bluuebunny/crossref_metadata_2025 huggingface dataset:
Reconstruction of optical pathlength distributions from images obtained by a wide‐field differential interference contrast microscope
Authors: VAN MUNSTER E. B. , VAN VLIET L. J. , ATEN J. A.
Published: November 2003

An image processing algorithm is presented to reconstruct optical pathlength distributions from images of nonabsorbing weak phase objects, obtained by a differential interference contrast (DIC) microscope, equipped with a charge‐coupled device camera. The method is demonstrated on DIC images of transparent latex spheres and unstained bovine spermatozoa. The images were obtained with a wide‐field DIC microscope, using monochromatic light. After image acquisition, the measured intensities were converted to pathlength differences. Filtering in the Fourier domain was applied to correct for the typical shadow‐cast effect of DIC images. The filter was constructed using the lateral shift introduced in the microscope, and parameters describing the spectral distribution of the signal‐to‐noise ratio. By varying these parameters and looking at the resulting images, an appropriate setting for the filter parameters was found. In the reconstructed image each grey value represents the optical pathlength at that particular location, enabling quantitative analysis of object parameters using standard image processing techniques. The advantage of using interferometric techniques is that measurements can be done on transparent objects, without staining, enabling observations on living cells. Quantitative use of images obtained by a wide‐field DIC microscope becomes possible with this technique, using relatively simple means.
Dual‐colour microscopy of single fluorophores bound to myosin interacting with fluorescently labelled actin using anti‐Stokes fluorescence
Authors: SAITO K. , TOKUNAGA M. , IWANE A. H. , YANAGIDA T.
Published: November 2003

We have refined prismless total internal reflection fluorescence microscopy with extremely low background to visualize single fluorophores attached to protein molecules interacting with a filamentous biopolymer labelled with different colour fluorophores. By using Stokes and anti‐Stokes fluorescence, two different colour fluorescences from two different colour fluorophores excited with a single wavelength laser can be observed simultaneously. This microscopy was applied to visualize motor proteins, actin and myosin molecules. Single myosin molecules labelled with a tetramethylrhodamine‐5‐iodoacetamide interacting with a BODIPY FL‐labelled actin filament, a filamentous polymer of actin molecules, were observed clearly and simultaneously in aqueous solution. Individual hydrolysis reactions of Cy3‐labelled ATP by single myosin molecules and sliding of a BODIPY FL‐labelled actin filament along the myosin molecules could also be observed simultaneously. Thus, this technique is useful for observing single molecular processes of proteins interacting with a biological macromolecule such as an actin filament and a DNA.
Multivariate statistical analysis of STEM–EDX data from radiation‐induced sensitization in stainless steel
Authors: Titchmarsh J. M. , Dumbill S.
Published: November 2003

A series of energy‐dispersive X‐ray spectra acquired in a field‐emission gun scanning transmission electron microscope to generate compositional profiles across a grain boundary in a neutron‐irradiated stainless steel were subjected to a multivariate statistical analysis (MSA). The complex profiles generated by conventional spectrum processing were factored by MSA into two independent components of information, one associated with both the pre‐irradiation heat treatment and radiation‐induced segregation (RIS) and the other with diffusion prior to precipitate formation. The two experimental components were separately modelled to deconvolute simple theoretical distributions of composition, which were then recombined to give the full variation in composition across the boundary. A new observation was that the commonly observed RIS enhancement of Ni and Si did not persist in the boundary plane, the composition of which was dominated by pre‐irradiation segregation. The potential benefits of MSA for segregation studies are discussed.
Highly spatially resolved electron energy‐loss spectroscopy in the bandgap regime of GaN
Authors: Bangert U. , Harvey A. J. , Freundt D. , Keyse R.
Published: November 2003

The possibilities of obtaining information about interband scattering processes in the bandgap regime of GaN from electron energy‐loss spectra, taken in a dedicated scanning transmission electron microscope (STEM), are investigated. With the help of precise simulations of the zero‐loss peak it is feasable to process, extract and analyse data in the extreme low‐loss regime of a few electronvolts. The accuracy of the results is restricted predominantly by instrumental broadening functions. By modelling these accurately, it is possible to eliminate the effects of the tail of the zero‐loss peak and to extract the low‐loss spectrum together with the correct value for the bandgap of GaN. Furthermore, differences in the shapes of the low‐loss spectra can be revealed, depending on the microstructural features, probed at different beam locations.
Dental gallium alloy composites studied by SEM and TEM
Authors: GUNNÆS A. E. , OLSEN A. , HERØ H.
Published: November 2003

Analytical scanning and transmission electron microscopy (SEM and TEM) studies of dental gallium alloys have been carried out. The Ga alloys were made by triturating a LU powder (Ag–Sn–Cu rich alloy powder) and a GF powder (Ag–Sn–Cu–Pd rich alloy powder) with a liquid Ga alloy containing Ga, In and Sn. The dental materials were found to be composites consisting of remaining, undissolved particles from the Ag‐based alloy powders in a matrix of reaction products with the Ga alloy. SEM studies have been carried out to give an overview of the composites. The distribution of the elements was found by the X‐ray mapping technique. The phases in the matrix and the remaining alloy particles have been identified by electron diffraction, high‐resolution electron microscopy and energy‐dispersive X‐ray spectroscopy. The following phases were identified in the LU alloy: orthorhombic Ag3Sn, cubic Ag9In4, tetragonal β‐Sn and hexagonal Ag2Ga. In addition to these well‐known phases Ga‐rich regions were observed consisting of an intergrowth of tetragonal CuGa2 and one of the cubic γ‐Cu9Ga4 phases. In addition to these phases cubic Ga7Pd3 was found in the GF alloy. The anomalous setting expansion of the GF alloy may be explained by the presence of Ga7Pd3.
Microstructural evolution of the latest generation of small‐diameter SiC‐based fibres tested at high temperatures
Authors: HOCHET N. , BERGER M. H. , BUNSELL A. R.
Published: November 2003

The new generation of silicon‐carbide‐based fibres made from organosilicon precursors, cross‐linked by electron irradiation, have been compared with the earlier fibres which have undergone cross‐linking in air. The latest fibres, known as Tyranno Lox‐E and Hi‐Nicalon, possess a lower oxygen content (≈5 wt% and ≈0.5 wt%) whereas the NLM202 fibres contain 12 wt% and the Tyranno Lox‐M 13 wt% of oxygen. The Tyranno fibres have been produced with a precursor similar to that used to produce the Nicalon fibres, but modified by the addition of titanium. All fibres possess a structure composed of β‐SiC grains, free carbon aggregates, with no crystallized titanium compounds in the Tyranno fibres and an oxygen‐rich intergranular phase, except in the Hi‐Nicalon fibre. The Hi‐Nicalon fibre has the largest grain size and its free carbon content is higher than in the NLM202 fibres. For all the fibres, the β‐SiC grains grow when the temperature increases, whilst the strengths and Young's moduli decrease. The NLM202 shows the least change in grain size and tensile properties. The Hi‐Nicalon is stiffer and stronger than the others at high temperature. TEM results show that grain growth is isotropic, even during creep tests. The growth depends on the nature and amount of the intergranular phase. Mechanical changes as a function of temperature can be explained by external oxidation during tensile tests in air and internal oxidation facilitated by the nanoporosity, which is greater in the Tyranno than in the Nicalon fibres. The presence of the oxygen‐rich phase in the three fibres containing the most oxygen decreases the creep resistance. Titanium does not improve the mechanical properties and the creep resistance beyond 1523 K and does not have any positive influence in limiting the SiC grain growth. Tyranno fibres are less well stabilized than the Nicalon fibres. The Hi‐Nicalon fibres have been shown to possess consistently better mechanical properties at all temperatures, including creep resistance, than the other fibres studied. All the fibres are sensitive to external oxidation at high temperature.
Three‐dimensional microscopic biopsy of in vivo human skin: a new technique based on a flexible confocal microscope
Authors: Masters B. R. , Gonnord G. , Corcuff P.
Published: November 2003

A new noninvasive microscopic technique of three‐dimensional optical biopsy from in vivo human skin based on real‐time confocal microscopy and computer reconstruction is demonstrated. A tandem scanning confocal microscope is a prototype of a mobile, flexible design for the in‐depth microscopic exploration of the skin on the human body. The various skin layers were observed in real‐time, at the subcellular level down to a depth of 200 μm with a vertical resolution of 2 μm. Rapid video recording of the Z‐series through the ventral aspect of the forearm avoided shifts caused by subject movement and blood flow pulsations. Two video frames were averaged, and the average was digitized, providing a stack of 64 optical sections in 1‐μm vertical steps. Three‐dimensional reconstructions of in vivo human skin were obtained with sets of orthogonal slices, and slices at arbitrary planes through a volume containing the stack of slices. This method clearly shows the spatial relationships between the different cell layers. The use of orthogonal cutting planes is preferred because of its analogy with classical vertical sections of histopathology. Linear structures (surface lines) within the stratum corneum are described and their global orientations were determined by the use of Fourier transform analysis. En face optical sections constitute unusual views of this tissue, since typical pathohistological studies are based on sagittal (vertical) slices. The noninvasive optical microscopic technique provides a three‐dimensional optical biopsy of in vivo human skin.
Biological imaging using fast laser scanning heterodyne differential phase confocal microscopes
Authors: Garside J. R. , Somekh M. G. , See C. W.
Published: November 2003

Differential phase microscopy has proved invaluable in the study of live, unstained, thin biological samples because of its ability to image changes in refractive index and topography. Similarly, because of its optical sectioning capability, confocal microscopy is now a well‐established technique in the study of relatively thick live biological samples. This paper describes the development and application of two differential phase heterodyne confocal microscopes, and compares their performance. The use of these systems for imaging in‐vitro cell and tissue cultures is considered and compared with confocal reflection microscopy. It is demonstrated that the differential phase capability reveals subcellular structural information not readily seen in the confocal reflection images. This technique opens up the possibility of imaging thick unstained live tissues, avoiding cell damage and artefacts associated with staining procedures. Furthermore, the differential phase images can be used to provide a visual frame within which stained features can be located.
Sex Differences in Cytosolic Androgen Receptors in Gonadectomized Male and Female Rats
Authors: McGinnis Marilyn Y. , Katz Sarah E.
Published: November 2003

The purpose of this experiment was to determine if there are sex differences in cytosolic androgen receptors (AR) in individual brain nuclei. Bilateral 500 μ diameter samples from 300 μ thick frozen brain sections were micropunched from males and females 2–3 weeks following gonadectomy. Tissue samples were taken from 12 brain nuclei: lateral septum, bed nucleus of the stria terminalis, medial preoptic nucleus, anterior hypothalamic area, arcuate nucleus, corticomedial nucleus of the amygdala, lateral preoptic area, parietal cortex, medial nucleus of the amygdala, dorsomedial nucleus of the hypothalamus, ventromedial nucleus of the hypothalamus, and dorsal hippocampus. Cytosolic ARs were higher in males than in females in each of these 12 areas, but this sex difference was significant only in the first 6. Sex differences in ARs were found in brain regions involved in the neuromodulation of androgen dependent responses. These data suggest that behavioral differences in male reproductive responses may be the result of a sexually dimorphic distribution of androgen receptor containing neurons.
Dynamic Changes in Gonadotropin Releasing Hormone Receptor mRNA Content in the Mediobasal Hypothalamus during the Rat Estrous Cycle
Authors: Jennes Lothar , McShane Teresa , Brame Blake , Centers Adrian
Published: November 2003

The purpose of the present study was to determine if GnRH receptor mRNA levels in the rat brain undergo changes during the estrous cycle. We focused on the arcuate and ventromedial nuclei of the hypothalamus and on the hippocampus which are sites in the rat central nervous system that have been shown to contain measurable amounts of GnRH receptor mRNA. Groups of regularly cycling female rats were decapitated at 08.00 and 17.00 h of each day of the estrous cycle, trunk blood was collected for radioimmunoassay analysis of circulating LH levels, and the brains were processed for ‘in situ’ hybridization. A cDNA probe encoding the rat pituitary GnRH receptor was transcribed ‘in vitro’ in the presence of 33P‐alpha UTP and used under saturating conditions to label GnRH receptor mRNA. The results show that in the arcuate and ventromedial nuclei GnRH receptor mRNA levels are relatively high during diestrus 1, they decline slightly during diestrus 2 before they rise to the highest levels at 08.00 h of proestrus. By 17.00 h of proestrus, GnRH receptor mRNA levels had declined to the lowest levels of the estrous cycle where they remain through the morning of estrus. The GnRH receptor mRNA levels rise again sharply during the afternoon of estrus. The changes in the hippocampus follow a similar pattern in that a decline in GnRH receptor mRNA levels to its lowest levels occurs between 08.00 and 17.00 h of proestrus. However, the changes in the hippocampus did not reach statistical significance.
A Rapid Suppressive Effect of Estrogen in the Paraventricular Nucleus on Pulsatile LH Release in Fasting‐Ovariectomized Rats
Authors: Nagatani Shoji , Tsukamura Hiroko , Murahashi Kumiko , Maeda Kei‐Ichiro
Published: November 2003

The paraventricular nucleus (PVN) and A2 are novel estrogen feedback sites where estrogen may modulate the neural response to adrenergic inputs during fasting. In the present study, the effects of local estradiol (E2) perfusion through a microdialysis probe placed in the PVN or A2 on pulsatile luteinizing hormone (LH) secretion and on norepinephrine (NE) release in the PVN were examined in 48‐h fasting ovariectomized (OVX) rats to determine whether local estrogen administered in the PVN or A2 rapidly inhibits LH secretion during fasting and whether this inhibition is mediated by an increase of NE release in the PVN. Five days after ovariectomy, animals (n=5 per group) stereotaxically implanted with a guide cannula for microdialysis in the PVN (experiment 1) or both PVN and A2 (experiment 2) were deprived of food for 48 h. Blood samples and dialysates were then collected every 6 min for 3 h and every 12 min (experiment 1) or 20 min (experiment 2) for 3 h, respectively. The PVN or A2 was perfused with E2 (5 ng/ml in artificial cerebrospinal fluid) through a microdialysis probe after the first hour of sampling. E2 perfusion in the PVN caused a rapid and significant suppression of mean plasma LH levels and LH pulse frequency in fasting rats but no changes in unfasting animals. NE release in the PVN was not affected by the local E2 perfusion of the PVN in either fasting or unfasting groups. This perfusion in A2, however, did not cause any apparent changes in plasma LH and perfusate NE levels in the PVN and A2. The present results indicate that estrogen feedback action at the PVN suppresses LH secretion rapidly during fasting and does not involve an increase of NE release in the PVN.
GHRP6‐Stimulated Hormone Secretion in Somatotrophs: Involvement of Intracellular and Extracellular Calcium Sources
Authors: Bresson‐Bépoldin L. , Dufy‐Barbe L.
Published: November 2003

GHRP6 is a synthetic hexapeptide which stimulates growth hormone (GH) secretion from the pituitary in vivo and in vitro. We have previously shown that in identified somatotrophs, GHRP6 induces a biphasic increase in cytosolic Ca2+ concentration ([Ca2+]i) consisting of an abrupt increase (first phase) followed by a sustained plateau of elevated [Ca2+]i (second phase). The first phase corresponds to mobilization of intracellular Ca2+ pools and the second phase to influx of extracellular Ca2+ ions through voltage‐sensitive Ca2+ channels. In these experiments, we investigated the specific role of each of these two phases in the hormone response to GHRP6. We found that inhibition by thapsigargin of the intracellular Ca2+ mobilization phase significantly inhibited the hormone response to the peptide during 30 min incubations. Inhibition of the extracellular Ca2+ influx phase by nifedipine, a blocker of voltage‐sensitive Ca2+ channels, resulted in a 53% reduction of the secretory response to 10−5 M GHRP6. Antagonism of PKC by phloretin, a flavonoid which prevents PKC activation, and PKC depletion induced by a 24 h treatment with 10−6 M PMA, completely inhibited the response to GHRP6. Somatostatin, which also inhibits the second phase of the Ca2+ response, suppressed the secretory response to GHRP6. We conclude that, Ca2+ is the main second messenger and both Ca2+ mobilization and Ca2+ entry play a role in the response to GHRP6. However, experiments with PKC depletion and SRIF suggest that other messengers are implicated in GHRP6 signalling in somatotrophs.
Compression of the Pituitary Stalk Elicits Chronic Increases in CSF Vasopressin, Oxytocin as well as in Social Investigation and Aggressiveness
Authors: Haller J. , Makara G.B. , Barna I. , Kovács K. , Nagy J. , Vecsernyés M.
Published: November 2003

The neurochemical and behavioural effects of a novel stereotaxic surgical method developed for interrupting the nerve fibres running through the rat pituitary stalk to the posterior pituitary gland was studied. The cerebrospinal fluid (CSF) vasopressin (AVP) and oxytocin (OT) content as well as changes in aggressiveness were measured in rats one week and one month after the surgical intervention. The main results are as follows: (1) the compression of the pituitary stalk elicits a chronic increase in water consumption, as well as in CSF vasopressin and oxytocin content; (2) the surgical intervention increased the frequency of clinch fighting after one week. The increase in aggressiveness accentuated after one month and, in addition, operated animals showed reduced scores of resting while exploratory and social behaviours increased; (3) there was a strong positive correlation between water consumption, vasopressin, and aggressiveness; (4) oxytocin changes showed a positive correlation with variation in social behaviour. The surgical intervention may serve as a model for lesions of the pituitary stalk and formation of ectopic neurohypophyses in humans.
Food or Water Deprivation Modulate Nitric Oxide Synthase (NOS) Activity and Gene Expression in Rat Hypothalamic Neurones: Correlation with Neurosecretory Activity?
Authors: O'Shea R. D. , Gundlach A. L.
Published: November 2003

Nitric oxide (NO) is produced by the enzyme NO synthase (NOS) and may be involved in the regulation of nutrient and endocrine homeostasis via actions on neurones of the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei. The effects of water deprivation or food deprivation for 4 days on the abundance of messenger RNA encoding NOS in these nuclei in rats were examined using in situ hybridization. Water deprivation markedly increased the abundance of NOS mRNA in both the SON and PVN (225±11% of control, P<0.05 and 261±34% of control, P<0.01 respectively). NOS mRNA abundance also appeared to be increased in magnocellular accessory nuclei. Food deprivation decreased NOS mRNA abundance in the SON and PVN (42±6% and 52±7% of control respectively, both P<0.05), while withdrawal of both food and water produced no significant net changes in the abundance of NOS mRNA. Treatment‐induced alterations in NOS mRNA abundance were reflected by changes in NOS activity, as assessed by NADPH‐diaphorase histochemistry, and NADPH‐diaphorase staining was observed in neurones both positive and negative for oxytocin‐like immunoreactivity. These findings suggest that NOS mRNA abundance, NOS enzymatic activity and presumably NO production are modulated in an activity‐dependent manner in hypothalamic (magnocellular and parvocellular) neurones by alterations in fluid and nutrient homeostasis, and support data from other studies suggesting a role for NO in the central regulation of water and food intake in the rat.
Hypothalamic Cellular and Molecular Mechanisms Helping to Satisfy Axiomatic Requirements for Reproduction
Authors: Pfaff D. W. , Kow L.‐M. , Zhu Y.‐S. , Scott R. E. M. , Wu‐Peng S. X. , Dellovade T.
Published: November 2003

In the absence of universal equations expressing neurobiological findings, the safest theoretical approach for the neuroendocrinologist is to start from axiomatic requirements for biologically adaptive neural mechanisms, in our case for reproduction. From this emerge two themes: the likely importance of interactions between internal (hormonal) and external signals in controlling gene expression relevant to reproductive functions; and, second, the vision of molecular interactions on DNA subserving environmental impacts on reproduction. The first theoretical notion has so far yielded data showing a role for synaptic inputs during the onset of estradiol actions for the hormone's induction of enkephalin mRNA, a finding which parallels earlier behavioral results. As well, noxious somatosensory inputs interact with estrogens and progesterone in their influence on enkephalin gene expression. The second theme led to novel investigations of thyroid influences on reproductive molecular biology and behavior, including the ability of exogenous or endogenous thyroid hormones to reduce female mating responses. Since elevated thyroid hormone levels could signal environmental cold, our experiments offer the possibility of explaining ethological facts at a molecular level. More generally, nuclear hormone receptor interactions on the surface of DNA may offer a new level of neural integration revealed first by hormone effects in neuroendocrine cells.
Signalling Pathways Mediating Secretory and Mitogenic Responses to Galanin and Pituitary Adenylate Cyclase‐Activating Polypeptide in the 235‐1 Clonal Rat Lactotroph Cell Line
Authors: Hammond P. J. , Smith D. M. , Akinsanya K. O. , Mufti W. A. , Wynick D. , Bloom S. R.
Published: November 2003

The neuropeptides galanin and pituitary adenylate cyclase‐activating peptide (PACAP) have been implicated in the physiological regulation of lactotroph function. Using the 235‐1 clonal lactotroph rat cell line we have studied the signalling pathways mediating the secretory and mitogenic responses to galanin and PACAP. Both peptides stimulated prolactin release to a similar maximal extent. PACAP (100 nM) stimulated an increase in the proliferation rate of 235‐1 cells, but was significantly less effective than 100 nM galanin (161.8±2.3%vs 296.1±9.1% of control). PACAP stimulated cAMP accumulation with an ED50 of 3.2 nM, and a maximal effect of almost two‐fold at a concentration of 100 nM. Galanin depleted cAMP, by 30% at a concentration of 100 nM. The aminosteroid phospholipase C (PLC) inhibitor U‐73122 virtually abolished maximal peptide stimulated prolactin release. Depletion of inositol phosphates or downregulation of protein kinase C reduced maximal peptide stimulated prolactin release from about 260% to about 160% of unstimulated release. Both peptides at a concentration of 100 nM caused a sustained increase in intracellular calcium when incubated with cells for 30 min. These results demonstrate that both peptides stimulate prolactin release and the proliferation rate of 235‐1 cells. The most important signalling pathway for prolactin release activated by both peptides is via PLC, although they also regulate cAMP levels, which are increased by PACAP and decreased by galanin. Despite maximal peptide stimulated prolactin release being equal, galanin has a greater mitogenic effect on 235‐1 cells than PACAP, raising the possibility that it activates an additional mitogenic signalling pathway.
Photoperiodic Modulation of Monoamines and Amino‐Acids Involved in the Control of Prolactin and LH Secretion in the Ewe: Evidence for a Regulation of Tyrosine Hydroxylase Activity
Authors: Viguié Catherine , Thibault Jean , Thiéry Jean‐Claude , Tillet Yves , Malpaux Benoît
Published: November 2003

Several neurotransmitters are implicated in the photoperiodic regulation of prolactin and luteinising hormone (LH) secretion in the ewe. This work investigated whether catecholamines, γ‐amino butyric acid (GABA), excitatory amino acids and serotonin diencephalic contents are affected by photoperiod and how such changes relate to the seasonal effects of photoperiod on LH and prolactin secretions. Moreover, to determine whether photoperiod can influence catecholamine biosynthesis, the activity of its rate limiting enzyme, tyrosine hydroxylase (TH) was also investigated. TH activity and the tissue content of the monoamines and their metabolites were measured in stalk‐median eminence (SME), preoptic area (POA) and the mediobasal, mediodorsal and laterobasal aspects of the hypothalamus. Investigation of excitatory amino acids and GABA was limited to the POA and the SME. Ovariectomized ewes were initially maintained in long days (LD) for 70 days. Thereafter half the ewes remained exposed to long days and the other half were transferred onto short days (SD) for 63 to 66 days to induce a stimulation of LH secretion and an inhibition of prolactin secretion. In each photoperiodic regime, half the ewes were treated with a subcutaneous oestradiol implant (+E) and half were not (−E). As expected, short days induced a decrease in prolactin and an increase in pulsatile LH secretion. These neuroendocrine changes were associated with a decrease in the TH activity of the SME in both oestradiol treated and non treated animals (146.5±24.1, 167.6±26.5 U TH/g of tissue in LD‐E and LD+E vs 83.5±12.4 and 95.0±30.2 U TH/g of tissue in SD‐E and SD+E animals; P≤0.01). A similar and parallel short day‐induced decrease was observed in the tissue content of dopamine and its metabolite, 3,4‐dihydroxy‐phenylacetic acid (SD level were 55% of LD levels, P<0.05). In POA, a short day‐induced decrease in dopamine (18%; P≤0.05) and GABA (16.4%; P≤0.05) content and an oestradiol‐induced decrease in aspartate (15.6%; P≤0.05) content were found. This study provides the first report of a photoperiodic control of the synthesis activity of catecholaminergic neurones of the SME in the ewe. The photoperiod‐induced changes in dopaminergic activity at the level of the SME were associated with changes in LH and prolactin secretion indicating that TH activity of dopaminergic neurones of the SME could be a critical component of the photoperiodic regulation of LH and/or prolactin secretion. In particular, this finding is in agreement with the hypothesis that photoperiod can control a dopaminergic pathway inhibitory of LH secretion and which ends in the median eminence.
Brainstem Hemisection Decreases Corticotropin‐Releasing Hormone mRNA in the Paraventricular Nucleus but not in the Central Amygdaloid Nucleus
Authors: Pacak Karel , Palkovits Miklos , Makino Shinya , Kopin Irwin J. , Goldstein David S.
Published: November 2003

Corticotropin‐releasing hormone (CRH) neurons in the paraventricular nucleus (PVN) of the hypothalamus and in the central nucleus of the amygdala (ACE) participate in neurohumoral and behavioral responses to stress. To understand better the central regulation of CRH, the present study assessed the effects of ipsilateral surgical hemisection of the brainstem on expression of CRH mRNA in the PVN and the ACE. In situ hybridization was used to demonstrate PVN CRH mRNA expression in hemisected, sham‐operated or intact rats before and after 3 h of immobilization (IMMO). In addition, hypothalamic‐pituitary‐adrenocortical (HPA) axis activity at baseline and during IMMO was assessed by measurements of plasma concentrations of ACTH and corticosterone. IMMO markedly increased CRH mRNA expression in the PVN in all experimental groups. Rats with brainstem hemisections had lower PVN CRH mRNA expression ipsilateral to the lesion and markedly blunted responses after IMMO, compared to values in sham‐operated rats. In contrast, neither hemisection nor IMMO affected CRH mRNA expression in the ACE. Lesioned and SHAM‐operated groups did not differ in baseline or IMMO‐induced increases in plasma ACTH or corticosterone levels. The present results indicate that baseline levels and IMMO‐induced increments in CRH mRNA expression in the PVN, but not in the ACE, depend on ipsilaterally ascending medullary tracts and that IMMO‐induced HPA activation does not depend on these pathways.
Production of a prolactin releasing factor by the ovine pars tuberalis
Authors: Hazlerigg D. G. , Hastings M. H. , Morgan P. J.
Published: November 2003

The anterior pituitary hormone prolactin is involved in the regulation of a wide variety of processes including mammary growth, lactation, reproduction and pelage growth (1). Correspondingly, in seasonal breeders, pronounced photoperiod‐driven, seasonal changes occur in the levels of prolactin secretion (2). These effects are thought to be mediated by the pineal hormone melatonin, which acts as a humoral indicator of photoperiod (3). Melatonin is thought to act through hypothalamic sites to control the gonadotrophic axis (4–6), but the sites through which melatonin modulates prolactin remain to be established. One possibility is that melatonin acts at the level of the hypothalamus to modulate the release of the hypothalamic prolactin inhibitory factor, dopamine (7). However, recent evidence from hypothalamo‐pituitary disconnection experiments performed in the ram suggests that the photoperiodic modulation of prolactin secretion can occur independently of the hypothalamus, presumably due to direct effects of melatonin on the anterior pituitary (8). The only identified site of melatonin receptor expression within the ovine pituitary is the pars tuberalis (PT) (9), the cells of which are almost completely non‐lactotrophic (10). Thus the possibility exists that factors released by the PT regulate the activity of lactotrophs in the pars distalis (PD). We have investigated this hypothesis using a range of co‐culture and medium‐conditioning experiments on primary cultures of ovine PT and PD cells, and here we report that PT cells secrete an unidentified factor that is a potent stimulus of prolactin secretion by PD cells.
Effect of CRH on the Preovulatory LH and FSH Surge in the Cyclic Rat: a Role for Arginine Vasopressin?
Authors: Roozendaal M. M. , Swarts J. J. M. , Wolbers W. B. , Threels A. , Wiegant V. M. , Mattheij J. A. M.
Published: November 2003

The effect of intracerebroventricular (i.c.v.) injection or infusion of various doses of corticotropin‐releasing hormone (CRH) on the LH and FSH surge was studied in pro‐oestrous rats supplied with a jugular vein and an i.c.v. cannula. Additionally, we investigated if arginine vasopressin (AVP) was involved in the CRH‐induced alterations to the surge of gonadotropins. I.c.v. injection of 10 μg CRH given 5 min before the presumed onset of the LH surge caused a strong inhibition of the LH surge and a slight inhibition of the FSH surge. Three to four h after CRH injection, its inhibitory effect diminished. A 6 h i.c.v. infusion of CRH started 1 h before the presumed onset of the LH surge, caused a dose‐related inhibition of the LH and FSH surge. Infusion of 1 μg/h CRH did not suppress the surge of both hormones while infusion of 5 or 10 μg/h CRH inhibited the LH surge. Infusion of 10 μg/h CRH caused a strong suppression of plasma LH during the first 3 h of the LH surge. Despite continuation of CRH infusion, the inhibitory effect disappeared and plasma LH increased to similar levels as in controls at corresponding points of time of the LH surge. The FSH surge was also suppressed by infusion of 10 μg/h CRH. The surge of LH and FSH was not affected by a 9‐h infusion of 10 μg/h CRH started 4 h before the presumed onset of the LH surge. This observation also indicates that the inhibitory effect of CRH may last for only 3–4 h. The surge of LH and FSH was not affected by i.c.v. injections of AVP‐antiserum. However, pretreatment with AVP‐antiserum prolonged the inhibitory effect of CRH on the LH surge. In conclusion, CRH can inhibit the pro‐oestrous LH and to a lesser extent the FSH rise for only 3–4 h after the beginning of CRH administration. AVP may play a role in limiting the inhibitory effect of CRH on LH to 3–4 h.
Tissue Specific Expression of Different Human ReceptorTypes for Pituitary Adenylate Cyclase Activating Polypeptideand Vasoactive Intestinal Polypeptide: Implications fortheir Role in Human Physiology
Authors: Wei Yang , Mojsov Svetlana
Published: November 2003

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are two structurally related peptides with pleiotropic physiological effects. Biochemical and cloning experiments have demonstrated that there are two structurally distinct receptors which recognize PACAP and VIP peptides with similar affinities (PACAP/VIP R‐1, PACAP/VIP R‐2), as well as a receptor that is specific for the PACAP peptide (PACAP‐Type 1 receptor). Using a homology‐based cloning strategy we have identified PACAP/VIP R‐2 receptor in human adipocytes, a tissue which was not previously identified as a target for PACAP and VIP action. This receptor type recognizes PACAP‐38 and VIP with similar affinity with inhibition concentrations of IC50=6.2±4.8 nM for PACAP‐38 and IC50=9.4±4.6 nM for VIP. Like the other two PACAP receptor types, PACAP/VIP R‐2 is coupled to cAMP‐mediated signal transduction pathway with effective doses ED50=3.2±1.6 nM and ED50=2.2±0.9 nM for PACAP‐38 and VIP respectively. Transcripts of the common PACAP/VIP R‐2 are also found in human brain and a number of peripheral tissues, such as pancreas, muscle, heart, lung, kidney, stomach and low levels in the liver. Comparison of the tissue distribution of the human PACAP/VIP R‐2 to that of the other two types of human PACAP receptors (PACAP‐Type 1 and the other common PACAP/VIP R‐1) by RNase protection showed that each of the three PACAP receptors is expressed in a unique set of human peripheral tissues. RNA transcripts for all three PACAP receptor types are found in human heart, brain and adipose tissue, while PACAP/VIP R‐2 is the only one of these three receptor types that is expressed in the pancreas and skeletal muscle. These results suggest a novel and not yet characterized role for PACAP and VIP peptides in the neuroendocrine regulation of insulin‐glucose homeostasis.
Deficiency in Mouse Oxytocin Prevents Milk Ejection,but not Fertility or Parturition
Authors: Young III W. Scott , Shepard Emily , Amico Janet , Hennighausen Lothar , Wagner Kay‐Uwe , LaMarca Mary E. , McKinney Cindy , Ginns Edward I.
Published: November 2003

Oxytocin is a nonapeptide hormone that participates in the regulation of parturition and lactation. It has also been implicated in various behaviors, such as mating and maternal, and memory. To investigate whether or not oxytocin (OT) is essential for any of these functions, we eliminated, by homologous recombination, most of the first intron and the last two exons of the OT gene in mice. Those exons encode the neurophysin portion of the oxytocin preprohormone which is hypothesized to help in the packaging and transport of OT. The homozygous mutant mice have no detectable neurophysin or processed oxytocin in the paraventricular nucleus, supraoptic nucleus or posterior pituitary. Interestingly, homozygous mutant males and females are fertile and the homozygous mutant females are able to deliver their litters. However, the pups do not successfully suckle and die within 24 h without milk in their stomachs. OT injection into the dams restores the milk ejection in response to suckling. These results indicate an absolute requirement for oxytocin for successful milk ejection, but not for mating, parturition and milk production, in mice.
Growth Hormone (GH)‐Releasing Peptide and GH Releasing Hormone Stimulate GH Release from Subpopulations of Somatotrophs in Rats
Authors: Mitani Maki , Kaji Hidesuke , Abe Hiromi , Chihara Kazuo
Published: November 2003

The synthetic hexapeptide GH‐releasing peptide (His‐D‐Trp‐Ala‐Trp‐D‐Phe‐Lys‐NH2; GHRP‐6) and GH releasing hormone (GHRH) are both potent stimulators of GH release in rats. Using reverse hemolytic plaque assay (RHPA), we have compared the effects of human GHRH and GHRP‐6 on GH release from the dispersed individual cells of rat anterior pituitary.

In a single RHPA, we quantified the percentage of plaque forming cells (% PFC) and their mean plaque area (MPA) after 30 min‐incubation, and calculated a total secretion index (TSI) by multiplying % PFC and MPA. 10 nM GHRH and 100 nM GHRP‐6 each caused a significant increase in % PFC (%) (GHRH 39.15, GHRP‐6 29.4, vs vehicle 24.3, P<0.01), MPA (×10−2 μm2) (GHRH 124.04, GHRP‐6 94.80, vs vehicle 44.57, P<0.01) and TSI (×10−2) (GHRH 54.46, GHRP‐6 32.87, vs vehicle 10.84, P<0.01). Simultaneous addition of both secretagogues caused a further increase in GH release (%PFC 46.4, MPA 142.55, TSI 69.82, P<0.01 vs vehicle), although the effect was additive but not synergistic. Somatostatin analog, SMS201–995 (SMS) partially suppressed all parameters in GH secretion after stimulation by GHRH and/or GHRP‐6.

A double RHPA was then performed to test whether all somatotrophs respond equally to GHRH and GHRP‐6 or some cells formed plaques only by either GHRH or GHRP‐6. There were somatotrophs responsive to only GHRH (23.3%vs control 6.2%, P<0.01), those responsive to only GHRP‐6 (11.9%vs control 6.1%, P<0.01), and those responsive to both GHRH and GHRP‐6 (7.8%vs control 0.2%, P<0.01).

These results confirmed the previous findings that GHRP‐6 and GHRH directly but independently stimulate GH release from the pituitary cells, and further suggest the presence of at least three functionally distinct somatotroph subpopulations concerning the responsiveness to GHRP‐6 and GHRH in rats.
Glutamate and N‐Methyl‐D‐Aspartate Stimulate Rat Hypothalamic Corticotropin‐Releasing Factor Secretion in vitro
Authors: Joanny Pierre , Steinberg Jean , Oliver Charles , Grino Michel
Published: November 2003

It is known that in vivo excitatory amino acids (EAA) stimulate the hypothalamo‐pituitary‐adrenal axis. However their site of action is not fully understood. We investigated the possibility of a direct action of EAA on the secretion of the major adrenocorticotropin hormone (ACTH) secretagogue: corticotropin‐releasing factor (CRF) from incubated rat hypothalamic slices. N‐methyl‐D‐aspartic acid (NMDA) or L‐glutamate (1×10−7 to 1×10−3 M) stimulated in a dose‐dependent fashion CRF release. The maximal effect was obtained at a concentration of 1×10−4 M for both drugs. The IC50 was 1.3×10−5 M and 3.3×10−5 M for NMDA and L‐glutamate, respectively. Incubation with 2.5×10−4 M D‐2‐amino‐5‐phosphonovalerate (a NMDA receptor antagonist) or 2‐amino‐4‐phosphonobutyrate (a metabotropic receptor antagonist) was without significant effect on basal CRF secretion and completely blocked the increase in CRF release induced by 5×10−5 M NMDA or L‐glutamate, respectively. Incubation with 1×10−4 M kainate or 0.5×10−4 M AMPA did not change basal CRF secretion. Incubation with 2×10−4 M γ‐D‐glutamylglycine (a specific antagonist of kainate and AMPA receptor) had no effect under basal conditions or during exposure to kainate or AMPA. Our data demonstrate that EAA could stimulate directly CRF secretion, by an action through NMDA and metabotropic receptors, but not kainate or AMPA receptors. These findings may be relevant to the regulation of the hypothalamo‐pituitary adrenal axis, both under basal conditions and during exposure to stress.
Duality of Glutamatergic and Gabaergic Control of Pulsatile GnRH Secretion by Rat Hypothalamic Explants: I. Effects of Antisense Oligodeoxynucleotides using Explants Including or Excluding the Preoptic Area
Authors: Bourguignon Jean‐Pierre , Gérard Arlette , Purnelle Gentiane , Czajkowski Vincent , Yamanaka Chutaro , Lemaître Marc , Rigo Jean‐Michel , Moonen Gustave , Franchimont Paul
Published: November 2003

Using antisense oligodeoxynucleotides we aimed to study the role of N‐methyl‐D‐aspartate (NMDA) and γ‐aminobutyric acid (GABA) receptors in the mechanism of Gonadotrophin‐releasing hormone (GnRH) secretion in vitro. Since GnRH cell bodies are located in the rat preoptic hypothalamus while most GnRH terminals are in the retrochiasmatic hypothalamus, we compared the effects of oligodeoxynucleotides on explants of the whole (preoptic area included) or retrochiasmatic hypothalamus. When GnRH secretion is evoked by muscimol and NMDA, a time‐related reduction of GnRH secretion is caused by antisense oligodeoxynucleotides for the β subunit of the GABAA receptor and the NR2A subunit of the NMDA receptor, respectively. After 6–7 h, binding studies of tritiated ligands show a decrease in GABA‐ and NMDA‐receptor expression. While these antisense effects are observed using whole explants, no such effects are seen using retrochiasmatic explants, indicating that the facilitatory GABAA and NMDA receptors are encoded in the preoptic area. Using several missense oligodeoxynucleotides or antisense for the NR2B and NR2C subunits of the NMDA receptor, the muscimol‐ and NMDA‐evoked release of GnRH is not affected. When spontaneous pulsatile GnRH secretion is studied, the NR2A antisense oligodeoxynucleotides cause an increase of the interpulse interval. This increase is seen using whole but not retrochiasmatic explants. In contrast, the GABAA and NR2C antisense oligodeoxynucleotides result in a reduction of GnRH interpulse interval. Such a reduction is seen using whole as well as retrochiasmatic explants, indicating that the GABAA and NMDA receptors which mediate inhibition of GnRH pulsatility are encoded in the retrochiasmatic hypothalamus. We conclude that NMDA receptors (NR2A subunit) encoded in the preoptic hypothalamus mediate a facilitatory effect on GnRH pulsatility while GABAA and NMDA (NR2C subunit) receptors encoded in the retrochiasmatic hypothalamus mediate an inhibition of GnRH pulsatility. Pulsatile GnRH secretion is affected differently than the agonist‐evoked release of GnRH suggesting that the GnRH secretory neurons and the GnRH pulse generator consist of different cellular entities.
Effects of Pituitary Adenylate Cyclase‐Activating Polypeptide (PACAP) and Vasoactive Intestinal Polypeptide (VIP) onHormone Secretion from Sheep Pituitary Cells in vitro
Authors: Sawangjaroen K. , Anderson S. T. , Curlewis J. D.
Published: November 2003

Although vasoactive intestinal polypeptide (VIP) is thought to be a prolactin releasing factor, in vivo studies on sheep suggest that it is inactive in this species. Recent studies, based primarily on the rat, suggest that the related pituitary adenylate cyclase‐activating polypeptide (PACAP) is also a hypophysiotrophic factor but again in sheep, this peptide has no in vivo effects on hormone secretion despite being a potent activator of adenylate cyclase in vitro. This lack of response to either peptide in vivo in sheep could be due to the low concentration of peptide that reaches the pituitary gland following peripheral injection. In the present study we therefore adopted an alternative approach of evaluating in vitro effects of these peptides on GH, FSH, LH or prolactin secretion from dispersed sheep pituitary cells. In a time‐course study, PACAP (1 μmol/l) increased GH concentrations in the culture medium between 1 and 4 h and again at 12 h but had no effect in the 6 and 24 h incubations. Prolactin, LH and FSH were not affected by PACAP. The response to various concentrations of PACAP (1 nmol/l–1 μmol/l) were then evaluated using a 3 h incubation. Again prolactin and LH were not affected by PACAP and there was a small increase in GH concentrations but only at high concentrations of PACAP (0.1 and 1 μmol/l; P<0.05). PACAP also stimulated FSH secretion in cells from some animals although this effect was small. The GH response to PACAP was inhibited by PACAP(6–38), a putative PACAP antagonist, but not by (N‐Ac‐Tyr1, D‐Arg2)‐GHRH(1–29)‐NH2, a GH‐releasing hormone (GHRH) antagonist. The cAMP antagonist Rp‐cAMPS was unable to block the GH response to PACAP suggesting that cAMP does not mediate the secretory response to this peptide. At incubation times from 1–24 h, VIP (1 μmol/l) had no effects on prolactin, LH or GH secretion and, in a further experiment based on a 3 h incubation, concentrations of VIP from 1 nmol/l–1 μmol/l were again without effect on prolactin concentrations. Interactions between PACAP and gonadotrophin releasing hormone (GnRH), GHRH and dopamine were also investigated. PACAP (1 nmol/l–1 μmol/l) did not affect the gonadotrophin or prolactin responses to GnRH or dopamine respectively. However, at a high concentration (1 μmol/l), PACAP inhibited the GH response to GHRH. In summary, these results show that PACAP causes a modest increase in FSH and GH secretion from sheep pituitary cells but only at concentrations of PACAP that are unlikely to be in the physiological range. The present study confirms that VIP is not a prolactin releasing factor in sheep.
Long‐Term Adrenalectomy can Decrease or Increase Hippocampal Dentate Gyrus Volumes
Authors: Conrad Cheryl D. , Leone Danny , Nemivant Ravi R. , Roy Edward J.
Published: November 2003

Male and female Long‐Evans adult rats were adrenalectomized and sacrificed 6 weeks later to determine whether dentate gyrus damage would differ in females and males. A subset of adrenalectomized rats of both sexes had significantly reduced dentate gyrus volumes compared to the same sex SHAM operated rats. The remainder of the male and female adrenalectomized rats which did not have clear dentate gyrus damage had significantly larger dentate gyrus volumes compared to the same sex SHAM rats. The dentate gyrus volumes of all adrenalectomized rats were significantly correlated with two indices of residual hormonal levels (Na+/K+ ratios and body weight gain 6 weeks after surgery), indicating that endogenous corticosterone levels may be a determining factor in the response of the dentate gyrus to adrenalectomy. These dentate gyrus volumetric changes could not be attributed to tissue shrinkage as there were no changes in CA3 volumes in any of the groups. These results suggest that long‐term adrenalectomy can result in either increased or decreased dentate gyrus volumes and that the adrenal steroid levels of each individual adrenalectomized rat may be the factor determining the direction of the dentate gyrus volumetric response.