bluuebunny/crossref_metadata_2025 huggingface dataset:
Three‐dimensional spectral imaging by Hadamard transform spectroscopy in a programmable array microscope
Authors: Hanley  , Verveer  , Arndt‐Jovin  , Jovin
Published: March 2003

We report the acquisition and deconvolution of three‐dimensional spectrally resolved images in a programmable array microscope implementing a Hadamard transform fluorescence spectroscopy system with adjustable spectral resolution. A stack of 16 two‐dimensional spectral images was collected at 400 nm intervals along the optical axis. The specimen consisted of a polytene chromosome spread from Drosophila melanogaster doubly labelled for the Polyhomeotic protein by indirect immunofluorescence labelling with Alexa594 and for DNA with YOYO‐1. The resulting four‐dimensional data set consisted of the xyz spatial dimensions (898 × 255 × 16) with a 26‐point spectrum at each spatial location. The total exposure time to the sample was 34 min. The system requires the acquisition of multiple images, and thus works best with fluorophores that are resistant to photobleaching. Image deconvolution reduced the amount of out‐of‐focus blur by up to a factor of 8, resulting in a dramatic improvement in the visualization of the chromosome backbone and localization of the specific Polyhomeotic domains.
Compact water‐window transmission X‐ray microscopy
Authors: Berglund  , Rymell  , Peuker  , Wilhein  , Hertz
Published: March 2003

We demonstrate sub‐100 nm resolution water‐window soft X‐ray full‐field transmission microscopy with a compact system. The microscope operates at λ = 3.37 nm and is based on a 100 Hz table‐top regenerative debris‐free droplet‐target laser‐plasma X‐ray source in combination with normal‐incidence multilayer condenser optics for sample illumination. High‐spatial‐resolution imaging is performed with a 7.3% efficiency nickel zone plate and a 1024 × 1024 pixel CCD detector. Images of dry test samples are recorded with exposure times of a few minutes and show features smaller than 60 nm.
A wavefront generator for complex pupil function synthesis and point spread function engineering
Authors: Neil  , Wilson  , Juskaitis
Published: March 2003

We describe a simple method to produce an arbitrary complex optical field using a ferroelectric liquid crystal spatial light modulator. The system is configured so as to act as a pupil plane filter in a confocal microscope. The ability to tune the complex pupil function permits the system to be used both to modify the imaging performance by effectively engineering the point spread function as well as to remove optical aberrations present in the optical system.
Atomic force microscopy studies of intercalation‐induced changes in plasmid DNA tertiary structure
Authors: Pope L. H. , Davies M. C. , Laughton C. A. , Roberts C. J. , Tendler S. J. B. , Williams P. M.
Published: March 2003

Structural transitions in the tertiary structure of plasmid DNA have been investigated using atomic force microscopy. Changes in superhelical stress were induced by ethidium bromide intercalation, and conformational effects monitored by recording topographic images from DNA complexes of various ethidium bromide : base pair stoichiometry. Significant changes in the tertiary structure of individual DNA molecules were observed with increasing ethidium bromide concentration. The first distinct conformational transition was from a predominantly relaxed structure to one consisting solely of toroidal supercoils. A further increase in ethidium bromide concentration resulted in the formation of regions of plectonemic supercoiling. The ratio of plectonemic : toroidal supercoiling gradually increased until an extremely tightly interwound structure of solely plectonemic supercoiling was finally adopted. The toroidal form of supercoiling observed in this study is unusual as both atomic force microscopy and electron microscopy techniques have previously shown that plectonemic supercoiling is the predominant form adopted by plasmid DNA.
Applications of gold cluster compounds in immunocytochemistry and correlative microscopy: comparison with colloidal gold
Authors: Robinson J. M. , Takizawa T. , Vandré D. D.
Published: March 2003

In this review, we discuss the immunocytochemical literature with respect to a comparison between conventional colloidal gold and gold cluster compounds as immunoprobes. The relative advantages and disadvantages of each of these types of particle for immunocytochemical applications are discussed. We present results from our own laboratories and those of others on the comparison of these immunoprobes in selected experimental situations. These results show the use of gold cluster compounds at both light and electron microscope levels. At the ultrastructural level, gold cluster compounds have been used in pre‐embedding labelling of cultured cells, and for labelling of ultrathin cryosections and freeze‐fracture preparations. Recently, fluorescently tagged gold cluster compounds have become available. Using ultrathin cryosections of human neutrophils as a model system, we demonstrate that a single immunoprobe (i.e. a fluorescently tagged gold cluster compound) is a robust probe for correlative fluorescence and electron microscopy.
Low‐temperature near‐field spectroscopy of CdTe quantum dots
Authors: Brun M. , Huant S. , Woehl J. C. , Motte J.‐F. , Marsal L. , Mariette H.
Published: March 2003

A near‐field optical microscope has been developed for operation at low temperature. This microscope is used to study the photoluminescence of CdTe‐based quantum dots. Spectra collected upon approaching the optical tip into the near‐field region of the sample reveal the evolution from a broad far‐field luminescence band − that is typical for a large ensemble of dots − to a near‐field structure made up of a few sharp peaks originating from individual dots. Experiments carried out in the excitation‐collection mode through the optical tip allow study of the effect of an increase in excitation power on the near‐field spectra. It is found that upon increasing the excitation by two orders of magnitude, a spatially resolved spectrum progressively transforms back into a broad ‘far‐field‐like’ spectrum. Photoluminescence images taken by scanning the sample under the tip are used to discriminate various contributions coming from individual dots.
Quantitative serial sectioning analysis
Authors: Alkemper J. , Voorhees P. W.
Published: March 2003

A method for serial sectioning is presented that allows one to take about 20 sections per hour with spacings in the range 1–20 µm between sections. The alignment of the cross‐sections is done with a linear variable differential transformer; it is thus independent of the microstructure of the sample and does not rely upon markers implanted in the sample. The alignment errors as well as tilts and rotation errors between sections associated with the new method are found to be negligible. Once all the sections are captured in a computer a three‐dimensional image can be constructed. This image can be viewed interactively and rotated, thus allowing the direct observation of three‐dimensional shapes. It can further be used to determine a vast array of microstructural parameters including those that cannot be determined from planar sections. The technique is illustrated through the reconstruction of the microstructure of a cast standard aluminium alloy specimen.
Optical near‐field harmonic demodulation in apertureless microscopy
Authors: Maghelli N. , Labardi M. , Patanè S. , Irrera F. , Allegrini M.
Published: March 2003

Spatial derivatives of the optical fields scattered by a surface can be investigated by apertureless near‐field optical microscopy by modulating sinusoidally the probe to sample distance and detecting the optical signal at the first and higher harmonics. Demodulation up to the fifth harmonic order has been accomplished on a sample of close‐packed latex spheres by means of the silicon tip of a scanning interference apertureless microscope. The working principles of such microscope are reviewed. The experimental configuration used comprises a tuning‐fork‐based tapping‐mode atomic force microscope for the distance stabilization, and a double‐modulation technique for complete separation of the topography tracking from the optical detection. Simple modelling provides first indications for the interpretation of experimental data. The technique described here provides either artefact‐free near‐field optical imaging, or detailed information on the structure of the near fields scattered by a surface.
Time‐domain whole‐field fluorescence lifetime imaging with optical sectioning
Authors: Cole M. J. , Siegel J. , Webb S. E. D. , Jones R. , Dowling K. , Dayel M. J. , Parsons‐Karavassilis D. , French P. M. W. , Lever M. J. , Sucharov L. O. D. , Neil M. A. A. , Juškaitis R. , Wilson T.
Published: March 2003

A whole‐field time‐domain fluorescence lifetime imaging (FLIM) microscope with the capability to perform optical sectioning is described. The excitation source is a mode‐locked Ti:Sapphire laser that is regeneratively amplified and frequency doubled to 415 nm. Time‐gated fluorescence intensity images at increasing delays after excitation are acquired using a gated microchannel plate image intensifier combined with an intensified CCD camera. By fitting a single or multiple exponential decay to each pixel in the field of view of the time‐gated images, 2‐D FLIM maps are obtained for each component of the fluorescence lifetime. This FLIM instrument was demonstrated to exhibit a temporal discrimination of better than 10 ps. It has been applied to chemically specific imaging, quantitative imaging of concentration ratios of mixed fluorophores and quantitative imaging of perturbations to fluorophore environment. Initially, standard fluorescent dyes were studied and then this FLIM microscope was applied to the imaging of biological tissue, successfully contrasting different tissues and different states of tissue using autofluorescence. To demonstrate the potential for real‐world applications, the FLIM microscope has been configured using potentially compact, portable and low cost all‐solid‐state diode‐pumped laser technology. Whole‐field FLIM with optical sectioning (3D FLIM) has been realized using a structured illumination technique.
Detection of mitochondrial DNA in living animal cells with fluorescence microscopy
Authors: Dellinger M. , Gèze M.
Published: March 2003

The detection of mitochondrial DNA (mtDNA) in living human cells could be useful for understanding mitochondrial behaviour during cellular processes and pathological mtDNA depletions. However, until now, human mtDNA has not been visualized in living cells with fluorescence microscopy, although it has been easily detected in organisms with larger mtDNA. Previous reports have stated that mtDNA staining results in homogeneous fluorescence of mitochondria or that animal mitochondria are refractory to DAPI staining. This paper shows that mtDNA of cultured green monkey kidney CV‐1 can be stained using a very low concentration of DAPI, then detected by a cooled Photometrics CCD camera with 14‐bit resolution detection. Indeed, under these conditions CV‐1 cells have small fluorescent spots in the cytoplasm that colocalize with mitochondria, even after mitochondrial movements, uncoupling by carbonyl cyanide p‐(trifluoromethoxy)phenylhydrazone and swelling. These observations have been reproduced for the human fibroblast foreskin cell line HS68. These results and known properties of DAPI as a specific DNA stain strongly suggest that mtDNA can be detected and visualized by fluorescence microscopy in human living cells, with potential developments in the study of mtDNA in normal and pathological situations.
Single‐section plane assessment in grain boundary engineered brass
Authors: Davies H. , Randle V.
Published: March 2003

The present paper reports a comparative analysis of Σ3 (in the coincidence site lattice notation) grain boundary types, in two grain boundary engineered brass specimens, by use of electron backscatter diffraction (EBSD) data coupled to the measurement of boundary traces in a single section. Although most of the data were analysed using the new single‐section technique, an analysis of boundary plane orientations in three dimensions was made in a subset of the data in order to validate the single section methodology. The single‐section trace analysis procedure, coupled with EBSD, is a viable and robust tool for analysis of Σ3 grain boundary planes. The procedure provides data which suggest that part of the enhanced strain‐to‐failure in specimen B compared to specimen A is the result of an increased proportion of mobile Σ3 boundaries, i.e. those which are displaced from the {111} symmetrical tilt configuration.
The ELF®‐97 phosphatase substrate provides a sensitive, photostable method for labelling cytological targets
Authors: Paragas V. B. , Kramer J. A. , Fox C. , Haugland R. P. , Singer V. L.
Published: March 2003

We compared fluorescent signals obtained with fluorescein conjugates and the ELF‐97 (enzyme‐labelled fluorescence) phosphatase substrate [2‐(5′‐chloro‐2‐phosphoryloxyphenyl)‐6‐chloro‐4(3H)‐quinazolinone] in labelling cytological structures requiring high spatial resolution. Enzymatic cleavage of the ELF‐97 phosphatase substrate yields an extremely fine precipitate that remains well localized to the site of enzymatic activity. This precipitate fluoresces bright yellow‐green, with maximal excitation at ~360 nm and maximal emission at ~530 nm. The ELF substrate was used with streptavidin–alkaline phosphatase, to fluorescently label site‐specific probes bound to their targets, including cell‐surface sites, cytoplasmic organelles, nuclear antigens and cytoskeletal networks. All targets were labelled successfully with both the ELF substrate and fluoresceinated probes or protein conjugates. However, the ELF method was frequently more sensitive, with lower background fluorescence, allowing detection of more lysosomes, actin filaments, microtubules and nuclear targets than were visible with corresponding fluoresceinated probes. The ELF substrate was also used with antifluorescein–alkaline phosphatase to amplify fluorescein signals. We found that the ELF signal was in all cases brighter and more photostable than fluorescein signals, permitting shorter film exposures and allowing more time for examining samples. Surprisingly, relative brightness and photostability depended on the target, rather than being a general phenomenon related to the choice of dye alone.
Quantitative X‐ray projection microscopy: phase‐contrast and multi‐spectral imaging
Authors: Mayo S. C. , Miller P. R. , Wilkins S. W. , Davis T. J. , Gao D. , Gureyev T. E. , Paganin D. , Parry D. J. , Pogany A. , Stevenson A. W.
Published: March 2003

We outline a new approach to X‐ray projection microscopy in a scanning electron microscope (SEM), which exploits phase contrast to boost the quality and information content of images. These developments have been made possible by the combination of a high‐brightness field‐emission gun (FEG)‐based SEM, direct detection CCD technology and new phase retrieval algorithms. Using this approach we have been able to obtain spatial resolution of < 0.2 µm and have demonstrated novel features such as: (i) phase‐contrast enhanced visibility of high spatial frequency image features (e.g. edges and boundaries) over a wide energy range; (ii) energy‐resolved imaging to simultaneously produce multiple quasi‐monochromatic images using broad‐band polychromatic illumination; (iii) easy implementation of microtomography; (iv) rapid and robust phase/amplitude‐retrieval algorithms to enable new real‐time and quantitative modes of microscopic imaging. These algorithms can also be applied successfully to recover object–plane information from intermediate‐field images, unlocking the potentially greater contrast and resolution of the intermediate‐field regime. Widespread applications are envisaged for fields such as materials science, biological and biomedical research and microelectronics device inspection. Some illustrative examples are presented. The quantitative methods described here are also very relevant to projection microscopy using other sources of radiation, such as visible light and electrons.
Comparative evaluation of retrospective shading correction methods
Authors: Tomaževič D. , Likar B. , Pernuš F.
Published: March 2003

Because of the inherent imperfections of the image formation process, microscopical images are often corrupted by spurious intensity variations. This phenomenon, known as shading or intensity inhomogeneity, may have an adverse affect on automatic image processing, such as segmentation and registration. Shading correction methods may be prospective or retrospective. The former require an acquisition protocol tuned to shading correction, whereas the latter can be applied to any image, because they only use the information already present in an image. Nine retrospective shading correction methods were implemented, evaluated and compared on three sets of differently structured synthetic shaded and shading‐free images and on three sets of real microscopical images acquired by different acquisition set‐ups. The performance of a method was expressed quantitatively by the coefficient of joint variations between two different object classes. The results show that all methods, except the entropy minimization method, work well for certain images, but perform poorly for others. The entropy minimization method outperforms the other methods in terms of reduction of true intensity variations and preservation of intensity characteristics of shading‐free images. The strength of the entropy minimization method is especially apparent when applied to images containing large‐scale objects.
Cloning, Expression, Pharmacology and Tissue Distribution of the Mouse Somatostatin Receptor Subtype 5
Authors: Baumeister Hans , Kreuzer Oliver J. , Roosterman Dirk , Schäfer Judith , Meyerhof Wolfgang
Published: March 2003

The gene encoding the mouse somatostatin receptor subtype 5 has been isolated from a genomic library and the mRNA start point mapped to position −95 relative to the translational start codon. The promoter region is devoid of TATA and CAAT boxes but contains putative binding sites for AP‐1, AP‐2 and SP1 and response elements for glucocorticoids (GRE) and phorbol esters (TRE). The encoded receptor protein with a predicted molecular weight of 42.5 kDa is comprised of 385 amino acids and thus contains 22 and 21 amino acids more than rat and human counterparts. The extra amino acids are caused by another translational initiation codon located further upstream. In the region of overlap the mouse somatostatin receptor subtype 5 displays 96.7% sequence identity to the rat and 81.7% to the human homologue. Application of somatostatin‐14 and −28 to human embryonic kidney cells expressing the recombinant receptor resulted in the inhibition of forskolin‐stimulated adenylyl cyclase with comparable EC50 values. Consistent with the observed sequence relationship, the mouse somatostatin receptor subtype 5 displays a pharmacological profile that resembles the rat homologue more closely than the human counterpart. mRNA for the mouse somatostatin type 5 receptor has been detected in pituitary, kidney, spleen and ovary and, to a lesser extent, in brain, stomach, intestine and thymus but was not observed in heart, pancreas and liver.
Dissociation of Early Glucocorticoid Inhibition of ACTH Secretion and Glucose Uptake in Mouse AtT20 D16:16 Corticotrophs
Authors: Booth Chloe , Tian Lijun , Shipston Michael J
Published: March 2003

Adrenal glucocorticoid hormones rapidly exert powerful effects on neurons, immune and neuroendocrine cells through induction of de novo protein synthesis. In this study, we investigated, using mouse clonal anterior pituitary AtT20 D16:16 corticotrophs, whether (i) glucocorticoids rapidly inhibit glucose transport and (ii) whether this inhibition of glucose transport is directly correlated with early inhibition of ACTH secretion. Glucose uptake in AtT20 D16:16 cells was Na+‐independent because the Na+‐independent glucose transport inhibitor phloretin (100 μM) completely inhibited specific 14C‐deoxygluose (DoG) uptake and replacement of extracellular Na+ with N‐methyl
d‐glucamine+ had no effect. Furthermore, the Na+‐independent glucose transporters, GLUTs 1 and 3 were expressed in AtT20 D16:16 cells. The synthetic type II glucocorticoid receptor agonist dexamethasone, rapidly, within 2 h, inhibited DoG uptake into AtT20 D16:16 cells through a mechanism that was dependent on de novo mRNA synthesis. Glucocorticoid inhibition of glucose transport was not correlated with early inhibition of ACTH secretion because removal of glucose from the external medium had no effect on CRF‐stimulated ACTH secretion or the efficacy of early glucocorticoid inhibition of ACTH release. Although the Na+‐independent glucose transport inhibitor phloretin significantly inhibited CRF‐stimulated ACTH release, this effect of phloretin was a result of its potent activation of large conductance calcium‐activated potassium (BK) channels. These data suggest that different molecular pathways and/or glucocorticoid induced proteins underlie the mechanism(s) of early glucocorticoid inhibition of glucose uptake and ACTH release, respectively.
Identification of α1b Adrenergic Receptor Protein in Gonadotropin Releasing Hormone Neurones of the Female Rat
Authors: Hosny Somaya , Jennes Lothar
Published: March 2003

Noradrenaline is an important neurotransmitter which regulates GnRH release from the median eminence in the female rat during both basal GnRH secretion and the preovulatory or steroid hormone‐induced GnRH‐mediated LH surge. However, it is not clear at which sites in the brain this predominantly stimulatory influence is exerted nor is it known which adrenergic receptor subtype(s) mediate(s) the effects of noradrenaline. In order to determine if the GnRH neurones in the septum‐diagonal band‐preoptic area and/or their axon terminals in the median eminence are direct targets for noradrenaline, immunohistochemical triple‐labelling studies were conducted to localize simultaneously GnRH peptide, dopamine‐β‐hydroxylase and α1B adrenergic receptor protein. The results show that about 80% of all GnRH neurones examined contained patches of immunoreactive α1B adrenergic receptor protein at or near the plasma membrane and that some of these α1B adrenergic receptors were adjacent to dopamine‐β‐hydroxylase containing axons. The GnRH neurones which did not contain α1B adrenergic receptors were preferentially located in the rostral portion of the septum and diagonal band while all GnRH neurones in the caudal septum, diagonal band and in the preoptic area expressed α1B adrenergic receptors. In the median eminence, a few α1B adrenergic receptor patches were seen in the external layer and these receptors were only rarely observed to be associated with GnRH containing axon terminals. The results suggest that the effects of noradrenaline on GnRH release are, at least in part, mediated by the activation of α1B adrenergic receptors which are located on most GnRH perikarya while the median eminence is not a likely site at which GnRH release is regulated by α1B adrenergic receptors.
Proopiomelanocortin mRNA Levels in Ovine Hypothalamus Are Not Reduced at the Time of the Preovulatory Luteinising Hormone Surge
Authors: Walsh John P. , Rao Alexandra , Simmons Donna M. , Clarke Iain J.
Published: March 2003

A reduction in inhibition (disinhibition) of gonadotropin‐releasing hormone (GnRH) secretion by endogenous opioid systems in the hypothalamus is thought to be permissive of the preovulatory surge of GnRH and luteinising hormone (LH). In rats, proopiomelanocortin (POMC) mRNA levels in the arcuate nucleus are reduced at the time of the LH surge, and this is thought to be part of the neural mechanism of oestrogen positive feedback. There are no studies of POMC mRNA levels at the time of the LH surge in other species. POMC mRNA levels were measured by in‐situ hybridization using a 35S‐labelled cRNA probe and computer‐assisted grain counting in the arcuate nucleus of ovary‐intact ewes (n=4) killed on day 10 of the luteal phase or 24 or 48 h into the follicular phase (experiment 1), and ewes killed on day 10 of the luteal phase or during the preovulatory LH surge (experiment 2). Grain counts per cell, the proportion of cellular area covered by grains and the number of labelled cells per section were not significantly different (P>0.1) between animals killed in the luteal phase and animals killed during the follicular phase or during the LH surge. We conclude that in sheep, POMC mRNA levels are not reduced at the time of the preovulatory LH surge, and reduced POMC gene transcription does not appear to be part of the neural mechanism of oestrogen positive feedback in this species.
Cytoarchitectonic Analysis of Fos‐immunoreactivity in Brainstem Neurones Following Visceral Stimuli in Conscious Rats
Authors: Mayne  , Armstrong  , Crowley  , Bealer
Published: March 2003

Visceral inputs to the brain make their initial synapses within the nucleus of the solitary tract (NTS), where information is relayed to other brain regions. These inputs relate to markedly different physiological functions and provide a tool for investigating the topography of visceral processing in brainstem nuclei. Therefore, Fos immunoreactivity was used to determine whether a gastric stimulus affects neurones within different or similar parts of the NTS, ventrolateral medulla (VLM) and parabrachial nucleus (PBN), compared to a baroreceptive stimulus. The contribution of catecholaminergic neurones in these areas was studied by combining Fos and tyrosine hydroxylase (TH) immunoreactivity. Conscious male rats received either cholecystokinin (CCK) intraperitoneally to activate gastrointestinal afferents, or were made hypertensive by intravenous infusion of phenylephrine (PE) to activate baroreceptors. Tissue sections were processed immunocytochemically for Fos and/or TH. Phenylephrine infusion and CCK injection elicited Fos expression in distinct and in overlapping regions of the NTS and the VLM. Cholecystokinin injections increased the number of Fos‐immunoreactive neurones in the area postrema (AP) and throughout the rostral‐caudal extent of the NTS, including commissural neurones and the medial subnuclei. Some reactive neurones in NTS were also positive for TH, but most were not, and most of the TH‐positive NTS neurones were not Fos‐positive. In contrast, PE infusion produced a more restricted distribution of Fos‐positive neurones in the NTS, with most neurones confined to a dorsolateral strip containing few TH‐positive neurones. The medial NTS at the level of the AP and the AP itself were largely unresponsive, but rostral to the AP the medial NTS was labelled, including some TH‐positive neurones. Both treatments produced labelling in the caudal and mid‐VLM, but PE infusion had a stronger effect in the rostral VLM. In the PBN, CCK elevated Fos expression in several subregions, whereas PE infusion failed to specifically alter any subdivision. The results suggest that stimulation of baroreceptor and gastric afferents evoke both overlapping and cytoarchitectonically distinct pathways in the brainstem.
Serotonergic 5‐HT2A Receptors Important for the Oestradiol‐Induced Surge of Lusteinising Hormone‐Releasing Hormone in the Rat
Authors: Fink  , Dow  , McQueen  , Bennie  , Carroll
Published: March 2003

Serotonin (5‐HT) plays a role in mediating the oestradiol‐induced surge of luteinising hormone (LH), but so far the 5‐HT receptor subtype involved has not been identified. Our previous in‐situ hybridization and pharmacological studies suggest that the action of 5‐HT involves the 5‐HT2A receptor. The aim of the present study was to investigate this possibility by the direct approach of determining whether 5‐HT2A receptor antagonists block the oestradiol‐induced surge of luteinising hormone releasing hormone (LHRH). Adult female Wistar rats, which had shown at least two consecutive 4‐day oestrous cycles, were ovariectomised under halothane anaesthesia in the morning of dioestrus and injected with vehicle (arachis oil) alone or oestradiol benzoate (OB). At 12.00 h of the next day, presumptive pro‐oestrus, the animals were injected intraperitoneally with one of three 5‐HT2A antagonists, a selective 5‐HT reuptake inhibitor (fluoxetine), or the appropriate vehicles; hypophysial portal blood was then collected under alphaxalone anaesthesia between 15.00 and 19.00 h. The amount of LHRH released into hypophysial portal blood during consecutive 30‐min periods was determined by radioimmunoassay. As expected, oestradiol, but not oil, triggered a surge of LHRH in hypophysial portal blood with a peak at about 16.00 h of presumptive pro‐oestrus. This oestradiol‐induced surge of LHRH was blocked by ketanserin, ritanserin and the highly selective 5‐HT2A receptor antagonist, RP62203, but not by fluoxetine. These results provide the first direct evidence that the 5‐HT2A receptor plays an important role in the oestradiol‐induced surge of LHRH.
Peripenduncular Lesion Alters Expression of c‐fos Induced in the Female Rat Brain by Male Mounting
Authors: Wedemeyer  , Vivas  , Carrer
Published: March 2003

In order to investigate the role of the peripeduncular nucleus (PP) in the control of lordosis in female rats, activation of neurons after mounts without intromission was investigated by means of FOS immunoreactivity (FOS‐IR). Ovariectomized rats were injected with estradiol and progesterone and submitted to ≈50 mounts by the male. The vaginal area was covered with masking tape to prevent intromission and vaginocervical stimulation. This limited stimulation produced FOS‐IR in the ventrolateral division of the ventromedial hypothalamic nucleus (VMHVL), in the lateral periaqueductal grey (LPAG), in the peripeduncular nucleus (PP), and in the posterior intralaminar thalamic nucleus (PIL). No significant differences were found in the anterodorsal or posterodorsal parts of the medial amygdaloid nucleus, in the medial part of the medial preoptic nucleus, in the dorsomedial periaqueductal grey and in the medial division of the posterointermediate part of the bed nucleus of the stria terminalis. The same experiment was performed in rats with unilateral lesion of the PP. Both VMHVL and LPAG activation were significantly reduced in the ipsilateral PP lesion side, leading to the conclusion that those structures are primary targets for the somatic stimuli that trigger lordotic reflexes and which are relayed in the PP. Taking into account what is known about the function of the target structures, it is proposed that afferences relayed in the PP reaching the VMHVL would contribute to control the long range level of sexual receptivity, whereas stimuli reaching the LPAG would serve to control lordotic responses in a moment to moment fashion.
Single Stress Induces Long‐Lasting Elevations in Vasopressin mRNA Levels in CRF Hypophysiotrophic Neurones, but Repeated Stress is Required to Modify AVP Immunoreactivity
Authors: Aubry¶  , Bartanusz¶  , Jezova  , Belin  , Kiss
Published: March 2003

Repeated stress is known to induce an increased vasopressin (AVP) expression in paraventricular corticotrophin‐releasing factor (CRF) neurones which is supposed to enhance the ACTH‐releasing capacity of these cells. To test the hypothesis that a single stress is sufficient to produce these changes, we used quantitative in‐situ hybridization analysis to measure steady state CRF and AVP mRNA. Moreover the colocalized AVP and CRF immunoreactive sites were assessed in the dense core vesicle compartment of CRF axon terminals in the external zone of the median eminence with quantitative immunoelectron microscopy. Acute immobilization produced a significant increase in the average AVP and CRF mRNA levels (145% and 65%, respectively, above control values) in the medial parvocellular subdivisions of the paraventricular nucleus (PVN), and these changes persisted for over 4 days after stress. In contrast to these changes in AVP mRNA levels, there were no concomitant changes in AVP immunostaining in CRF terminals and axons during the 4‐day period. However, when immobilization stress was repeated daily, the number of CRF terminals containing AVP increased progressively. Moreover, the ratio of AVP and CRF immunoreactivity in the dense core vesicle compartment was increased. Taken together, these results provide evidence that single stress experience can cause long‐lasting changes in AVP and CRF mRNA steady state expression that is not apparently accompanied by changes in peptide levels. They also suggest that repeated stress is required for developing progressive shifts in the neurohormone storage pattern of these neurones.
Immunoreactive POMC‐Derived Peptides and Cytokines in the Chicken Thymus and Bursa of Fabricius Microenvironments: Age‐Related Changes
Authors: Franchini  , Ottaviani
Published: March 2003

Changes from hatching to the involutive stage in the thymus and bursa of Fabricius of Gallus domesticus were studied. Pro‐opiomelanocortin (POMC)‐derived peptides and cytokines were also tested by immunocytochemical procedures. Thymic histological modifications appeared at 3 months and involved an increase in connectival argyrophilic reticular fibres, a proliferation of non‐epithelial cells in the reticulum network, the presence of mucous cells and small mucous cysts, and an increase in the number of eosinophilic and myoid cells. A clear distinction between the cortex and the medulla was lost. Immunoreactive POMC‐related molecules and cytokines were demonstrated in thymic cells from 4‐day‐old chicken, and their number increased with ageing. These molecules were expressed in a few single epithelial cells and in interdigitating cells. With ageing, the number of immunoreactive interdigitating cells also increased, and these appeared in an activated phase. Histological modifications in the bursa of Fabricius appeared at 2 months and concerned a folding of the interfollicular surface epithelium covering the bursal plicae and a reduction in lymphoid follicle‐associated epithelium. Fibrous tissue gradually increased, and large mucoid cysts were evident. The expression of POMC‐derived peptide‐and cytokine‐like molecules differed during the development and involution phases of the organ. Cells of follicle associated epithelium and dendritic reticular cells of lymphoid follicles were immunoreactive to β‐endorphin between 4 days and 2 months, while ACTH‐, α‐MSH‐ and cytokine‐like molecules were observed in follicles after 2 months. The findings indicate a physiological role of these molecules during the growth and involution of the two organs.
Metabolic Influences on Circadian Rhythmicity in Siberian and Syrian Hamsters Exposed to Long Photoperiods
Authors: Challet  , Kolker  , Turek
Published: March 2003

Calorie restriction and other situations of reduced glucose availability in rodents alter the entraining effects of light on the circadian pacemaker located in the suprachiasmatic nuclei. Siberian and Syrian hamsters are photoperiodic species that are sexually active when exposed to long summer‐like photoperiods, while both species show opposite changes in body mass when transferred from long to short or short to long days. Because metabolic cues may fine tune the photoperiodic responses via the suprachiasmatic nuclei, we tested whether timed calorie restriction can alter the photic synchronization of the light‐entrainable pacemaker in these two hamster species exposed to long photoperiods. Siberian and Syrian hamsters were exposed to 16 h:8 h light:dark cycles and received daily hypocaloric (75% of daily food intake) or normocaloric diet (100% of daily food intake) 4 h after light onset. Four weeks later, hamsters were transferred to constant darkness and fed ad libitum. The onset of the nocturnal pattern of locomotor activity was phase advanced by 1.5 h in calorie‐restricted Siberian hamsters, but not in Syrian hamsters. The lack of phase change in calorie‐restricted Syrian hamsters was also observed in individuals exposed to 14 h:10 h dim light:dark cycles and fed with lower hypocaloric food (i.e. 60% of daily food intake) 2 h after light onset. Moreover, in hamsters housed in constant darkness and fed ad lib., light‐induced phase shifts of the locomotor activity in Siberian hamsters, but not in Syrian hamsters were significantly reduced when glucose utilization was blocked by pretreatment with 500 mg/kg i.p. 2‐deoxy‐
d‐glucose. Taken together, these results show that the photic synchronization of the light‐entrainable pacemaker can be modulated by metabolic cues in Siberian hamsters, but not in Syrian hamsters maintained on long days.
Transcriptional Repression of the 5‐HT1A Receptor Promoter by Corticosterone Via Mineralocorticoid Receptors Depends on the Cellular Context
Authors: Meijer  , Williamson  , Dallman  , Pearce
Published: March 2003

The diverse effects of the corticosteroid hormones are mediated in large measure by the mineralocorticoid and glucocorticoid receptors, two closely related members of the nuclear receptor superfamily. In the brain, corticosteroids regulate neuronal excitability and responses to neurotransmitters in a cell type‐specific manner. The 5‐HT1A receptor, for example, is highly expressed in the hippocampus and raphe but transcription is repressed by corticosterone (the principal glucocorticoid in rodents) only in hippocampus. We have used transient transfection of cultured cells to study the transcriptional regulation of the 5‐HT1A receptor promoter by activators and repression by glucocorticoids. We find that transcription factors Sp1 and NF‐kB subunit p65, both of which are coexpressed in hippocampus with the 5‐HT1A receptor in vivo, synergistically activate a reporter driven by receptor 5′‐flanking region. Primer extension data suggest that the multiple transcription initiation sites used in reporter gene transcription correlate with those used in transcription of the endogenous gene which has a TATA‐less promoter. Repression of transcription by corticosteroids was found to be mediated by both mineralocorticoid and glucocorticoid receptors, but not identically. While glucocorticoid receptors potently inhibited both p65‐ and p65/Sp1‐stimulated transcription, repression via mineralocorticoid receptors (MR) depended on the transcriptional activators that were present: p65‐stimulated reporter activity was not repressed via MR, whereas a similar level of transcription resulting from synergistic activation by p65/Sp1‐stimulation was repressed via MR. The context‐dependence of these MR‐mediated effects provides a model for the cell‐type and state‐dependent actions of corticosterone in the brain.
Expression of mRNA for Voltage‐Dependent and Inward‐Rectifying K Channels in GH3/B6 Cells and Rat Pituitary
Authors: Wulfsen  , Hauber  , Schiemann  , Bauer  , Schwarz
Published: March 2003

The expression of mRNA for voltage‐dependent (Kv) and inward‐rectifying K channels (Kir) was studied in clonal rat somato‐mammotroph cells (GH3/B6 cells) and rat pituitary using reverse transcription‐polymerase chain reaction (RT‐PCR). In GH3/B6 cells transcripts for 16 different Kv channel α‐subunits (seven Shaker‐related: Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv3.2, Kv4.1, Kv5.1; six EAG: eag1, erg1, erg2, elk1–elk3; three KCNQ: KCNQ1–KCNQ3) and for five different Kir channel α‐subunits (Kir1.1, Kir2.3, Kir3.2, Kir3.3, Kir6.2) were found. In addition, transcripts for a short isoform of Kvβ2 and transcripts for Kvβ3 subunits were present. In rat pituitary transcripts for 21 different Kv channel α‐subunits (11 Shaker‐related: Kv1.3, Kv1.4, Kv1.6, Kv2.1, Kv2.2, Kv3.2, Kv3.4, Kv4.1, Kv4.2, Kv4.3, Kv6.1; seven EAG: eag1, erg1–erg3, elk1–elk3; three KCNQ: KCNQ1–KCNQ3) and nine Kir channel α‐subunits (Kir1.1, Kir2.2, Kir3.1–Kir3.4, Kir4.1, Kir6.1, Kir6.2) were found. In addition, all tested auxiliary subunits (Kvβ1–Kvβ3, minK, SUR1, SUR2) are expressed in the pituitary. The results indicate that the macroscopic K currents in GH3/B6 and pituitary cells are presumably mediated by K channels constructed by a larger number of K channel α‐subunits and auxiliary β‐subunits than previously distinguished electrophysiologically and pharmacologically.
The Immune–Endocrine Interaction Varies with the Duration of the Inflammatory Process in Cardiac Surgery Patients
Authors: Roth‐Isigkeit A. , Dibbelt L. , Schmucker P. , Seyfarth M.
Published: March 2003

The present study investigated the perioperative course of cytokine release and hypothalamic‐pituitary‐adrenal (HPA) axis activation in relation to the duration of the inflammatory response in cardiac surgery patients. Twelve male patients scheduled for elective coronary artery bypass grafting surgery with cardiopulmonary bypass and general anaesthesia were divided into two study groups: group 1 (n=6) underwent surgery at 13.00 h±30 min, group 2 (n=6) at 08.30 h±50 min. Blood samples were collected preoperatively and up to the first postoperative day. Postoperatively, on the day of surgery, serum concentrations of the proinflammatory cytokines interleukin (IL)‐6, IL‐1β and tumour necrosis factor (TNF)‐α were not significantly different between the two groups, while blood concentrations of cortisol, adrenocorticotrophic hormone (ACTH) and β‐endorphin in group 2 patients were significantly higher than in group 1 patients. Postoperatively, on the day of surgery, ACTH and cortisol concentrations in group 1 patients were positively correlated to the blood concentrations of IL‐1β, IL‐6 and TNF‐α. By contrast, group 2 patients showed no significant relationship between cytokine release and activation of HPA axis at this time. Our results suggest that in patients undergoing cardiac surgery, the cytokine response is initiated before the HPA axis is fully activated. In the early postoperative period, cytokines appear to be involved in the activation of the HPA axis, while in the later postoperative period, high cortisol concentrations may inhibit the release of IL‐6.
The Effects of Acute and Chronic Stresses on Vasotocin Gene Transcripts in the Brain of the Rainbow Trout (Oncorhynchus mykiss)
Authors: Gilchriest B. J. , Tipping D. R. , Hake L. , Levy A. , Baker B. I.
Published: March 2003

Secretion of adrenocorticotropic hormone (ACTH) from the fish pituitary, which occurs in times of stress, is stimulated by several hypothalamic neuropeptides, one of which is arginine vasotocin (AVT). This study investigates whether gene expression for AVT is up‐regulated during acute or chronic stress. Rainbow trout (Oncorhynchus mykiss) were subjected to one of two forms of acute stress—either 2 h confinement followed by 2 h recovery, or capture and transfer to low water for 2 min followed by 4 h recovery in their home tank before autopsy. In other experiments, these stresses were repeated daily for 5 or 6 days (chronic stress). Quantification of AVT transcript prevalence in the parvocellular and magnocellular neurones of the preoptic nucleus after in situ hybridization was used as a monitor of the AVT gene response to stress. The results showed that acute confinement, but apparently not brief low‐water stress, significantly increased AVT transcript prevalence in a group of parvocellular perikarya. When applied repeatedly, both forms of stress caused habituation, such that the AVT hybridization signal remained at control or even lower levels despite elevated pro‐opiomelanocortin transcripts in the corticotropes and raised plasma cortisol concentrations. The AVT hybridization signal in the magnocellular perikarya showed no significant response to either acute or chronic stress. The results support the idea that these parvocellular AVT neurones are involved in ACTH stimulation during acute stress, and that the system habituates to chronic stresses.
Area Postrema Ablation Attenuates Activation of Neurones in the Paraventricular Nucleus in Response to Systemic Adrenomedullin
Authors: Shan J. , Krukoff T. L.
Published: March 2003

Adrenomedullin (ADM) is a potent vasodilator in the periphery which also acts centrally to increase blood pressure and inhibit drinking, feeding and salt appetite. This study was designed to study the effects of circulating ADM on neuronal activation in autonomic centres in the rat brain and to examine whether neuronal nitric oxide (NO) may participate in these processes. We identified activated neurones 1 h after intravenous (i.v.) injections of ADM (2 nmol/kg) using immunohistochemistry for Fos. The nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐d) histochemical reaction was used to localize putative NO‐producing neurones and double labelling for Fos and NADPH‐d was used to identify activated NO producing neurones. To separate baroreceptor‐induced neuronal activation in autonomic centres by ADM from other effects which it may have, i.v. infusions of sodium nitroprusside (NP) were used to mimic the hypotensive effects of ADM in control rats. Significantly greater numbers of activated neurones were found in the paraventricular nucleus of the hypothalamus (PVN) and especially in the dorsolateral medial parvocellular division, the nucleus of the solitary tract, and the area postrema (AP) of ADM‐treated rats compared to control rats. In addition, the number of activated NO‐producing neurones in the PVN was significantly higher in ADM‐treated rats compared to rats treated with NP. To determine whether AP is one of the possible routes through which systemic ADM enters the brain to exert its central effects, the APs of rats were ablated by aspiration. One hour after i.v. injections of ADM, significantly fewer PVN neurones were activated in AP ablation rats compared to AP sham ablation rats. Similarly, the number of activated NO‐producing neurones in the PVN was significantly lower in AP ablation rats compared to AP sham ablation rats. In conclusion, our results suggest that systemic ADM gains access to the brain through the AP to regulate neuronal activity in autonomic centres and that neuronal NO might be involved in central autonomic and/or neuroendocrine regulation by ADM.
Oestrogen Prevention of Neural Cell Death Correlates with Decreased Expression of mRNA for the Pro‐Apoptotic Protein Nip‐2
Authors: Meda C. , Vegeto E. , Pollio G. , Ciana P. , Patrone C. , Pellicciari C. , Maggi A.
Published: March 2003

We have recently identified nip‐2 as a gene target for 17β‐oestradiol activity in the neuroblastoma SK‐ER3 cells expressing the oestrogen receptor (ER) α. Here we show 17β‐oestradiol treatment of neuroblastoma and rat embryo neurones in culture blocks the increase in nip‐2 mRNA induced by apoptotic stimuli and prevents cell death as indicated by cell counting, 3,(4,5‐dimethylthiazol‐2‐yl)2,5‐diphenil‐tetrazoliumbromide and DNA fragmentation assays. Neither of these effects are observed in the presence of the specific ER antagonist ICI 182,780, and are absent in neuroblastoma cells not expressing ER. We propose that nip‐2 plays a relevant role in neural cell apoptosis and that a decrease in its expression is instrumental for the oestrogen anti‐apoptotic effect described here. The experimental evidence presented supports the recent hypothesis of a protective role of oestrogens in neurodegenerative diseases such as Alzheimer's disease and highlights the importance of the development of new ER ligands for the prevention of neural cell damage.