bluuebunny/crossref_metadata_2025 huggingface dataset:
Automatic image acquisition, calibration and montage assembly for biological X‐ray microscopy
Authors: Loo  , Meyer‐Ilse  , Rothman
Published: March 2003

We describe a system for the automatic acquisition and processing of digital images in a high‐resolution X‐ray microscope, including the formation of large‐field high‐resolution image montages. A computer‐controlled sample positioning stage provides approximate coordinates for each high‐resolution subimage. Individual subimages are corrected to compensate for time‐varying, non‐uniform illumination and CCD‐related artefacts. They are then automatically assembled into a montage. The montage assembly algorithm is designed to use the overlap between each subimage and multiple neighbours to improve the performance of the registration step and the fidelity of the result. This is accomplished by explicit use of recorded stage positions, optimized ordering of subimage insertion, and registration of subimages to the developing montage. Using this procedure registration errors are below the resolution limit of the microscope (43 nm). The image produced is a seamless, large‐field montage at full resolution, assembled automatically without human intervention. Beyond this, it is also an accurate X‐ray transmission map that allows the quantitative measurement of anatomical and chemical features of the sample. Applying these tools to a biological problem, we have conducted the largest X‐ray microscopical study to date.
Morphological and structural study of pseudowollastonite implants in bone
Authors: De Aza  , Luklinska  , Martinez  , Anseau  , Guitian  , De Aza
Published: March 2003

In vitro experiments show that pseudowollastonite (α‐CaSiO3) is a highly bioactive material that forms a hydroxyapatite surface layer on exposure to simulated body fluid and also to human parotid saliva. This finding is very significant, as it indicates that the pseudowollastonite can be physically and chemically integrated into the structure of living bone tissue, and therefore could be suitable for repair or replacement of living bone.  The physical and chemical nature of the remodelled interface between the pseudowollastonite implants and the surrounding bone has been studied after in vivo implantation of 20 pseudowollastonite cylinders into rat tibias. The interfaces formed after 3, 6, 8 and 12 weeks of implantation were examined histologically using an optical microscope and also by analytical scanning electron microscopy.  SEM and X‐ray elemental analysis showed that the new bone was growing in direct contact with the implants. Other examinations found that the bone was fully mineralized. The ionic exchange taking place at the implant interface with the body fluids was essential in the process of the implant integration through a dissolution–precipitation–transformation mechanism. The study found the interface biologically and chemically active over the 12‐week implantation period. The rate of new bone formation decreased after the first 3 weeks and reached constant value over the following 9 weeks. The osteoblastic cells migrated towards the interface and colonized the surface at the contact areas with the cortical regions and also bone marrow.
Charge effect in point projection images of carbon fibres
Authors: Prigent M. , Morin P.
Published: March 2003

Nanometre‐sized carbon fibres across holes have been observed in a lensless point projection field‐emission microscope operating between 100 and 300 eV. At sufficiently high magnification fringe patterns appear; with the help of simulations we show that they are strongly dependent on the charge density of the fibres. These patterns are characterized by an odd number of fringes with a central fringe that becomes very bright as the charge increases. Average diameter and linear charge density have been obtained with remarkable precision from analysis of fringes. Charge distribution from the middle to the edge of fibres has been investigated as well as narrowings at localized places on the fringe pattern. From these two examples, the limits of the models used for the simulations and those of the data acquisition system are discussed. Finally, this work emphasizes the fact that the fringe pattern masks the actual form of the fibre and that it is necessary to take account of the charge effect to interpret this diffraction pattern.
Transmission electron microscopy of semiconductor quantum dots
Authors: Liu C.‐P. , Miller P. D. , Henstrom W. L. , Gibson J. M.
Published: March 2003

We report on plan‐view transmission electron microscopy techniques, by which the size, the actual shape and the strain of a coherent quantum dot in semiconductor heterostuctures can be measured very accurately. The bright‐field suppressed‐diffraction imaging condition where no strong diffracted beam is excited in the sample provides reliable size measurement. Using suppressed‐diffraction imaging condition, the intensity contour in a coherent island is related to the height, and thus the detailed shape and the aspect ratio can be extracted. The strain contrast of a coherent island imaged using an exact two‐beam dynamical diffraction condition is useful for strain measurement and the corresponding features is related to the shape of an island. The physical origins and accuracy of interpretation of the image contrast are discussed, using the simulations and experimental examples.
Multiphoton microscopy in life sciences
Authors: König K.
Published: March 2003

Near infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three‐dimensional fluorescence imaging based on non‐resonant two‐photon or three‐photon fluorophor excitation requires light intensities in the range of MW cm−2 to GW cm−2, which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi‐gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non‐invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depth‐resolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non‐invasive fluorophore loading into single living plant cells.

Non‐destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis‐like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two‐photon excitation process rather than a one‐photon or three‐photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two‐photon vital cell studies. In labelled cells, additional phototoxic effects may occur via photodynamic action. This has been demonstrated for aminolevulinic acid‐induced protoporphyrin IX and other porphyrin sensitizers in cells. When the light intensity in NIR microscopes is increased to TW cm−2 levels, highly localized optical breakdown and plasma formation do occur. These femtosecond NIR laser microscopes can also be used as novel ultraprecise nanosurgical tools with cut sizes between 100 nm and 300 nm. Using the versatile nanoscalpel, intracellular dissection of chromosomes within living cells can be performed without perturbing the outer cell membrane. Moreover, cells remain alive. Non‐invasive NIR laser surgery within a living cell or within an organelle is therefore possible.
Monitoring age‐related changes of collagen content and vascularity in ganglia using unbiased stereological methods
Authors: Boyd‐Clark L. C. , Galea M. P. , Briggs C. A. , Opeskin K.
Published: March 2003

We describe for the first time application of unbiased stereological techniques to estimate total volume and volume fractions of interest in individual dorsal root ganglia (DRG). Volume estimates were obtained using a two‐stage sampling design. Sections were systematically sampled following a random start, from DRG which were embedded in methacrylate and exhaustively sectioned. We further examined the efficiency of point counting irregular volume fractions housed in a regular reference volume. We found that the precision of volume estimates was relatively unaffected by exhaustive sampling, and that the magnitude of error was, in large part, determined by object shape.
Transmission electron microscopy of the interface between bone and pseudowollastonite implant
Authors: De Aza P. N. , Luklinska Z. B. , Anseau M. R. , Guitian F. , De Aza S.
Published: March 2003

This paper reports on the structural morphology of the interface in vivo between implants composed of bioactive synthetic pseudowollastonite ceramic and bone in rat tibias. Thin sections of the interfaces were examined after 6 and 8 weeks of implantation period in a high resolution transmission electron microscope up to the lattice plane resolution level.

The interfaces developed normal biological and chemical activities and remained reactive over the 8‐week period. The regions showing direct bone tissue bonding to the implant contained nanocrystals of hydroxyapatite‐like phase growing epitaxially across the interface in the [002] direction. The nanocrystals were also identified in the bone tissue formed in the interfacial area.

The reactivity of the implant caused in the first instance formation of an amorphous woven type of bone, which transformed into a crystalline lamellar type containing collagen fibres. The Ca/P ratio of the interfacial region was found to be between 1.67 in the mature bone tissue formed about 5 µm from the interface, and 2.06 in the regions right at the interface.
Pure optical contrast in scattering‐type scanning near‐field microscopy
Authors: HillenbRand R. , Knoll B. , Keilmann F.
Published: March 2003

We have enhanced the apertureless scattering‐type scanning near‐field optical microscope by two improvements which together achieve a recording of the true near field without any height‐induced artefact. These are the use of interferometric detection of the scattered light on one hand, and the use of higher‐harmonic dither demodulation of the scattered signal on the other. Here we present the basic rationale for these techniques, and give examples measured with two different experiments, one in the infrared (10 µm wavelength), the other in the visible (633 nm). The latter operates in a fully heterodyne mode and displays simultaneous images of optical near‐field phase and amplitude, at below 10 nm resolution.
Near‐field fluorescence imaging and simultaneous observation of surface potentials
Authors: Kwak K. J. , Hosokawa T. , Yamamoto N. , Muramatsu H. , Fujihira M.
Published: March 2003

We present a new detection method to measure simultaneously surface potential and fluorescence intensity distributions using a combined scanning near‐field optical microscope‐atomic force microscope (SNOM‐AFM). A surface potential image of phospholipid monolayers was obtained in non‐contact mode using the SNOM‐AFM with a thin‐step etched optical fibre probe. For applying this technique, a phospholipid of dipalmitoylphosphatidylethanolamine labelled at the head with a nitrobenzoxadiazole group was used as a fluorescent and single component Langmuir–Blodgett film. It is well known that aggregation of the lipid molecules and their fluorescence intensities are very sensitive to its environmental conditions such as humidity and temperature. We demonstrated for the first time the near‐field optical imaging and simultaneous observation of surface potentials with Maxwell stress microscopy.
Mapping the distribution of emissive molecules in human ocular lipofuscin granules with near‐field scanning optical microscopy
Authors: Krogmeier J. R. , Clancy C. M. R. , Pawlak A. , Rozanowska M. , Sarna T. , Simon J. D. , Dunn R. C.
Published: March 2003

Several high resolution imaging techniques are utilized to probe the structure of human ocular lipofuscin granules. Atomic force microscopy reveals typical granule sizes to be about one micrometre in diameter and hundreds of nanometres in height, in agreement with previous electron microscopy results. For issues concerning the role of lipofuscin in age‐related macular degeneration, recent attention has focused on the orange‐emitting fluorophore, A2E. Confocal microscopy measurements are presented which reveal the presence of a highly emissive component in the granules, consistent with the presence of A2E. It is shown, however, that the interpretation of these results is complicated by the lack of structural details about the particles. To address these issues, near‐field scanning optical microscopy (NSOM) measurements are presented which measure both the lipofuscin fluorescence and topography, simultaneously. These measurements reveal distinct structure in the fluorescence image which do not necessarily correlate with the topography of the granules. Moreover, direct comparison between the NSOM fluorescence and topography measurements suggests that A2E is not the major component in lipofuscin. These measurements illustrate the unique capabilities of NSOM for probing into the microstructure of lipofuscin and uncovering new insights into its phototoxicity.
Precursor effects of martensitic transformations in Ti‐based alloys studied by electron microscopy with energy filtering
Authors: Murakami Y. , Shibuya H. , Shindo D.
Published: March 2003

Precursor effects of martensitic transformations in two well‐known shape memory alloys, Ti50Ni48Fe2 and Ti50Pd34Fe16, were studied extensively by energy‐filtered electron microscopy, including in‐situ observations, and high‐resolution electron microscopy. Energy‐filtered dark‐field images, where weak diffuse scattering was utilized, clearly showed the microstructure in the premartensitic state of Ti50Ni48Fe2. Tiny domains observable in this state were close to spherical rather than thin and slender, and the temperature dependence of the domain‐like structure was clarified by the in‐situ observations. It was found that Ti50Pd34Fe16 exhibited a domain‐like structure similar to that of Ti50Ni48Fe2, which was attributed to a transverse lattice displacement. High‐resolution images of Ti50Pd34Fe16 showed that a domain was coupled with other ones with different orientations of distortion, so as to reduce the total strain due to their formations. Furthermore, effects of including a fundamental reflection, in addition to the diffuse scattering, on dark‐field images were discussed based on the observations and the image processing.
Simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object
Authors: Paganin D. , Mayo S. C. , Gureyev T. E. , Miller P. R. , Wilkins S. W.
Published: March 2003

We demonstrate simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object. Subject to the assumptions explicitly stated in the derivation, the algorithm solves the twin‐image problem of in‐line holography and is capable of analysing data obtained using X‐ray microscopy, electron microscopy, neutron microscopy or visible‐light microscopy, especially as they relate to defocus and point projection methods. Our simple, robust, non‐iterative and computationally efficient method is applied to data obtained using an X‐ray phase contrast ultramicroscope.
Lamellar subcomponents of the cuticular cell membrane complex of mammalian keratin fibres show friction and hardness contrast by AFM
Authors: Smith J. R. , Swift J. A.
Published: March 2003

There is a substantial body of information indicating that 18‐methyleicosanoic acid (18‐MEA) is covalently linked to the outer surface of all mammalian keratin fibres and also forms the outer β‐layer of the cuticular cell membrane complex (CCMC) which separates the cuticle cells from each other. Low cohesive forces are expected between the lipid‐containing outer β‐layer and the δ‐layer of the CCMC, thus providing a weak point for cuticular delamination and presenting a fresh layer of 18‐MEA to the newly exposed surface.

We have used lateral force microscopy and force modulation atomic force microscopy (AFM) to examine human hair fibres in which the non‐covalently linked fatty acids have been removed. Examination of the lateral force images of new cuticle surfaces revealed by the attrition of overlying cuticle layers showed three separate zones of clearly defined frictional contrast. These are thought to correspond with the δ‐layer, the proteinaceous epicuticle and outer β‐layers of the CCMC. The δ‐layer was found to have a thickness of 16 nm (SD = 1 nm, n = 25), comparable to the 18.0 nm thickness measured from transverse cross‐sections of fibres with transmission electron microscopy. Force modulation AFM showed that the outer β‐layer was softer than the epicuticle and the δ‐layer. The frictional contrast was removed following treatment with methanolic KOH (0.1 mol dm−3) at 25 °C for 30 min, suggesting the hydrolysis of the thioester linkage and removal of 18‐MEA from the surface.
Stimulation of Ca2+ Entry in Lactotrophs and Somatotrophs from Immature Rat Pituitary by N‐Terminal Fragments of Proopiomelanocortin
Authors: Lorsignol Anne , Himpens Bernard , Denef Carl
Published: March 2003

We have previously shown that 10–12 kDa N‐terminal fragments of rat proopiomelanocortin (POMC) and human POMC1–76 stimulate mitosis and/or differentiation in lactotrophs of early postnatal rat pituitary. A truncated form, POMC1–26, mimics the differentiation‐inducing but not the mitogenic action of the former peptides. To further characterize these two biological responses, the present study compared changes in the intracellular free calcium concentration ([Ca2+]i) in response to POMC1–76 and POMC1–26 in isolated pituitary cells from 14‐day‐old female rats. Calcium (Ca2+) responses were also used as a guide to determine whether the responsive cells belong to the lactosomatotroph lineage. Application of POMC1–76 or POMC1–26 induced a maintained oscillating [Ca2+]i increase in a small population of cells. Increasing doses of the peptides did not affect the magnitude and the frequency of [Ca2+]i oscillations but clearly augmented the number of responding cells. Approximately 2% of the cells responded at 0.1 nM POMC1–76 or 5 nM POMC1–26, and 11–13% of the cells responded at 10 nM and 500 nM of the respective peptides. About one‐third of the cells responsive to these peptides also showed a [Ca2+]i increase in response to growth hormone‐releasing peptide‐6 (GHRP‐6) while, in a small number of responsive cells, [Ca2+]i was depressed by dopamine, suggesting that the former cells are somatotrophs and the latter lactotrophs. This interpretation was confirmed by immunocytochemical identification of prolactin and growth hormone (GH) in the cells. Of the cells showing Ca2+ response to POMC1–76, approximately one‐third contained GH and another third prolactin. The remainder contained neither GH nor prolactin. Comparable results were obtained with POMC1–26. The rise of [Ca2+]i induced by the N‐terminal POMC peptides persisted after depletion of intracellular Ca2+ stores by thapsigargin. Removal of Ca2+ from the extracellular medium or addition of cadmium completely abolished both the POMC1–76‐

and POMC1–26‐induced [Ca2+]i increase. Nifedipine inhibited the Ca2+ response to both peptides,

although only in 55% of the responsive cells. Depletion of some isoforms of protein kinase C by preincubation with the phorbol ester PMA for 24 h did not modify the Ca2+ responses. In contrast, blockade of the protein kinase A pathway with Rp‐cAMPs partially inhibited the POMC1–76‐ or

POMC1–26‐induced [Ca2+]i increase. The present data show that, in immature pituitary cells, POMC1–76

induces an increase in [Ca2+]i through extracellular Ca2+ influx, possibly mediated in part by

protein kinase A activation. The active domain of POMC1–76 seems to comprise its N‐terminal moiety. The data support the hypothesis that POMC1–76 exerts a specific function in the development of different members of the lactosomatotroph lineage and that the peptide mobilizes different subsets of cells within this lineage, by a mechanism determined by its concentration.
Ionotropic and Metabotropic Glutamate Receptor Agonist‐Induced [Ca2+]i Increase in Isolated Rat Supraoptic Neurons
Authors: Hattori Yukio , Shibuya Izumi , Tanaka Keiko , Kabashima Narutoshi , Ueta Yoichi , Yamashita Hiroshi
Published: March 2003

In the present study, the effects of glutamate and of agonists for ionotropic and metabotropic glutamate receptors on intracellular Ca2+ concentration ([Ca2+]i) were investigated in neurons of the rat supraoptic nucleus (SON). We used the intracellular Ca2+ imaging technique with fura‐2, in single magnocellular neurons dissociated from the SON of rats. Glutamate (10−6−10−4 M) evoked a dose‐dependent increase in [Ca2+]i. The glutamate agonists exerted similar effects, although with some differences in the characteristics of their responses. The [Ca2+]i response to NMDA was smaller than those of glutamate or the non‐NMDA receptor agonists, AMPA and kainate, but was significantly enhanced by the removal of extracellular Mg2+. Glutamate, as well as quisqualate, an agonist for both ionotropic and metabotropic glutamate receptors, evoked a [Ca2+]i increase in a Ca2+‐free condition, suggesting Ca2+ release from intracellular Ca2+ stores. This was further evidenced by [Ca2+]i increases in response to a more selective metabotropic glutamate receptor agonist, t‐ACPD, in the absence of extracellular Ca2+. Furthermore, the quisqualate‐induced Ca2+ release was abolished by the selective metabotropic glutamate receptor antagonist, (S)‐4‐carboxyphenylglycine. The results suggest that metabotropic glutamate receptors as well as non‐NMDA and NMDA receptors are present in the SON neurons, and that activation of the first leads to Ca2+ release from intracellular Ca2+ stores and the activation of the latter two types induces Ca2+ entry. These dual mechanisms of Ca2+ signalling may play a role in the regulation of SON neurosecretory cells by glutamate.
Ovarian Steroid‐Independent Diurnal Rhythm in Cyclic GMP/Nitric Oxide Efflux in the Medial Preoptic Area: Possible Role in Preovulatory and Ovarian Steroid‐Induced LH Surge*
Authors: Pu Shuye , Kalra Pushpa S. , Kalra Satya P.
Published: March 2003

The aim of this study was to evaluate the relationship between cyclic LH hypersecretion and nitric oxide (NO) release in the medial preoptic area (MPOA), the hypothalamic site implicated in induction of LH hypersecretion. The MPOA extracellular cyclic GMP (cGMP) efflux (an index of NO release), was monitored by microdialysis. Quite unexpectedly, we observed a daily afternoon rise in the MPOA cGMP efflux in cycling female rats on proestrus and diestrus II, in ovariectomized (ovx) rats and in ovx rats treated with ovarian steroids to induce the LH surge. The daily rise in cGMP efflux occurred earlier in diestrous and in estradiol benzoate (EB)‐treated ovx rats than in ovx rats. Progesterone (P) injection to estrogen‐primed ovx rats further advanced the onset of the rise close to the earliest time of rise as seen on proestrus. The afternoon increase in the cGMP efflux in proestrous rats was abolished by pentobarbital treatment that blocked the LH surge. Intracerebroventricular (icv) injection of 1H‐[1,2,4]oxadiazo[4,3‐a]quinoxalin‐one (ODQ), a selective inhibitor of soluble guanylyl cyclase, suppressed the P‐induced LH surge in EB‐primed ovx rats, but not basal LH secretion in unprimed ovx rats. Analysis of brain NOS (bNOS) levels in the POA by Western blotting showed that the morning bNOS levels were higher in the POA of EB‐treated rats than in unprimed ovx rats. Further, with the exception of ovx rats treated with sequential EB and P treatment, the POA bNOS levels rose significantly in the afternoon in unprimed ovx and EB‐treated ovx rats. Collectively, these findings reveal a diurnal rhythm in the MPOA cGMP/NO efflux that is ovarian steroid‐independent. Ovarian steroids apparently shift the timing of the afternoon rise in cGMP/NO efflux to synchronize with the activation of steroid‐dependent neuronal systems responsible for the LH surge.
Day and Night Dysfunction in Intraretinal Melatonin and Related Indoleamines Metabolism, Correlated with the Development of Glaucoma‐Like Disorder in an Avian Model
Authors: Dkhissi  , Chanut  , Versaux‐Botteri  , Trouvin  , Repérant  , Nguyen‐Legros
Published: March 2003

As previous studies have suggested that melatonin and serotonin may be involved in the regulation of intraocular pressure, retinal concentrations of melatonin, 5‐HT, and related indoleamines measured at day and at night were studied during the development of a glaucoma‐like disorder with increased intraocular pressure in thealmutant quail. Indoleamine levels were determined by HPLC with electrochemical detection in 1‐month‐, 3‐month‐, and 7‐month‐oldalmutant and control quails. Morphology and numbers of melatonin‐synthesizing and 5‐HT‐containing cells, labelled immunohistochemically with an anti‐hydroxyindol‐0‐methyltransferase (HIOMT) antibody and an anti‐5‐HT antibody, respectively, were studied. Major findings were that: (1) no significant changes in morphology of melatonin‐synthesizing cells or in the morphology and density of 5‐HT‐containing amacrine cells were observed during the d velopment of glaucoma: (2) 5‐HT metabolism was modified during the night at 1 month of age and during the day after 3 months; and (3) melatonin metabolism was modified during the night at 7 months and during the day after 3 months. These results demonstrate a relationship between the temporal evolution of this avian glaucoma and a dysfunction in indoleamine retinal metabolism.
Actions of Prostaglandin E2 on Rat Supraoptic Neurones
Authors: Sutarmo Setiadji  , Shibuya  , Kabashima  , Ibrahim  , Harayama  , Ueta  , Yamashita
Published: March 2003

Prostaglandins (PGs) have been implicated in the regulation of vasopressin (VP) and oxytocin (OT) release in response to various stimuli. To examine the site and mechanism of actions of PGs, we studied effects of PGE2 and PG‐receptor agonists on supraoptic nucleus (SON) neurones of rat hypothalamic slice preparations using extracellular recording and whole‐cell patch‐clamp techniques. PGE2 modulated the electrical activity of more than 80% of the neurones studied. The effects of PGE2 on both phasic and non‐phasic neurones were mostly excitatory, and dose‐dependent. The effects of PGE2 were mimicked by PGF2α or the FP agonist, fluprostenol, whereas PGD2 or the selective EP, IP or TP agonist was less effective or had no effect. The effects of PGE2 were unaffected by the EP1 antagonist, SC‐51322, but reduced to 80% of control by the EP1/FP/TP antagonist, ONO‐NT‐012, which reduced the effects of fluprostenol to 32% of control. Moreover, some neurones responsive to PGE2 did not respond to fluprostenol. Patch‐clamp analysis in SON slice preparations revealed that PGE2 at 10−6 M depolarized the membrane potential by 3.9±0.3 mV from the resting membrane potential of −58.4±2.2 mV in the current‐clamp mode. In the voltage‐clamp mode, PGE2 induced inward currents at a holding potential of −70 or −80 mV, while it did not affect spontaneous excitatory postsynaptic currents. PGE2 induced currents also in dissociated SON neurones and the reversal potential of the currents was −35.5±0.9 mV, which was similar to that of currents induced by fluprostenol. These results suggest that SON neurones possess at least two types of PG receptors, FP receptors and EP receptors of a subclass different from EP1, EP2, or EP3, and that activation of these receptors leads to the opening of nonselective cation channels, membrane depolarization and increase of the action potential discharge.
Correlation of Hypothalamic Somatostatin mRNA Expression and Peptide Content with Secretion: Sexual Dimorphism and Differential Regulation by Gonadal Factors
Authors: Murray  , Simonian  , Herbison  , Gillies
Published: March 2003

Sex differences in growth hormone (GH) secretion in the rat are thought to be determined, to a large extent, by gonadal steroid‐dependent sex differences in somatostatin (SRIH) secretion from neurones in the periventricular nucleus (PeN) which project to the median eminence (ME). The present study aimed to obtain direct evidence for sex differences and gonadal regulation of SRIH release within this pathway and to determine the relationships between SRIH mRNA expression, SRIH peptide content and SRIH secretion in the adult rat. Somatostatin mRNA expression in the PeN and peptide content in both PeN and ME were higher in males than females (P<0.05). However, both basal and 56 m m K+‐stimulated SRIH release in vitro from hypothalamic explants incorporating the PeN–ME pathway were higher (P<0.01) in females. The gonadectomy of female rats resulted in significantly reduced basal levels of SRIH release equivalent to that of males but had no effect on SRIH mRNA/peptide content or K+‐stimulated release. In contrast, gonadectomy of male rats reduced SRIH mRNA and peptide contents and elevated K+‐stimulated secretion (P<0.01) to levels similar to that seen in intact females, without affecting basal release. In summary, these results demonstrate that in the PeN‐ME of the adult rat: (1) SRIH mRNA and peptide content is well correlated and sexually dimorphic but dependent on gonadal factors in the male only; (2) SRIH secretion is sexually dimorphic and dependent on gonadal factors; but (3) differences in mRNA/peptide content do not reflect secretory capacity; and (4) gonadal factors differentially modulate SRIH secretory dynamics in males and females.
Ontogeny of Diazepam Binding Inhibitor/Acyl‐CoA Binding Protein mRNA and Peripheral Benzodiazepine Receptor mRNA Expression in the Rat1
Authors: Bürgi  , Lichtensteiger  , Lauber  , Schlumpf
Published: March 2003

The Diazepam Binding Inhibitor/Acyl‐CoA Binding Protein (DBI/ACBP) has been implicated in different functions, as acyl‐CoA transporter and as an endogenous ligand at the GABAA receptor and the peripheral benzodiazepine receptor (PBR). The latter is thought to be involved in control of steroidogenesis. We studied the ontogeny of DBI/ACBP and PBR mRNA expression in embryos and offspring of time‐pregnant Long Evans rats by in‐situ hybridization with 33P‐endlabelled oligonucleotides. Both mRNAs were present in embryo and placenta at gestational day (G)11, the earliest stage studied. DBI/ACBP mRNA was strongly expressed from embryonic through mid‐foetal stages in central nervous system (maximum in neuroepithelium), cranial and sympathetic ganglia, anterior pituitary, adrenal cortex, thyroid, thymus, liver and (late foetal) brown adipose tissue, moderately in testis, heart, lung and kidney. In brain, a late foetal decrease of DBI/ACBP mRNA was followed by an increase at postnatal day 6. Peripheral benzodiazepine receptor mRNA expression started very low and increased to moderate levels in adrenal cortex and medulla, testis, thyroid, brown adipose tissue, liver, heart, lung, salivary gland at mid‐ to late‐foetal stages. Data suggest a significant role of DBI/ACBP at early developmental stages. Both proteins may be involved in the control of foetal steroidogenesis. However, differences in developmental patterns indicate that additional functions may be equally important during ontogeny, such as the involvement in lipid metabolism in the case of DBI/ACBP.
Oestrogen Receptor‐α‐Immunoreactive Neurones Project to the Suprachiasmatic Nucleus of the Female Syrian Hamster
Authors: De La Iglesia  , Blaustein  , Bittman
Published: March 2003

Ovarian steroid hormones regulate circadian period and phase, but classical receptors for these hormones are absent in the circadian pacemaker localized in the suprachiasmatic nucleus of the hypothalamus (SCN). In order to determine whether effects of oestrogen may be exerted through steroid‐binding systems afferent to the SCN we have performed double label immunocytochemistry for oestrogen receptor‐α(ER‐α) and the retrograde tracer cholera toxin B subunit (CtB) after its application to the SCN. Most of the areas that contain ER‐α‐immunoreactive (ERα‐ir) cells also contained cells afferent to the SCN. The percentage of neurones afferent to the SCN which show ERα‐immunoreactivity varies between areas. As many as one‐third of the neurones afferent to the SCN in some parts of the preoptic area and the corticomedial amygdala are ERα‐ir. Very few of the afferent neurones from the septum and the central grey are ERα‐ir, whereas an intermediate proportion of afferents from the bed nucleus of the stria terminalis and the arcuate nucleus are ERα‐ir. Our retrograde tracing results were compared with results of anterograde tracing from some of the sites containing SCN afferents. Using a combined retrograde and anterograde tracing technique we tested the possibility that single ERα‐ir neurones afferent to the SCN could receive reciprocal innervation by SCN efferents. Although we found SCN input to some SCN afferent neurones, we found no evidence of reciprocity between single ERα‐ir cells and the SCN. Our results indicate the existence of oestrogen binding systems afferent to the SCN. These neuroanatomical pathways may mediate effects of gonadal steroid hormones on circadian rhythms.
The Role of Ascending Neuronal Pathways in Stress‐Induced Release of Noradrenaline in the Hypothalamic Paraventricular Nucleus of Rats
Authors: Palkovits  , Baffi  , Pacak
Published: March 2003

Central catecholaminergic pathways carrying pain‐related signals to the hypothalamic paraventricular nucleus (PVN) were investigated in laboratory rats. Four per cent formalin injected subcutaneously was employed as a stressful stimulus. Neuronal activity in brainstem catecholaminergic and paraventricular neurones was assessed by Fos immunohistochemistry. Stress‐induced noradrenaline (NE) release from nerve terminals in the PVN was measured in extracellular fluid by in‐vivo microdialysis. Within 30 min, formalin elicited a four‐ to sixfold increase in plasma ACTH and corticosterone concentrations and intense Fos‐like activity was seen in the superficial zones of the lumbar spinal cord ipsilateral to the side of the formalin injection. In brainstem catecholaminergic neurones, the PVN, and midline thalamic nuclei, formalin‐induced Fos‐immunopositivity was equally present in the ipsi‐ and contralateral sides of the injection. An immediate elevation (4–5 times higher than baseline levels) of NE levels was measured in both the right and left PVN after a formalin injection into the right paw. Unilateral surgical transections at the medulla‐spinal cord junction failed to affect formalin‐induced elevations in NE levels in the PVN independently of the side of the formalin injection or the knife cut. Thus, this observation clearly shows that fibres carrying pain‐evoked signals ascend bilaterally from the spinal cord to the brainstem and forebrain. Hemisections of the medulla oblongata between the level of A1–A2 NE cell groups and the locus coeruleus reduced but did not eliminate formalin‐induced NE release from the PVN ipsilateral to the knife cut. This effect was independent of the side of the formalin injection. In the contralateral PVN, high and similar NE levels were measured in response to a formalin injection into the right or the left leg. The present study indicates that formalin‐induced pain signals are carried by sensory fibres to the ipsilateral spinal cord. From there, axons of different dorsal horn neurones reach noradrenergic cells on both sides of the medulla oblongata. The majority of noradrenergic fibers ascend on the same side and innervate the ipsilateral PVN. Since formalin administration resulted in a moderate elevation of NE levels in the PVN on the operated side, the role of other ascending noradrenergic (from the locus coeruleus) or noncatecholaminergic fibres that could modulate NE release from the PVN should be considered.
Localisation and Semi‐Quantitative Measurement Of Lipocortin 1 In Rat Anterior Pituitary Cells By Fluorescence‐Activated Cell Analysis/Sorting and Electron Microscopy
Authors: Christian  , Flower  , Morris  , Buckingham
Published: March 2003

Lipocortin 1 (LC1, also called annexin 1), a Ca2+‐ and phospholipid‐binding protein, is an important mediator of glucocorticoid action in the anterior pituitary gland. Previous studies based on immunoprecipitation and Western blot analysis suggest that LC1 is found intracellularly both in the cytoplasm and in association with membranes and also on the cell surface where it attaches to the membrane by a Ca2+‐dependent mechanism. However, as yet it is unclear which anterior pituitary cell types express the protein. Accordingly, we have developed a method based on a combination of fluorescence activated cell (FAC) analysis/sorting and electron microscopy to detect and quantify intracellular LC1 in rat anterior pituitary cells and to identify the cell types in which it is expressed. In addition, we have measured cell surface LC1 and examined the influence of glucocorticoids on the cellular disposition of the protein. Anterior pituitary cells were dispersed with collagenase. For experiments measuring intracellular LC1, three cell fixation/permeabilisation methods were examined initially, i.e. (1) Zamboni’s fluid (30 min) and Triton‐X‐100 (0.12%, 1 or 12 h); (2) paraformaldehyde (2%, 1 h) and Triton‐X‐100 (0.2%, 10 min); and (3) paraformaldehyde (0.2%, 15 min) and saponin (0.1%, 5 min). The protocol using paraformaldehyde/Triton‐X‐100 provided optimal preservation of cell ultrastructure and of LC1 immunoreactivity (ir‐LC1) while also effectively permeabilising the cells; it was therefore used in subsequent studies. Using an anti‐LC1 monoclonal antibody as a probe, 82±5% of the secretory cells in the heterogeneous anterior pituitary cell preparation were shown by FAC analysis to display specific fluorescence for intracellular ir‐LC1. Morphological analysis and immunogold‐histochemistry of cells separated by FAC sorting identified corticotrophs, lactotrophs, somatotrophs and gonadotrophs in the population displaying LC1 immunofluorescence. LC1 was also detected on the surface of anterior pituitary cells by FACS analysis. Incubation of anterior pituitary cells with dexamethasone or corticosterone (0.1 and 1.0 μM) prior to fixation and analysis produced a significant, concentration‐dependent decrease in intracellular ir‐LC1 and a concomitant increase in the amount of ir‐LC1 detected on the surface of the cells; the effects of the two steroids were indistinguishable quantitatively. In conclusion, we report a novel method which permits (1) the detection and semi‐quantitative measurement of intracellular and surface LC1 in anterior pituitary cells; and (2) the identification of the cell types in which the protein is found.
The Hypothalamic‐Pituitary‐Adrenal Axis Response to Endotoxin is Attenuated During Lactation
Authors: Shanks  , Windle  , Perks  , Wood  , Ingram  , Lightman
Published: March 2003

Pregnancy and lactation are times of prolonged physiological changes affecting the neuroendocrine and immunological systems. One well‐characterized change is the neuroendocrine hyporesponsiveness to acute stressful stimuli. We have now designed studies to see whether there is an alteration in the response of the hypothalamic‐pituitary‐adrenal (HPA) axis to an immunological inflammatory challenge and to ascertain whether lactating animals show altered neural and endocrine responses to inflammatory stimuli. Lactating (day 9–12 postpartum) or virgin control Sprague‐Dawley female rats were injected with either 200 μg of endotoxin (lipopolysaccharide, LPS ) or sterile saline given i.p. Trunk blood or jugular blood was collected from the animals at 2 h or hourly over 6 h after injection. Both plasma adrenocorticotropic hormone (ACTH) and corticosterone concentrations were significantly higher in saline treated lactating animals compared with the virgin group. LPS significantly elevated circulating levels of plasma ACTH and corticosterone in both virgin and lactating animals compared with saline controls, however, hormone responses to LPS were significantly reduced in lactating animals relative to virgin controls. Corticosterone‐binding globulin concentrations were lower in lactating animals compared to virgin animals and LPS decreased concentrations in virgin, but not lactating rats. Analysis of cfos mRNA in the paraventricular nucleus (PVN) of the hypothalamus revealed that 2 h following injection there was a increase in cfos expression only in the virgin animals treated with LPS, compared to all other treatment conditions. Corticotropin‐releasing hormone (CRH) mRNA expression was overall greater in virgin animals, but was increased to similar extent in both virgin and lactating animals treated with LPS. Primary arginine vasopressin (AVP) mRNA transcripts were increased 2 h following LPS injection, but a greater increase in expression was seen in virgin animals. These data demonstrate that there is a lower level of free circulating glucocorticoid in response to inflammatory stimuli and suggests that communication between the immune and endocrine systems may be altered during lactation.
Interactions Between the Stimulated Hypothalamic‐Pituitary‐Adrenal Axis and Leptin in Humans
Authors: Nye E. J. , Bornstein S. R. , Grice J. E. , Tauchnitz R. , Hockings G. I. , Strakosch C. R. , Jackson R. V. , Torpy D. J.
Published: March 2003

Leptin, produced by adipocytes, has homeostatic effects on body fat mass through inhibition of appetite and stimulation of the sympathetic nervous system. Several studies have reported that high‐dose exogenous glucocorticoids increase circulating leptin concentrations in humans. Conversely, leptin has inhibitory effects on the hypothalamic‐pituitary‐adrenal (HPA) axis, both at the hypothalamic and adrenal levels. We hypothesized that acute hypercortisolism, in the physiological range, may not alter leptin secretion. Four stimuli of the HPA axis were administered to eight healthy male volunteers in a placebo‐controlled study. On separate afternoons, in a randomised order, fasting subjects received i.v. injections of saline, naloxone (125 μg/kg); vasopressin (0.0143 IU/kg); naloxone and vasopressin in combination; or insulin (0.15 U/kg; a dose sufficient to induce hypoglycaemia). Plasma concentrations of adrenocorticotrophic hormone (ACTH), cortisol and leptin were measured before and for 120 min after the injection. The cortisol secretory response was greatest after insulin‐hypoglycaemia, this response was significantly greater than that following naloxone, naloxone/vasopressin, or vasopressin alone. Despite the cortisol release, leptin concentrations were not increased after any stimulus. Insulin‐hypoglycaemia was associated with a decrease in leptin concentration at 60 and 90 min, while naloxone did not alter leptin concentrations. However, basal leptin concentrations were positively correlated with integrated ACTH and cortisol responses to naloxone, but did not correlate with ACTH or cortisol responses to the other stimuli. Thus acute elevations of plasma cortisol, in the physiological range, do not appear to influence plasma leptin concentrations. The fall in plasma leptin concentration after insulin‐induced hypoglycaemia may reflect catecholamine secretion after this stimulus.
Brain Oxytocin Inhibits Basal and Stress‐Induced Activity of the Hypothalamo‐Pituitary‐Adrenal Axis in Male and Female Rats: Partial Action Within the Paraventricular Nucleus
Authors: Neumann  , Wigger  , Torner  , Holsboer  , Landgraf
Published: March 2003

Oxytocin is a classic reproductive neuropeptide in the female mammal, but its functions in the brain of the male have been less well studied. As stress induces intracerebral oxytocin release independently of gender, we postulated that central oxytocin may play a role in the control of stress responses. In both male and virgin female rats, oxytocin receptor blockade in the brain by intracerebral infusion of a selective oxytocin antagonist (des Gly‐NH2 d(CH2)5 [Tyr(Me)2, Thr4] OVT; 0.75 μg/5 μl increased the activity of the hypothalamo‐pituitary‐adrenal (HPA) axis as indicated by a significantly enhanced basal and stress‐induced (exposure to the elevated plus‐maze, forced swimming) secretion of corticotropin (ACTH) and corticosterone into blood. The anxiety‐related behaviour on the plus‐maze was not altered by the antagonist in either males or females. Infusion of the oxytocin antagonist into the hypothalamic paraventricular nucleus by reversed microdialysis resulted in a significant increase in basal release of ACTH in both male and virgin female rats. These results demonstrate a novel, gender‐independent physiological function of endogenous brain oxytocin in the regulation of neuroendocrine stress responses. Under basal conditions, the inhibition of the HPA axis occurs, at least in part, within the paraventricular nucleus.
Cyanamide‐Induced Activation of the Hypothalamo‐Pituitary‐Adrenal Axis
Authors: Kinoshita  , Jessop  , Finn  , Harbuz
Published: March 2003

The present study investigated the effects of acute administration of cyanamide (a potent inhibitor of aldehyde dehydrogenase used to treat alcoholics), on the hypothalamo‐pituitary adrenal (HPA)‐axis. Cyanamide resulted in a significant increase in arginine vasopressin mRNA and corticotrophin releasing factor (CRF) mRNA in the parvocellular cells of the paraventricular nucleus and pro‐opiomelanocortin (POMC) mRNA in the anterior pituitary. Plasma corticosterone concentrations were elevated by a range of doses of cyanamide which were maintained in the high dose group at 4 h following administration. These results suggest that cyanamide is able to activate the HPA axis at all levels of the axis. Arginine vasopressin mRNA, in the parvocellular cells of the paraventricular nucleus is an important component of the stress response. Silver grain counting of emulsion dipped slides is commonly used for its evaluation following in‐situ hybridization. This method is however, not entirely satisfactory and very time‐consuming. We compared this method with a film autoradiographic method, and show that the film autoradiographic method is valid for the determination of arginine vasopressin mRNA in the parvocellular cells of the paraventricular nucleus.