual inspection and ultrasonic inspection.

Visual inspection revealed the gross effects of ballistic testing. These primarily included deformation and delamination of the composite plate. The result obtained corresponded with the predicted impact event and, for high velocity bullet impact, corresponded well to the images obtained using high speed photography. Optical, stereo and electron microscopy allowed for fibre and matrix characterization. Fibre displacement and pull‐out were also observed on impact and exit faces.
The reliability of cryoSEM for the observation and quantification of xylem embolisms and quantitative analysis of xylem sap in situ
Authors: Mccully  , Shane  , Baker  , Huang  , Ling  , Canny
Published: March 2003

The reliability of cryoSEM for visualizing gas embolisms in xylem vessels of intact, functioning roots is examined and discussed. The possibility that these embolisms form as a result of freezing water columns under tension is discounted by a double‐freeze experiment. Two regions of the same root, one frozen under tension, the other isolated from the tension by the first freeze, had the same percentage of embolisms, as did also long pieces of root frozen simultaneously along their length. The reliability of energy‐dispersive X‐ray analysis to measure xylem sap concentration in situ in frozen tissue was established by measurement of KCl standard solution frozen on stubs, and within xylem vessels. Solute heterogeneity within the vessels varied with freezing procedure; deep‐freeze > LN2 > cryopliers > liquid ethane, but only the deep‐freeze method gave unsatisfactory estimates of concentration for the standard solution. It is concluded that cryoanalytical SEM is useful for direct observation of gas and liquid‐filled compartments, and for solute analyses at depth within intact plant organs.
Measurement of glutathione levels in intact roots of Arabidopsis
Authors: Fricker M. D. , May M. , Meyer A. J. , Sheard N. , White N. S.
Published: March 2003

Levels of glutathione were measured for different cell types in roots of intact Arabidopsis seedlings after labelling with monochlorobimane to give fluorescent glutathione S‐bimane (GSB) and imaging using confocal laser scanning microscopy with excitation at 442 nm. Labelling increased to a plateau in most cell types after about 15–20 min and the GSB accumulated rapidly in the vacuole. Formation of GSB in the cytoplasm was not affected by treatment with sodium azide; however, vacuolar transport of GSB was substantially inhibited under these conditions. We infer that vacuolar sequestration was mediated by a tonoplast glutathione S‐conjugate pump. Quantitative estimates of the cytoplasmic glutathione concentration involved correction for the loss in fluorescence signal with depth into the specimen using an empirically determined model derived in situ from a permeabilized root. Correction for the dilution experienced on transport into the vacuole also required an estimate of the amount of cytoplasm present in each cell type. This was achieved in two stages: first, the levels of protein were mapped after fixation, permeabilization and labelling with fluroescein isothiocyanate. Second, the corresponding cytoplasmic volume was determined as 40% for epidermal cells in the elongation zone by manual segmentation of the cytoplasm in serial optical sections. Values of relative cytoplasmic volume for other cells were extrapolated in proportion to their protein content. Using this approach, cytoplasmic glutathione concentrations were found to be 2–3 mm in most cell types. There was a marked difference between the central cells and the neighbouring, rapidly dividing initials, and between the columella cells and the outermost cells of the root cap. In the latter case, the difference was equalized in the presence of azide. This might indicate that additional cell–cell movement and preferential sequestration of GSB can occur during the detoxification process in an intact system.
Advantages of stereo imaging of metallic surfaces with low voltage backscattered electrons in a field emission scanning electron microscope
Authors: Richards R. G. , Wieland M. , Textor M.
Published: March 2003

High emission current backscattered electron (HC‐BSE) stereo imaging at low accelerating voltages (≤ 5 keV) using a field emission scanning electron microscope was used to display surface structure detail. Samples of titanium with high degrees of surface roughness, for potential medical implant applications, were imaged using the HC‐BSE technique at two stage tilts of + 3° and − 3° out of the initial position. A digital stereo image was produced and qualitative height, depth and orientation information on the surface structures was observed. HC‐BSE and secondary electron (SE) images were collected over a range of accelerating voltages. The low voltage SE and HC‐BSE stereo images exhibited enhanced surface detail and contrast in comparison to high voltage (> 10 keV) BSE or SE stereo images. The low voltage HC‐BSE stereo images displayed similar surface detail to the low voltage SE images, although they showed more contrast and directional sensitivity on surface structures. At or below 5 keV, only structures a very short distance into the metallic surface were observed. At higher accelerating voltages a greater appearance of depth could be seen but there was less information on the fine surface detail and its angular orientation. The combined technique of HC‐BSE imaging and stereo imaging should be useful for detailed studies on material surfaces and for biological samples with greater contrast and directional sensitivity than can be obtained with current SE or BSE detection modes.
X‐ray microanalysis of cultured keratinocytes: methodological aspects and effects of the irritant sodium lauryl sulphate on elemental composition
Authors: Grängsjö A. , Pihl‐Lundin I. , Lindberg M. , Roomans G. M.
Published: March 2003

Irritant substances have been shown to induce elemental changes in human and animal epidermal cells in situ. However, skin biopsies are a complicated experimental system and artefacts can be introduced by the anaesthesia necessary to take the biopsy. We therefore attempted to set up an experimental system for X‐ray microanalysis (XRMA) consisting of cultured human keratinocytes. A number of methodological aspects were studied: different cell types, washing methods and different culture periods for the keratinocytes. It was also investigated whether the keratinocytes responded to exposure to sodium lauryl sulphate (SLS) with changes in their elemental composition. The concentrations of biologically important elements such as Na, Mg, P and K were different in HaCaT cells (a spontaneously immortalized non‐tumorigenic cell line derived from adult human keratinocytes) compared to natural human epidermal keratinocytes. The washing procedure and time of culture influenced the intracellular elemental content, and rinsing with distilled water was preferred for further experiments. Changes in the elemental content in the HaCaT cells compatible with a pattern of cell injury followed by repair by cell proliferation were seen after treatment with 3.33 µm and 33 µm SLS. We conclude that XRMA is a useful tool for the study of functional changes in cultured keratinocytes, even though the preparation methods have to be strictly controlled. The method can conceivably be used for predicting effects of different chemicals on human skin.
Two new microscopical variants of thermomechanical modulation: scanning thermal expansion microscopy and dynamic localized thermomechanical analysis
Authors: Hammiche A. , Price D. M. , Dupas E. , Mills G. , Kulik A. , Reading M. , Weaver J. M. R. , Pollock H. M.
Published: March 2003

We describe two ways in which thermomechanical modulation may be used in conjunction with scanning thermal microscopy, in order to distinguish between different components of an inhomogeneous sample. The sample is subjected to a modulated mechanical stress, and the heating is supplied locally by the probe itself.

Scanning thermal expansion microscopy is an imaging mode, in which an imposed localized temperature modulation is used to generate thermal expansion, which in turn produces mechanical strain and gives thermal expansion contrast images. We present results using two types of active thermal probe. For polymer/resin samples, the depth of material contributing to the measured thermal expansion is typically a few micrometres. Under certain conditions we observe a reversal in contrast as the frequency of the temperature modulation is increased.

In dynamic localized thermomechanical analysis, the modulated stress is applied directly, and accompanied by a localized temperature change, as used in other forms of localized thermal analysis. The resulting modulated lateral force signals are obtained. The glass transition of polystyrene is detected, and shows a significant variation with frequency. The amplitude or phase signal may be used to obtain image contrast for inhomogeneous samples.
An efficient algorithm for measurement and correction of chromatic aberrations in fluorescence microscopy
Authors: Kozubek M. , Matula P.
Published: March 2003

Even the best optical microscopes available on the market exhibit chromatic aberrations to some extent. In some types of study, chromatic aberrations of current optics cannot be neglected and a software correction is highly desirable. This paper describes a novel method of chromatic aberration measurement and software correction using sub‐resolution bead imaging and computer image analysis. The method is quick, precise and enables the determination of both longitudinal and lateral chromatic aberrations. Correction function can be computed in about half an hour, including image acquisition. Using this approach, chromatic aberrations can be reduced to 10–20 nm laterally and 10–60 nm axially depending on the type of optical set‐up. The method is especially suitable for fluorescence microscopy, where a limited number of wavelengths are observed.
Accumulation of in‐vivo fatigue microdamage and its relation to biomechanical properties in ageing human cortical bone
Authors: Zioupos P.
Published: March 2003

Bone matrix accumulates microdamage in the form of microcracks as a result of everyday cyclic loading activities. In two very recent studies, which used conventional histological stains and light microscopy techniques, the amount of this in‐vivo microdamage in the cortices of long bones has been shown to increase with age. These articles have suggested that in‐vivo microcracks may have an effect on the material properties of the tissue. However, a precise quantitative relationship between the number of microcracks and the mechanical properties of these same bones has not been produced before, and in particular the way the microcracks may affect the stiffness, the strength or possibly the toughness of the tissue. This article presents an examination of the in‐vivo microdamage in human bones by the use of laser scanning confocal microscopy, which offers better discrimination and allows examination of the cracks in‐situ. Quantification of in‐vivo fatigue microcracks was performed by counting the microcrack numerical density and surface density in specimens for which we have previously derived a full set of mechanical properties as a function of age. It is shown that bone microdamage relates more to the toughness (measured by three different measures) of ageing bone tissue than to its stiffness and strength. The result allows us (i) to re‐evaluate the fragility of ageing human bone and put more emphasis on its energy‐related resistance to fracture than perhaps on its stiffness or strength and also (ii) to understand more fully the causal relationship and interactions between microcracks and tissue toughness.
Probe design optimization for a high‐resolution scattering‐type scanning near‐field optical microscope
Authors: Sasaki Y. , Sasaki H.
Published: March 2003

The scattering‐type scanning near‐field optical microscope (SNOM) has a probe with a sharp tip for use in high resolution imaging. As sharp a tip as possible is generally considered ideal for the observations, but actually, a sharp tip does not always provide a high resolution SNOM image. We numerically examined the scattering property of the SNOM probe by the three dimensional finite difference time domain method. In this paper, we show the criterion for the ideal scattering probe which satisfies the simple relation between radius and taper angle of the tip.
On the quantum physical relation between photon tunnelling and near‐field optics
Authors: Keller O.
Published: March 2003

The standard theory of optical tunnelling based on a mathematical analogy between the time‐independent one‐particle Schrödinger equation describing electron tunnelling and the Helmholtz equation for the macroscopic electric field is criticized. In a classical perspective photons are related to the dynamics of the transverse part of the electromagnetic field, and with the assumption that the electrons in a coupled photon‐atom system are driven in a linear fashion by the field, a rigorous integro‐differential equation for the transverse field is established. In the near‐field zone of matter a transverse self‐field exists and this may cause the appearance of superluminality in optical tunnelling. In the wake of a brief review of the space‐time dynamics of free photons, a first‐quantized description of the birth process of a single polychromatic photon in the near‐field region of an active atom (molecule, mesoscopic particle) is presented, and afterwards the link between photon localizability, Einstein causality and near‐field photon tunnelling is discussed. On the basis of a new one‐photon quantum theory of near‐field scattering from a microscopic (or mesoscopic) object, it is shown that photon tunnelling always appears in photon‐atom scattering. A first‐order Born approximation cannot capture the phenomenon, however. The emergence of the energy wave function of the scattered photon is followed in space and time.
3D light scanning macrography
Authors: Huber D. , Keller M. , Robert D.
Published: March 2003

The technique of 3D light scanning macrography permits the non‐invasive surface scanning of small specimens at magnifications up to 200×. Obviating both the problem of limited depth of field inherent to conventional close‐up macrophotography and the metallic coating required by scanning electron microscopy, 3D light scanning macrography provides three‐dimensional digital images of intact specimens without the loss of colour, texture and transparency information. This newly developed technique offers a versatile, portable and cost‐efficient method for the non‐invasive digital and photographic documentation of small objects. Computer controlled device operation and digital image acquisition facilitate fast and accurate quantitative morphometric investigations, and the technique offers a broad field of research and educational applications in biological, medical and materials sciences.
Misorientation distributions in hot deformed NaCl using electron backscattered diffraction
Authors: Pennock G. M. , Drury M. R. , Trimby P. W. , Spiers C. J.
Published: March 2003

EBSD orientation mapping has been used to derive subgrain boundary misorientation distributions in a series of hot deformed and etched NaCl samples. The main objective of this study has been to examine the influence of data processing, noise caused by angular resolution limits and step size on the subgrain misorientation distributions in hot deformed NaCl. Processing of non‐indexed EBSD patterns increased the average misorientations in etched NaCl. Noise contributed significantly to low angle misorientation peaks for step sizes less than the minimum subgrain size. Orientation data collected using a step size larger than the average subgrain size cumulated misorientations across individual subgrains and effectively measured an orientation gradient between steps. Orientation gradient distributions were not influenced by noise. Average misorientation values calculated from large step data correlated well with average misorientation from small step size data. Average misorientations showed a power law relationship with strain. Three types of substructures were identified using scanning electron microscopy and EBSD mapping, equiaxed subgrains, long subgrain boundaries and a core‐mantle subgrain arrangement.
Quantitative phase‐amplitude microscopy I: optical microscopy
Authors: Barone‐Nugent E. D. , Barty A. , Nugent K. A.
Published: March 2003

In this paper, the application of a new optical microscopy method (quantitative phase‐amplitude microscopy) to biological imaging is explored, and the issue of resolution and image quality is examined. The paper begins by presenting a theoretical analysis of the method using the optical transfer function formalism of Streibl (1985). The effect of coherence on the formation of the phase image is explored, and it is shown that the resolution of the method is not compromised over that of a conventional bright‐field image. It is shown that the signal‐to‐noise ratio of the phase recovery, however, does depend on the degree of coherence in the illumination.

Streibl (1985) notes that partially coherent image formation is a non‐linear process because of the intermingling of amplitude and phase information. The work presented here shows that the quantitative phase‐amplitude microscopy method acts to linearize the image formation process, and that the phase and amplitude information is properly described using a transfer function analysis. The theoretical conclusions are tested experimentally using an optical microscope and the theoretical deductions are confirmed.

Samples for microscopy influence both the phase and amplitude of the light wave and it is demonstrated that the new phase recovery method can separate the amplitude and phase information, something not possible using traditional phase microscopy. In the case of a coherent wave, knowledge of the phase and amplitude constitutes complete information that can be used to emulate other forms of microscopy. This capacity is demonstrated by recovering the phase of a sample and using the data to emulate a differential interference contrast image.
Direct modulation of the effective sensitivity of a CCD detector: a new approach to time‐resolved fluorescence imaging
Authors: Mitchell A. C. , Wall J. E. , Murray J. G. , Morgan C. G.
Published: March 2003

In this paper a novel approach to frequency‐domain fluorescence lifetime imaging (FLIM) is described. In a CCD camera a single pixel is defined by a charge pattern on a group of electrodes. By modulation of the pattern of voltages defining the pixel structure it is possible to modulate the sensitivity of the CCD at radio frequency. The modulation enhances the noise performance of the CCD, in contrast to the deterioration in performance seen when an intensifier stage is similarly modulated. The new technology has potential applications to a wide range of assays as well as in conventional FLIM applications. Unlike intensifier‐based systems, the directly modulated CCD is physically small, inexpensive, robust and offers superior resolution and noise performance.
Measurement of magnetic domain wall width using energy‐filtered Fresnel images
Authors: Lloyd S. J. , Loudon J. C. , Midgley P. A.
Published: March 2003

Magnetic domain walls in Nd2Fe14B have been examined using a series of energy‐filtered Fresnel images in the field emission gun transmission electron microscope (FEGTEM). We describe the changes in the intensity distribution of the convergent wall image as a function of defocus, foil thickness and domain wall width. The effect of tilted domain walls and beam convergence on the fringe pattern is also discussed. A comparison of the experimental intensity profile with that from simulations allows the domain wall width to be determined. Measurement of very narrow walls is made possible only by using a relatively thick foil, which necessitates energy‐filtering to allow quantitative comparison with simulations. The magnetic domain wall width in Nd2Fe14B was found to be 3 ± 2 nm.
Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser
Authors: Karpova T. S. , Baumann C. T. , He L. , Wu X. , Grammer A. , Lipsky P. , Hager G. L. , McNally J. G.
Published: March 2003

One manifestation of fluorescence resonance energy transfer (FRET) is an increase in donor fluorescence after photobleaching the acceptor. Published acceptor‐photobleaching methods for FRET have mainly used wide‐field microscopy. A laser scanning confocal microscope enables faster and targeted bleaching within the field of view, thereby improving speed and accuracy. Here we demonstrate the approach with CFP and YFP, the most versatile fluorescent markers now available for FRET. CFP/YFP FRET imaging has been accomplished with a single laser (argon) available on virtually all laser‐scanning confocal microscopes. Accordingly, we also describe the conditions that we developed for dual imaging of CFP and YFP with the 458 and 514 argon lines. We detect FRET in a CFP/YFP fusion and also between signalling molecules (TNF‐Receptor‐Associated‐Factors or TRAFs) that are known to homo‐ and heterotrimerize. Importantly, we demonstrate that appropriate controls are essential to avoid false positives in FRET by acceptor photobleaching. We use two types of negative control: (a) an internal negative control (non‐bleached areas of the cell) and (b) cells with donor in the absence of the acceptor (CFP only). We find that both types of negative control can yield false FRET. Given this false FRET background, we describe a method for distinguishing true positive signals. In summary, we extensively characterize a simple approach to FRET that should be adaptable to most laser‐scanning confocal microscopes, and demonstrate its feasibility for detecting FRET between several CFP/YFP partners.
Reduced Noradrenergic Tone to the Hypothalamic Paraventricular Nucleus Contributes to the Stress Hyporesponsiveness of Lactation
Authors: Toufexis Donna J. , Thrivikraman  , Plotsky Paul M. , Morilak David A. , Huang Ning , Walker Claire‐Dominique
Published: March 2003

Lactation in mammals is accompanied by a marked decrease in stress responsiveness that we previously attributed, in part, to a reduction in noradrenergic (NA) innervation of hypothalamic paraventricular nucleus (PVN) neurons controlling neuroendocrine stress responses. In the present study, we compared in‐vivo PVN catecholamine secretion by microdialysis between nonlactating and lactating females and tested the effects of NA α–1 and α–2 receptor antagonists (corynanthine and idazoxan, respectively) on the acute stress response of lactating and virgin female rats. To determine if PVN α–adrenoreceptor density, affinity, or synthesis, changes as a function of lactation, we performed receptor autoradiography, Scatchard analysis and in situ hybridization of α–adrenoreceptors. Densitometric analysis of the α–adrenoreceptors in the supraoptic nucleus (SON) was used to evaluate changes in magnocellular neurons. Endogenous PVN norepinephrine release under basal conditions was lower in lactating females than in females who had their pups removed for 2 days, and microdialysate concentrations of adrenaline and MHPG were attenuated in lactating females. α–2 adrenoreceptor density in the PVN showed a significant decrease from lactation day 3 to lactation days 10–12 and a reduction to 40% of virgin controls on days 10–20 of lactation. A similar pattern was observed for the SON. The affinity of hypothalamic α–2 adrenoreceptors was reduced as a function of lactation. α–1 adrenoreceptor density in the PVN and in the hypothalamus rose as a function of lactation, although the affinity of these receptors was not altered. In contrast, α–1D adrenoreceptor subtype mRNA expression in the PVN decreased in middle lactating females (day 10) compared to virgins. Intracerebroventricular (icv) application of idazoxan, significantly increased the ACTH response to swim stress in virgin females, but had the opposite effect in lactating females. In contrast, icv corynanthine treatment significantly decreased the ACTH response in virgins, but not in lactating females. Overall, these data suggest that the secretion of NA in the PVN is reduced during lactation, and that the ability of PVN parvocellular neurons to respond to changes in synaptic NA levels (i.e. after stress) is also altered.
Immunohistochemical Distribution of Oestrogen Receptor and Luteinizing Hormone B Subunit in the Ovine Pituitary Gland During Foetal Development
Authors: Sheng  , McNeilly  , Brooks
Published: March 2003

The presence of oestrogen receptor in the developing hypothalamo‐hypophyseal system is an essential prerequisite for the development of sex‐steroid feedback on gonadotrophin secretion. We have used dual immunocytochemistry to examine the ontogeny and regional distribution of oestrogen receptor and LHβ subunit in the ovine pituitary gland during foetal development. At day 65 gestation (term=145 days) oestrogen receptor and LHβ immunopositive cells are found in a small region at the base of the anterior pituitary gland, and also in a band immediately adjacent to the neurointermediate lobe. By day 100 gestation there was a significant increase in the number of immunopositive LHβ cells accounting for around 12% of the total cell population, and these were widely distributed throughout the anterior pituitary gland. There was also a significant increase in the proportion of gonadotrophs which contain oestrogen receptor compared with day 65. By day 130 gestation the percentage of LH containing cells had declined to around 7% of the total population, but the proportion which also contained oestrogen receptor remained the same. There were no differences in the numbers or distribution of cells containing LH or oestrogen receptors between male and female foetuses, at any age. These data describing a parallel change in the number of oestrogen receptors and LHβ containing cells in the pituitary gland throughout gestation suggest that the development of pituitary sex‐steroid feedback is not solely dependent on changes in the numbers of oestrogen receptor containing cells alone.
Sleep Endocrine Effects of Megestrol Acetate in Healthy Men
Authors: Wiedemann Klaus , Hirschmann Margarete , Knaudt Kristina , Rupprecht Rainer , Eckart Seier Fried , Holsboer Florian
Published: March 2003

Synthetic and naturally occurring steroids exert a variety of neural effects that include modulation of nocturnal sleep. The present study focuses on the effect of progesterone receptor (PR) activation on the nocturnal sleep electroencephalogram (EEG) in male volunteers. As a PR ligand, the synthetic progesterone megestrol was used, which has the advantage over progesterone in that it is not metabolized into other steroid compounds which could cloud the progesterone‐mediated effects through their own neuroactive properties. Nine healthy male volunteers were investigated in a prospective single‐blind randomized study design. They received either placebo tablets or megestrol acetate dosages of 160, 320 or 480 mg at 14.00 h and 19.00 h. Blood samples were drawn half‐hourly from 22.00 h until 07.00 h. After 320 mg megestrol, plasma adrenocorticotropin secretion was lower and growth hormone secretion was higher than after 160 mg and 480 mg megestrol or placebo. Similarly, the reduction in the relative amount of rapid eye movement sleep was most pronounced after 320 mg. Thus, progesterone receptor activation, as reflected by the sleep EEG and associated pituitary hormone secretion, follows a nonlinear U‐shape dose dependency of a well‐defined PR ligand, which may explain the unresolved inconsistencies of neuroendocrine progesterone effects to date. Moreover, employing a CV1 cell line, contransfected with a human glucocorticoid receptor expression vector and a reporter gene‐based detection system for transcriptional activity, revealed that a PR agonist such as megestrol may also activate glucocorticoid receptors. This may account for some of the neuroendocrine effects of megestrol and other progestins.
Induction of Pituitary Cytokine Transcripts by Peripheral Lipopolysaccharide
Authors: Whiteside  , Quan  , Herkenharn
Published: March 2003

Systemically administered lipopolysaccharide (LPS) elicits profound changes in pituitary hormone secretion. Pro‐inflammatory cytokines have been proposed as mediators of these responses. In this study, we used in‐situ hybridization histochemistry to investigate LPS‐induced cytokine gene expression in the rat pituitary. After i.p. or i.v. injection of various doses of LPS, mRNA for the immediate‐early gene IκBu (an inhibitor of NF‐κB, a transcription factor that regulates the expression of many pro‐inflammatory cytokines) was induced in the anterior lobe as early as 0.5 h. The induced IκBα mRNA expression peaked at 1 h. In the posterior lobe, IκBα mRNA was first induced at 0.5 h and peaked at 2 h. A similar spatiotemporal pattern of interleukin‐1b (IL‐1) mRNA induction was observed. In addition, at 2 h after injection, TNFα, IL‐1β converting enzyme (ICE), and IL‐1 receptor antagonist (IL‐1RA) mRNAs were induced in both anterior and posterior lobes. Type 1 IL‐1 receptor (IL‐1R1) mRNA was constitutively expressed in the pituitary, and its expression level did not change after the LPS injection. Interestingly, the mRNA coding for glial fibrillary acidic protein (GFAP), an astrocyte marker, was selectively induced in the posterior lobe at 2 h after LPS injection, suggesting that LPS affects pituicyte function. Together, these results suggest that LPS acts directly on the pituitary to rapidly induce cytokine expression. Locally synthesized cytokines may activate cytokine receptor bearing cells to modulate the endocrine activities of the pituitary.
Effect of Corticotropin‐Releasing Hormone Antagonist on Oestrogen‐Dependent Glucoprivic Suppression of Luteinizing Hormone Secretion in Female Rats
Authors: Tsukahara  , Tsukamura  , Foster  , Maeda
Published: March 2003

Pharmacological reduction of glucose availability with 2‐deoxyglucose (2DG) suppresses pulsatile luteinizing hormone (LH) secretion in rats and growth‐retarded lambs. Gonadal steroids enhance the glucoprivic suppression of LH secretion in rats. The present study determined if corticotropin‐releasing hormone (CRH) plays a role in mediating oestrogen‐dependent and ‐independent glucoprivic suppression of LH secretion. The study was conducted in ovariectomized (OVX) rats some of which received Silastic implants containing oestradiol‐17β (OE2) dissolved in peanut oil at 20 μg/ml to produce a physiological plasma level of OE2 (30 pg/ml). Seven days after ovariectomy, the rats were stereotaxically implanted with a guide cannula into the third cerebral ventricle. Seven days later, blood samples were collected through an indwelling atrial cannula every 6 min for 3 h for LH pulse determination. After the first hour of blood sampling, a CRH antagonist, [
d‐Phe12, Nle21,38]hCRF‐(21–41), or vehicle was injected into the third cerebral ventricle through the implanted cannula before 2DG administration through the indwelling atrial cannula. Pulsatile LH secretion was suppressed by 2DG (200 mg/kg b.w.) in the vehicle‐treated rats bearing OE2 implants. The CRH antagonist (5.65 nmol) blocked the suppressive effect of 2DG on pulsatile LH secretion in the OE2‐treated OVX animals. On the other hand, in the absence of oestrogen, the effect of a twice greater dose of 2DG (400 mg/kg b.w.) was not blocked by five times greater amount of CRH antagonist (28.3 nmol). These results suggest the mechanisms mediating glucoprivic suppression of LH secretion involve two components: one is oestrogen‐dependent and the other oestrogen‐independent. CRH may be involved in the oestrogen‐dependent component of glucoprivic suppression of LH secretion but not the oestrogen‐independent one.
Persistent Effects of Maternal Deprivation on HPA Regulation Can Be Reversed By Feeding and Stroking, But Not By Dexamethasone
Authors: Van Oers  , De Kloet  , Levine
Published: March 2003

Maternal deprivation of neonatal rats for 24 h has immediate and persistent effects on hypothalamic‐pituitary‐adrenal (HPA) regulation. Immediately after deprivation corticosterone (CORT) is elevated. The primary purpose of our experiments was to determine if, by preventing this CORT elevation, the persistent effects could be reversed. In experiment 1, pups were injected with dexamethasone at the onset of the 24‐h deprivation period on postnatal day 11 to suppress the rise in CORT. In experiment 2 some aspects of maternal behaviour known to suppress CORT levels were mimicked during deprivation from postnatal days 11–12. The pups were either: (1) left undisturbed; (2) stroked periodically; or (3) stroked and episodically fed. At postnatal day 20 basal and stress‐induced adrenocorticotrophic hormone (ACTH) and CORT levels were measured as well as brain mineralocorticoid (MR) and glucocorticoid receptors (GR). Neonatal rats receiving dexamethasone prior to the onset of the deprivation on day 11 did not differ on day 20 from deprived pups that were exposed to elevated CORT levels. There were no detectable changes in the non‐deprived pups that were treated with dexamethasone. In contrast, feeding and stroking during the period of deprivation obliterated the persistent effects both with regard to the reduced ACTH response and the decreased GR mRNA in hippocampus and hypothalamus. Stroking alone appears to have no influence. In conclusion, the persistent reduction of the ACTH response to mild stress and the decrease of GR mRNA is not mediated by deprivation‐induced elevations in CORT, but appears to be reversible by reinstating specific aspects of the dam’s nurturing behaviour.
Glucocorticoids Decrease Phenylethanolamine N‐Methyltransferase mRNA Expression in the Immature Foetal Sheep Adrenal
Authors: Adams  , Ross  , Butler  , McMillen
Published: March 2003

This study examined the impact of a chronic physiological elevation of plasma cortisol levels on adrenal catecholamine synthetic enzyme and proenkephalin A mRNA expression in foetal sheep. Cortisol (2.5–3.0 mg.5 ml−1.24 h−1, n=9) or saline (0.9% saline, n=6) was infused into foetal sheep for 7 days between 109 days and 116 days gestation. Foetal plasma cortisol concentrations were higher (P<0.0005) in the cortisol infused foetuses when compared with the saline infused group (43.07±4.13 nmol.l−1 vs 1.67±0.10 nmol.l−1). There were no differences, however, in the plasma ACTH levels between the two groups. Using Northern blot analysis, adrenal phenylethanolamine N‐methyltransferase (PNMT) mRNA expression was found to be reduced (P<0.005) fivefold in the cortisol infused foetuses when compared with the controls, as was the relative area of the adrenal medulla which stained positively with anti‐PNMT (28.1±2.5%vs 44.8±4.8%, P<0.007). No effect of cortisol infusion was observed on adrenal tyrosine hydroxylase mRNA and protein expression or proenkephalin A mRNA expression. We conclude that before birth, adrenaline synthesis may be suppressed by a novel direct, or indirect, inhibitory effect of glucocorticoids on PNMT mRNA expression.
Sex Differences in the Parental Behaviour of Adult Virgin Prairie Voles: Independence From Gonadal Hormones and Vasopressin
Authors: Lonstein  , De Vries
Published: March 2003

Sexually and parentally experienced prairie voles display robust biparental care of pups that is similar between the sexes. Little is known, however, about possible sex differences in the parental behaviours of sexually inexperienced prairie voles. Parental behaviour of adult virgin male and female prairie voles was examined in sham‐operated and gonadectomized subjects treated with vehicle or oestradiol. Since arginine‐vasopressin (AVP) has been suggested to stimulate parental behaviour in sexually inexperienced males, neural AVP immunoreactivity (AVP IR) was quantified. Most sham‐operated and castrated males displayed high levels of parental behaviour (9/9 controls, 6/9 castrates) during a 15‐min exposure to pups 4 weeks after surgery, and few behavioural differences were seen between groups. Conversely, almost all gonadally intact (8/9) and gonadectomized (8/9) females attacked pups. Implantation of a 0.1‐mg pellet of oestradiol immediately after gonadectomy had little effect on males (9/9 parental), whereas most (5/9) oestradiol‐treated females acted maternally. AVP‐immunoreactive (AVP‐ir) fibre density in the lateral septum (LS) and lateral habenula (LHb), expressed by the number of pixels that covered AVP‐ir fibres during computerized optical density analysis, was greater in males than females, was non‐significantly reduced in castrated males, and doubled in the LS of oestradiol‐treated females. In a second experiment, males tested 8 weeks after similar manipulations remained highly parental though castrated males had almost no AVP‐ir fibres in the LS and LHb. Levels of AVP IR in males treated with oestradiol were similar to those observed in intact males. A dramatic sex difference therefore exists in the parental behaviour of adult sexually naive prairie voles which cannot be explained by sex differences in gonadal hormones. Because both castrated and intact males were highly parental, even though castrates had virtually no AVP‐ir in the LS or LHb, AVP does not appear to be crucial for their responsiveness toward pups.
Altered Pituitary Sensitivity to Corticotropin‐Releasing Factor and Arginine Vasopressin Participates in the Stress Hyporesponsiveness of Lactation in the Rat
Authors: Toufexis  , Tesolin  , Huang  , Walker
Published: March 2003

The regulation of the activity of the hypothalamic‐pituitary‐adrenal (HPA) axis is modified during lactation, wherein a blunted stress‐induced adrenocorticotropic hormone (ACTH) and glucocorticoid secretion is coupled with elevated basal secretion of these hormones. The involvement of pituitary modifications in lactation‐induced stress hyporesponsiveness has yet to be established. In this study we tested the hypothesis that the pituitary sensitivity to corticotropin‐releasing factor (CRF) and arginine vasopressin (AVP) is altered in lactation in the rat. We examined the effect of exogenous CRF (0.1–5 μg/rat), AVP (0.01–0.5 μg/rat), and AVP (0.01–0.5 μg/rat)+CRF (0.1 μg/rat) on the ACTH response of virgin, mid‐lactating (lactation day 10–12) females, as well as nursing females separated from their pups for 48 h. Additionally, to determine if changes in CRF‐ or AVP‐receptor densities might mediate alterations in pituitary sensitivity, we compared pituitary CRF‐ and AVP‐receptor binding by autoradiography in pregnant, mid‐lactating, and virgin female rats. While both virgin and lactating female rats exhibited significant ACTH responses to CRF, the responses to the highest doses of CRF (2.0 and 5.0 μg/rat) were greater in virgin than in lactating females. Separation of the litter for 48 h partially restored pituitary responsiveness to 2.0 μg of CRF. Conversely, whereas lactating females displayed robust ACTH secretion following a high dose of AVP or following a combination of AVP+CRF, the response of virgin females was much smaller. These modifications in pituitary responsiveness were not accompanied by significant differences in pituitary CRF‐and AVP‐receptors levels between female groups. Our results demonstrate that a reduction in pituitary sensitivity to CRF, but not to AVP occur during lactation in the rat which mediates, at least in part, the stress hyporesponsiveness of lactation.
Increased Prolactin Receptor Immunoreactivity in the Hypothalamus of Lactating Rats
Authors: Pi  , Grattan
Published: March 2003

Prolactin (PRL) exerts numerous effects in the brain including induction of maternal behaviour, increased food intake, and inhibition of GnRH secretion. Knowledge about the distribution of PRL receptors (PRL‐R) in the brain will be critical for investigating mechanisms of PRL–brain interactions during lactation. The present study aimed to investigate the distribution of PRL‐R in specific hypothalamic nuclei of lactating rats by immunohistochemistry and to compare this distribution with that in dioestrous rats. Rats were perfused with 2% paraformaldehyde and brains were cut into coronal sections (18 μm) for immunostaining. Immunoreactivity was detected by the avidin biotin complex method using mouse monoclonal antibody U5. In dioestrous rats, PRL‐R immunoreactivity was observed in the choroid plexus, three hypothalamic nuclei: medial preoptic, periventricular and arcuate, and in the median eminence. The number of labelled profiles per section in the medial preoptic and arcuate nuclei increased significantly (P<0.05) in lactating rats (days 7–10 to post partum) when compared with dioestrous rats. Furthermore, in lactating rats, PRL‐R immunoreactive neurons were identified in the cerebral cortex, substantia nigra and numerous additional hypothalamic nuclei including the ventromedial preoptic, ventrolateral preoptic, lateroanterior hypothalamic, ventrolateral hypothalamic, paraventricular hypothalamic, supraoptic, suprachiasmatic, and ventromedial hypothalamic nuclei. These observations assist our understanding of the multiple sites of PRL effects on brain function during lactation.