bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of low phosphorus contents in neurofilaments of squid axons by Image‐EELS contrast spectroscopy
Authors: Door R. , Richter K. , Martin R.
Published: November 2003

We show that Image‐EELS is suitable for detecting relatively low phosphorus concentrations in very small axoplasmic structures of squid axons. Imaging plates and a CCD camera were used as electron sensors. From image series spanning a certain energy‐loss range EELS (electron energy‐loss spectra) were derived by averaging read‐outs from many axoplasmic particles (APs). The ratio of these spectra to spectra of the background was plotted, showing the contrast modulation as a function of the energy loss. This new approach is called EELC (electron energy‐loss‐dependent contrast spectroscopy). A distinct phosphorus signal was found in APs of presynaptic terminals of the squid giant synapse, in the peripheral giant axon and, as controls, in ribosomes. Biochemical experiments supported this result. In neurofilament‐enriched pellets a phosphorus signal could be directly detected by serial EELS and in electron spectroscopic micrographs. After dephosphorylation of either the pellets or the extruded axoplasm with alkaline phosphatase, phosphorus signals in electron spectroscopic micrographs were absent or much reduced in size and intensity.

With Image‐EELS inherent limitations of traditional element detection modes in energy filtering transmission electron microscopy can be overcome. Compared with serial EELS, the selective analysis of small areas with irregular shape is possible with greatly improved signal‐to‐noise ratio. The identification of the element‐peak in Image‐EEL spectra directly proves the presence of the element within the region of interest. For small peaks, the visualization is facilitated by the contrast presentation (EELC). However, the background subtraction modes used for elemental mapping in electron spectroscopic imaging are subject to uncertainties when elemental ionization edges like the PL2,3 edge are examined.

Imaging plates are very sensitive electron sensors with a wide dynamic range. Unlike photographic emulsions, they allow acquisition of image series covering a large energy‐loss range without normalization of exposure times, and direct extraction of EEL spectra. Thus, the combination of Image‐EELS and imaging plates is proposed as an efficient new tool for analytical electron microscopy.
Evaluation of autofocus functions in molecular cytogenetic analysis
Authors: SANTOS A. , ORTIZ DE SOLÓRZANO C. , VAQUERO J. J. , PEÑA J. M. , MALPICA N. , DEL POZO F.
Published: November 2003

This work describes a systematic evaluation of several autofocus functions used for analytical fluorescent image cytometry studies of counterstained nuclei. Focusing is the first step in the automatic fluorescence in situ hybridization analysis of cells. Thirteen functions have been evaluated using qualitative and quantitative procedures. For the last of these procedures a figure‐of‐merit (FOM) is defined and proposed. This new FOM takes into account five important features of the focusing function. Our results show that functions based on correlation measures have the best performance for this type of image.
Interface reactions and fracture behaviour of fibre‐reinforced Mg/Al alloys
Authors: FELDHOFF A. , PIPPEL E. , WOLTERSDORF J.
Published: November 2003

In the composite system carbon fibre/magnesium alloy the interface reactivity was varied over a wide range by adding different amounts of the alloying element aluminium (alloys: AM20, AZ91) and by using carbon fibres of different surface properties (fibres: M40J, T300J). The structure and composition of interlayers in these composites down to the atomic scale as well as their effect on the mechanical properties were studied systematically by the combination of high‐voltage electron microscopy, high‐resolution electron microscopy, energy‐dispersive X‐ray spectroscopy and electron energy‐loss spectroscopy with scanning electron microscope in situ bending tests. As resulting microstructure and nanochemistry correlate with the micromechanical fracture behaviour of the metal matrix composites, the interface reactivity can be used as a parameter governing the composite properties.

In addition to precipitates of aluminium carbide, strongly influencing the fracture behaviour, there are also graphitic carbon ribbons and layers of nanocrystalline magnesium oxide at the fibre/matrix interface.

Increasing the reactivity of the composite system, three characteristic modes of fracture behaviour are observed: single fibre pullout, bundle fracture (the optimum composite) and brittle failure.
An investigation into microstructure and particle distribution of Ni–P/diamond composite thin films
Authors: BOZZINI B. , GIOVANNELLI G. , CAVALLOTTI P. L.
Published: November 2003

The present paper deals with a theoretical and experimental study of the co‐deposition of spheroidal particles in a metal matrix deposited by autocatalytic chemical deposition (ACD). The 3‐D particle size distribution — as measured by image analysis of SEM micrographs of metallographic cross‐sections — is related to the size distribution of the starting powder — as measured by photon autocorrelation spectroscopy — through a simple analytical model. The application of the model to the co‐deposition of diamond particles in an ACD Ni–P (9 wt.% P) amorphous matrix led to a correct prediction of average volume of embedded particles (V¯), coverage factor (CF) and number of co‐deposited particles per unit volume of deposit (NV).
A study of the initial growth of PtxCo(1−x) thin films on Si3N4
Authors: Casey S. , Hill E. , Miles J. , Sivasamy P. , Birtwistle K. , Middleton B. , Joyce D.
Published: November 2003

PtxCo(1−x) thin films where x =0.24 have been deposited onto Si3N4 windows and studied using transmission electron microscopy. The films are used in ultrahigh‐density recording studies and it was found that the surface of the substrate had a strong influence on the microstructure, crystallography and magnetic properties of the film. An investigation of the early growth of the film was made by studying films of different thickness between 100 and 300 Å. It was found that the grains were hexagonal in structure with a strong c‐axis orientation perpendicular to the surface.
Confocal imaging and second‐order stereological analysis of a liquid foam
Authors: Reed M. G. , Howard C. V. , Shelton C. G.
Published: November 2003

Foam structures are found in diverse fields of study; the structure of fire‐fighting foam, upholstery foams and even the head on a pint of beer all share detailed similarities in their microstructure and dynamics. Despite impressive developments in the theory of two‐dimensional foams the challenge in future will be to analyse and model the dynamics of three‐dimensional foams. However, the myriad of gas/liquid interfaces in an aqueous foam make direct imaging of their structure difficult. In this study we circumvent this problem by using fluorescence confocal microscopy to acquire three‐dimensional images of the structure of a coarsening aqueous foam.

A stable aqueous foam was created by mixing commercial shaving foam with ethanol and a small amount of fluorescein solution. The foam was imaged in fluorescence mode such that the liquid fraction of the foam, containing the fluorescent dye, could be seen in optical sections. These images could be acquired in three‐dimensional stacks of optical sections up to a depth of about 200 μm. Single images were also acquired as a time series.

The time series of single optical section images clearly show the dynamics of the foam. Early images show a structure made mainly of spherical bubbles; later images show the polyhedral structure of the foam which coarsens as a function of time. The polyhedral nature of the foams is shown particularly clearly in stereo pair images of the three‐dimensional image sets. The three‐dimensional images of the foams were also analysed using second‐order stereology (statistical summaries of spatial distribution). The x, y and z coordinates of the foam vertices were extracted from the images and used to compute the nearest neighbour (G‐function) and reduced second moment (K‐function) statistics. These statistics allow quantification of the range of length scales found in the foams. These results form part of an ongoing study of the coarsening of aqueous foams.
Effects of specimen refractive index on confocal imaging
Authors: Sheppard C. J. R. , To¨ro¨k P.
Published: November 2003

The aberrations introduced when focusing within a specimen with a refractive index equal to that of water using an oil‐immersion objective are investigated theoretically. The peak intensity in the confocal point spread function drops by a factor of two for focusing less than 10 μm into the specimen. The effects on scaling of dimensions in the resulting images are discussed. The image exhibits an axial stretching by a factor of about 1.12.
Induction of Galanin mRNA in GnRH Neurons by Estradiol and its Facilitation by Progesterone
Authors: Rossmanith Winfried G. , Marks Daniel L. , Clifton Donald K. , Steiner Robert A.
Published: November 2003

On the day of proestrus in the rat, rising plasma levels of estradiol (E) act in concert with progesterone (P) to trigger a preovulatory release of gonadotropins. Cellular levels of galanin mRNA in GnRH neurons are increased in association with the proestrous surge of gonadotropin secretion; however, the relative contribution made by E and P to the induction of galanin mRNA expression in GnRH neurons is unknown. We investigated the role of E and P in the induction of galanin gene expression in GnRH neurons by examining the effects of different combinations of E (estradiol benzoate; 50 μg and P (5 mg)) on the LH surge and the concomitant induction of galanin mRNA in GnRH neurons. We sacrificed ovariectomized adult rats after 1 of 4 treatments: Group 1: vehicle control (n=6); Group 2: P alone (n=7) Group 3: E alone (n=7); Group 4: combined E/P (n=6); the animals were killed at 18.00 h at the time of the LH surge. The brains from these animals were processed by double‐label in situ hybridization to allow measurement of galanin mRNA levels in GnRH neurons. GnRH neurons were identified with a digoxigenin‐labeled cRNA probe for GnRH mRNA, and galanin mRNA was detected and measured simultaneously with an 35S‐labeled cRNA probe coupled with computerized grain counting. Estimation of cellular levels of GnRH mRNA was accomplished with single‐label in situ hybridization, an 35S‐labeled GnRH cRNA probe and computerized grain counting. We observed a 3‐fold induction of galanin mRNA in the GnRH neurons of animals treated with E alone compared with those treated with the vehicle alone (vehicle: 13±2 vs E: 42±4 grains/cell (g/c); P<0.01); LH levels in the E‐treated animals were elevated, albeit moderately, with respect to the vehicle controls. Compared with vehicle‐treated animals, those treated with the combination of E and P showed a 5‐fold induction of galanin mRNA in GnRH neurons (68±9 g/c), which was significantly (P<0.01) greater than that observed in the animals treated with E alone; in addition, the magnitude of the LH surge was much greater (P<0.05) in the E/P‐treated group compared with the E alone group. In contrast, compared to the vehicle controls, animals treated with P alone (15±2 g/c) showed no discernable effect on galanin mRNA levels; moreover, no LH surge occurred in the P alone group. Neither the number of identified GnRH cells nor their content of GnRH mRNA differed significantly among the experimental groups (GnRH mRNA signal: vehicle controls: 153±6 vs E: 159±6 vs E/P: 153±3 vs P: 148±8 g/c). We conclude that while E is the primary ovarian signal inducing galanin mRNA expression in GnRH neurons and the LH surge itself, P plays a facilitatory role in both of these processes.
Effect of Vorozole, an Aromatase Enzyme Inhibitor, on Sexual Behavior, Aromatase Activity and Neural Immunoreactivity
Authors: Rissman Emilie F. , Harada Nobuhiro , Roselli Charles E.
Published: November 2003

Aromatase enzyme is essential for the expression of normal sexual behavior in many mammals and birds. Here we report that vorozole (R83842), a non‐steroidal aromatase inhibitor, blocks sexual behavior in the female musk shrew. In addition, vorozole treatment lowers aromatase activity in male and female preoptic area, and reduces plasma estradiol concentrations in females. Our findings confirm and extend results demonstrated in other species, conducted with the active enantiomer (R83842), or the racemic mixture (R76713, racemic vorozole). We also report that vorozole treatment affects the immunocytochemical distribution of aromatase immunoreactivity (AROM‐ir) in musk shrew brain. The histological identification of neurons that contain this enzyme has been difficult in mammals. Several aromatase enzyme antisera have been developed and used in brain, and each gives a different pattern of immunoreactivity. Moreover, despite the fact that aromatase activity is very high in the bed nucleus of the stria terminalis, several amygdala nuclei, the preoptic area and hypothalamus, AROM‐ir in these regions has been very limited. The distribution of AROM‐ir in female musk shrew brain tissues is modified by treatment with vorozole prior to sacrifice. Female musk shrew brains contain aromatase immunoreactive cell bodies, as reported previously, in the central amygdala, lateral septum and to a limited extent in the bed nucleus of the stria terminalis (BST). Brains of females treated with vorozole show additional immunoreactivity in the preoptic area, hypothalamus, and medial amygdala, and have a broad distribution of AROM‐ir in several subdivisions of the BST. Several sexual dimorphisms are apparent in musk shrews brains after treatment with vorozole. We have quantified this sexual dimorphism in the medial preoptic area (MPO) by counting immunoreactive cells. In both the rostral and caudal portions of the MPO, female brains contain significantly fewer AROM‐ir cell bodies than males. These data are in complete agreement with sex differences in biochemical analyses of aromatase activity in the MPO. At this time we do not know if these dimorphisms are the result of differences in circulating levels of steroids in males and females, and/or if the AROM‐ir nuclei regulate sexually dimorphic behaviors.
Ontogeny of Pituitary Transcription Factor‐1 (Pit‐1), Growth Hormone (GH) and Prolactin (PRL) mRNA Levels in Male and Female Rats and the Differential Expression of Pit‐1 in Lactotrophs and Somatotrophs
Authors: González‐Parra Silvia , Chowen Julie A. , Segura Luis Miguel García , Argente Jesús
Published: November 2003

Pituitary transcription factor‐1 (Pit‐1 or GHF‐1), a transcription factor specific to the anterior pituitary, is involved in the expression and regulation of the growth hormone (GH) and prolactin (PRL) genes. Post‐pubertally, the expression of both GH and PRL becomes sexually dimorphic with males having higher GH levels and females higher PRL levels; however, little is known about the postnatal regulation of their common transcription factor. Furthermore, whether the Pit‐1 gene is differentially expressed in somatotrophs and lactotrophs remains to be elucidated. In this study, we used in situ hybridization histochemistry to examine Pit‐1, GH and PRL mRNA levels in the anterior pituitaries of male and female rats throughout development (0, 5, 10, 20, 30, 40 and 60 days of age) to determine when GH and PRL production becomes sexually dimorphic and if this is accompanied by a dimorphism in Pit‐1 gene expression. In addition, the level of Pit‐1 mRNA was determined separately in both GH mRNA and PRL mRNA containing cells during the various developmental stages. We found that in both males and females the mRNA levels of Pit‐1, GH and PRL remain relatively unchanged until around the time of pubertal onset (30–40 days) when there is a significant increase in all three mRNA species, which is followed by a decrease to adult levels. Also around the time of puberty, both GH and PRL mRNA levels become sexually dimorphic, with males having higher levels of GH mRNA and females higher PRL mRNA levels. In contrast, at no time during development were overall Pit‐1 mRNA levels found to differ between the sexes. However, when Pit‐1 mRNA content was measured separately in specific cell types, significant differences between the sexes became evident. Throughout development Pit‐1 mRNA levels are higher in lactotrophs of females than in those of males, whereas in somatotrophs males have higher Pit‐1 mRNA levels than females. Furthermore, within a sex there is differential expression of Pit‐1 in the two cell types with females having significantly higher levels of Pit‐1 in lactotrophs than in somatotrophs and males having higher levels in somatotrophs than in lactotrophs. These data support the hypothesis that a sexual dimorphism exists in the expression of the pituitary specific transcription factor Pit‐1; however, this dimorphism is not manifest as a difference in overall mRNA levels, but in the differential expression of this gene in lactotrophs and somatotrophs.
Reduced NPY Induced Feeding in Diabetic but not Steroid‐Treated Rats: Lack of Evidence for Changes in Receptor Number or Affinity
Authors: Morgan David G. A. , Lambert Philip D. , Smith David M. , Wilding John P. H. , Bloom Stephen R.
Published: November 2003

Concentrations of the potent hypothalamic appetite stimulating peptide neuropeptide Y (NPY), and its mRNA, are increased in rats with experimental diabetes, suggesting a role in the hyperphagia of this disorder. The 2‐h feeding responses to intracerebroventricular (i.c.v.) injection of neuropeptide Y (NPY) (5, 10, and 15 μg doses) were measured in male Wistar rats treated with streptozotocin (55 mg/kg) to induce diabetes. Streptozotocin‐diabetic rats given i.c.v. NPY exhibited reduced feeding responses compared to controls (P<0.05). Dexamethasone treated rats exhibit similar changes in NPY content and mRNA in the hypothalamus to those seen in diabetes, but are not hyperphagic. Feeding repsonses were also measured in this model, to assess whether high levels of endogenous NPY might account for the reduced response in diabetes. In contrast, the feeding response to NPY in comparison to controls was unaltered in dexamethasone‐treated rats.
Expression of Fos‐Like Immunoreactivity in the Hypothalamus of the Ring Dove (Streptopelia risoria) at the Onset of Incubation
Authors: Sharp Peter J. , Li Qiushi , Georgiou George , Lea Robert W.
Published: November 2003

Fos immunocytochemistry was used in the ring dove (Streptopelia risoria) to identify areas in the forebrain and hypothalamus which might be involved in the expression of incubation behaviour and the associated changes in neuroendocrine function. In this species, both sexes participate in incubation which becomes fully established after laying the second egg in the two egg clutch. The eggs hatch after 14–15 days incubation. Observations were made in both sexes 16–18 h after laying the first egg, 3–5 h after laying the second egg, and after 12–14 days of incubation. Non‐breeding birds were used as controls. Fos‐labelled cells were observed in several forebrain areas in both sexes irrespective of reproductive status but their numbers did not change during incubation. In the hypothalamus, very few fos‐labelled cells were seen in non‐breeding birds but appeared in both sexes in the nucleus preopticus medialis after egg laying and after 12–14 days of incubation. Fos‐labelled cells also appeared in the nucleus tuberis (≡arcuate nucleus) in both sexes after laying the first or second egg and less abundantly, after 12–14 days incubation. Additionally, in males only, fos‐labelled cells were seen after the laying of the first and second eggs in the rostral area of the nucleus tuberis close to the walls of the third ventricle. These observations suggest that the fos‐labelled cells in the nucleus preopticus medialis may be involved in the expression of incubation behaviour while those in the nucleus tuberis may be involved in the regulation of gonadotrophin or prolactin secretion.
Androgens Modulate Glucocorticoid Receptor mRNA, but not Mineralocorticoid Receptor mRNA Levels, in the Rat Hippocampus
Authors: Kerr Janice E. , Beck Sheryl G. , Handa Robert J.
Published: November 2003

Androgen, mineralocorticoid and glucocorticoid receptors (AR, MR and GR, respectively) are ligand‐activated transcription factors that alter gene expression and have a wide variety of effects in the central nervous system. High levels of AR, MR and GR mRNA have been found in the CA1 pyramidal cell region of the rat hippocampus and all 3 of these proteins bind to a similar hormone response element in DNA suggesting the possibility of common receptor function or cross‐talk between these receptors at the level of transcription. To begin to investigate this hypothesis, we examined the regulation of AR, MR and GR mRNA expression in the rat hippocampus following treatment with androgens in combination with gonadectomy and/or adrenalectomy. Three‐month‐old male Sprague‐Dawley rats were either castrated for 3 weeks, castrated and immediately implanted with 2 Silastic capsules filled with the non‐aromatizable androgen, dihydrotestosterone, or left gonadally intact. Four days prior to sacrifice, these animals were either adrenalectomized or sham operated. GR, MR and AR mRNA were measured in the hippocampal subfields using in situ hybridization. In the CA1 region, dihydrotestosterone treatment of castrates decreased GR mRNA levels to 69 percent of levels found in gonadally intact rats and prevented the adrenalectomy‐induced increases in GR mRNA observed in the gonadally intact and castrated animals. No changes in GR mRNA were observed in the CA3 region or dentate gyrus, where AR expression is low or absent. There was no effect of androgen treatment on MR mRNA levels nor did gonadectomy or androgen replacement alter the increases in MR mRNA following adrenalectomy. AR mRNA levels in the CA1 region were unchanged across all treatment groups. In vitro binding studies revealed almost complete nuclear occupancy of hippocampal AR in dihydrotestosterone‐treated castrates. No appreciable in vitro binding of dihydrotestosterone to hippocampal MR or GR (Ki≈1500 nM) was observed which suggests that androgen regulation of GR mRNA in the hippocampus is occurring through AR binding. These data demonstrate a functional similarity of androgens and glucocorticoids in the regulation of GR mRNA levels in an area where AR and GR are colocalized. Androgen‐mediated downregulation of GR expression may prove to be an important event in the adaptive responses of CA1 pyramidal cells to hormonal stimuli.
The Modulatory Role of Estrogens on Corticotropin‐Releasing Factor Gene Expression in the Hypothalamic Paraventricular Nucleus of Ovariectomized Rats: Role of the Adrenal Gland
Authors: Paulmyer‐Lacroix Odile , Héry Micheline , Pugeat Michel , Grino Michel
Published: November 2003

In vitro, estrogens stimulate corticotropin‐releasing factor (CRF) gene transcription. In ovariectomized (OVX) rats, estrogens have a negative effect on CRF mRNA levels in the hypothalamic paraventricular nucleus. This suggests that the stimulatory influence of estrogens found in vitro may be masked in vivo by an inhibitory effect mediated through neural and/or humoral factors. Glucocorticoids may be involved in this phenomenon since estrogens increase circulating corticosterone levels in OVX rats. We studied the effect of 7‐day 17 β‐estradiol (E2) treatment on CRF gene expression in the paraventricular nucleus of 2‐week OVX mature rats after sham operation or adrenalectomy (ADX) with or without corticosterone replacement. In sham ADX animals, E2 administration increased plasma corticosterone concentrations, did not change the binding capacity or the affinity of circulating corticosteroid binding globulin, and decreased hypothalamic CRF gene expression. Following ADX, CRF mRNA levels increased and were normalized by corticosterone treatment. Estrogens induced a significant build up in CRF mRNA concentrations in both ADX or ADX corticosterone‐replaced animals. Our data demonstrate that the positive effect of estrogens on CRF gene expression found in vitro or in vivo after ADX is antagonized, in vivo when the adrenal glands are intact, by a negative influence. They strongly suggest that the increased circulating corticosterone levels induced by E2 administration mediate the inhibitory effect of estrogens on CRF mRNA levels. These observations may explain the gender related differences in the basal and stress‐induced hypothalamo‐pituitary‐adrenal activity.
Alteration of Gα Subunits mRNA Levels in Bromocriptine Resistant Prolactinomas
Authors: Caccavelli L. , Morange‐Ramos I. , Kordon C. , Jaquet P. , Enjalbert A.
Published: November 2003

Patients with prolactinoma are commonly treated with the D2 dopamine agonist bromocriptine, which in most cases, normalizes prolactin (PRL) levels. However, resistance to bromocriptine has been observed in 5 to 18% of tested prolactinomas and is associated to a decrease in both D2 receptor density and mRNA levels. In this study, we used quantitative RT‐PCR to investigate whether expression of Gα proteins could be also modified in bromocriptine resistant prolactinomas. No difference in Gαo mRNA levels or in the relative expression of Gαs between bromocriptine sensitive and bromocriptine resistant prolactinomas was observed. In contrast, the relative expression of Gαi2 was found to be decreased in bromocriptine resistant prolactinomas when compared to that of bromocriptine sensitive prolactinomas. Interestingly, the relative Gαi2 expression was correlated to both bromocriptine inhibition of in vitro PRL secretion and D2 receptor mRNA levels. Bromocriptine resistance could thus result from a decrease in D2 dopamine receptors associated with a decrease in Gαi2 expression.
The Effect of Pinealectomy on Osmotically Stimulated Vasopressin and Oxytocin Release and Fos Protein Production within the Hypothalamus of the Rat
Authors: Windle R. J. , Luckman S. M. , Stoughton R. P. , Forsling M. L.
Published: November 2003

Osmotically stimulated vasopressin and oxytocin release were measured in pinealectomized and sham operated male rats infused with hypertonic sodium chloride. Neuronal activation in the hypothalamic regions associated with oxytocin and vasopressin release was investigated by quantitative assessment of Fos protein production. The osmotically stimulated release of both vasopressin and oxytocin was significantly lower in pinealectomized animals as compared to sham operated controls. The slope of regression lines between plasma osmolality and hormone concentrations in the sham animals showed a 1.0±0.1 pmol per mosm/kg rise in vasopressin and 2.0±0.4 pmol per mosm/kg rise in oxytocin whilst in the pinealectomized animals these values were significantly lower at 0.4±0.1 pmol vasopressin per mosm/kg and 0.8±0.2pmol oxytocin per mosm/kg. The osmotic thresholds for hormone release were unaffected by pinealectomy. Fos production was also significantly lower in the supraoptic nucleus and organ vasculosum of the lamina terminalis in the pinealectomized rat at 62±20 and 59±9 Fos immunoreactive cells/section as compared to corresponding values of 202±31 and 123±20 Fos immunoreactive cells/section in the shams. These observations suggest that reduced hormone release in the pinealectomized animal is due to lowered responsiveness of central osmoregulatory mechanisms and that melatonin may therefore influence the activation of the magnocellular system.
Activation of Locus Coeruleus Somatostatin Receptors Induces an Increase of Growth Hormone Release in Male Rats
Authors: Mounier Françoise , Bluet‐Pajot Marie‐Thérèse , Moinard Florence , Kordon Claude , Epelbaum Jacques
Published: November 2003

Noradrenergic neurons of the locus coeruleus (LC) negatively regulate the endogenous rhythmicity of growth hormone (GH) secretion. These neurons express high concentrations of receptors for somatostatin (SRIH) and galanin (GAL), two neuropeptides which can affect electrical activity of LC neurons and also centrally modulate plasma GH levels. We thus investigated whether somatostatin and galanin receptors located in the LC are involved in GH regulation. Pulsatile patterns of endogenous GH secretion were monitored after unilateral infusion of the peptides into the lateral ventricle (ICV) or into the LC, after lesion of contralateral LC neurons by 6 hydroxydopamine. Neither unilateral LC lesions nor administration of saline affected GH release. When administered ICV, both SRIH (5 μg/μl/15 min) and GAL (1 μg/μl/15 min) resulted in a marked increase in GH secretion. Infusion of SRIH into the LC induced a significant but weaker stimulation of plasma GH as compared to ICV injections. In contrast, infusion of GAL into the LC was ineffective. These results indicate that somatostatin can exhibit direct effects on noradrenergic neurons of the LC involved in GH regulation, whereas central effects of galanin on the hormone are mediated by distinct structures.
Inhibition of Tuberoinfundibular Dopaminergic Neural Activity During Suckling: Involvement of μ and κ Opiate Receptor Subtypes
Authors: Callahan Phyllis , Baumann Michael H. , Rabii Jamshid
Published: November 2003

Previous studies have shown that mu (μ) and kappa (κ) opioid antagonists inhibit suckling‐induced prolactin release. Prolactin responses elicited by pup suckling or opioid administration are mediated, at least in part, by suppression of dopamine (DA) release from tuberoinfundibular dopaminergic (TIDA) neurons in the hypothalamus. We examined the effects of the μ opiate receptor antagonist, β‐funaltrexamine (β‐FNA), and the κ opiate receptor antagonist, nor‐binaltorphimine (nor‐BNI) on the activity of TIDA neurons in lactating rats. TIDA neuronal activity was determined by measuring DOPA accumulation in the caudate putamen (CP) and median eminence (ME). The effects of opioid antagonist treatment were determined in pup‐deprived (low circulating prolactin levels) or pup‐suckled rats (high circulating prolactin levels). The accumulation of 5‐hydroxytryptophan (5‐HTP) in the medial preoptic area (MPOA), the anterior hypothalamus (AH) and the median eminence (ME) was quantified as an index of serotonergic activity in the same animals for comparative purposes.
Hippocampal Responses to Corticosterone and Stress, One of which is the 35,000 Mr Protein, Glycerol Phosphate Dehydrogenase
Authors: Nichols N. R. , Dokas L. , Ting S.‐M. , Kumar S. , de Vellis J. , Shors T. J. , Uenishi N. , Thompson R. F. , Finch C. E.
Published: November 2003

Previously, the synthesis of a hippocampal 35,000 Mr protein increased in response to glucocorticoid treatment and a variety of stressors. We now show by immunoprecipitation that this cytosolic protein is glycerol 3‐phosphate dehydrogenase (E.C.1.1.1.8; GPDH). In addition, four polypeptides encoded by glucocorticoid‐induced mRNAs co‐migrated with hippocampal protein synthetic products on two‐dimensional polyacrylamide gels, including a 35,000 Mr protein of ∼pI 6.3, that had previously been identified as GPDH by hybrid‐selection with a GPDH cDNA clone. The 35,000 Mrin vitro translation product was also immunoprecipitated with the GPDH antibody. Using radiolabeled hippocampal slices and two‐dimensional gel analysis, a 35,000 Mr polypeptide of ∼pI 6.4 increased five‐fold after 30 min of intermittent tail‐shock. This protein was found predominantly in the 20,000×g pellet and did not immunoprecipitate with the GPDH antibody . However, a 35,000 Mr polypeptide was also found in the cytosol as a minor component after stress, which did immunoprecipitate with the GPDH antibody. Therefore, there are at least two shock‐induced 35,000 Mr proteins, one of which is GPDH. These results establish that increases in GPDH mRNA prevalence and protein synthesis occur in response to both glucocorticoids and stress in the adult rat hippocampus. Based on the increased enzyme activity seen in the nervous system in response to glucocorticoids, dietary restriction, and nerve injury, the induction of GPDH may have functional consequences in cellular adaptation to stress.
Blockade of Corticosterone Synthesis Reduces Serotonin Turnover in the Dorsal Hippocampus of the Rat as Measured by Microdialysis
Authors: Korte‐Bouws Gerdien A. H. , Korte S. Michiel , De Kloet E. Ronald , Bohus Bela
Published: November 2003

The influence of plasma corticosterone concentration on serotonin (5‐HT) turnover in the dorsal hippocampus was investigated. The experiments were performed in freely moving male Wistar rats in their home cage. Blood samples were taken via a permanent jugular vein catheter to determine plasma corticosterone levels. Extracellular levels of 5‐HT and its metabolite 5‐hydroxy‐indole acetic acid (5‐HIAA) were measured using in vivo microdialysis. The rats received an intravenous (i.v.) infusion of the steroid synthesis‐inhibitor metyrapone (150 mg/kg/ml) in order to manipulate circulating corticosterone levels. Three hours later, the monoamine oxidase inhibitor pargyline (15 mg/kg/2 ml i.v.) was administered to produce an accumulation of extracellular 5‐HT. Pargyline administration led to a four fold increase in 5‐HT levels, while reducing 5‐HIAA by 45%. Metyrapone pretreatment blocked the pargyline‐induced rise in plasma corticosterone to baseline levels and diminished the pargyline‐induced increase in 5‐HT, without affecting 5‐HIAA levels. Thus, the data suggest that a decrease in availability of corticosterone for its receptors by metyrapone diminished the 5‐HT synthesis rate. Since plasma corticosterone levels during this blockade are still low, it is assumed that brain glucocorticoid receptor occupation is reduced, while mineralocorticoid receptors are still substantially occupied. Therefore the present results support the hypothesis that corticosterone through glucocorticoid receptor activation enhances 5‐HT synthesis rate and release in the dorsal hippocampus.
Localization and Characterization of Dopamine D1 Receptors in Sheep Hypothalamus and Striatum
Authors: Colthorpe K. L. , Curlewis J. D.
Published: November 2003

Dopamine receptors are pharmacologically grouped as D1 and D2 receptors. Previous research in the ewe has shown that central D1 receptors may have a role in facilitating prolactin release. The aims of this study were therefore to localize and characterize D1 binding sites in the hypothalamus of sheep. For comparison, a known D1 receptor‐rich tissue (striatum) was also studied. The bioactivities of several D1 analogues were also assessed for their efficacy in sheep tissue. In vitro autoradiography with [125I]‐SCH23982 was used to localize D1 binding sites. The ventromedial hypothalamic nucleus (VMH) displayed moderate levels of specific binding, localized to the medial portion of the nucleus. Low levels of specific binding were seen in the preoptic area, supraoptic nucleus and anterior hypothalamic area. The suprachiasmatic nucleus, median eminence and arcuate nucleus did not show specific binding. As expected the striatum displayed high levels of specific binding. The VMH, preoptic area, median eminence, striatum and anterior pituitary were examined with radioligand binding studies to quantify and characterize D1 binding sites. Scatchard analysis gave KD 1.04 nM and Bmax 127.4 fmol/mg protein for VMH and KD 1.99 nM and Bmax 454.6 fmol/mg protein for striatum. While specific binding occurred in the preoptic area and median eminence this binding did not show saturation characteristics. Specific binding was not observed in the anterior pituitary. Affinities determined by competitive binding studies showed that the binding sites in both VMH and striatum have the characteristics of a D1 receptor, that is, high affinity for the D1 agonists and antagonists, low affinity for dopamine and the serotonergic antagonist ketanserin and extremely low affinity for the D2 agonists and noradrenaline. Adenylate cyclase studies showed that in the striatum dopamine and the D1 agonists, fenoldopam and SKF38393, were able to cause significant dose‐dependent increases in adenylate cyclase activity. In contrast the D1 agonist, SKF82958, was inactive in this system. The D1 antagonists SCH23390 and SCH39166, but not SKF83566, abolished the adenylate cyclase response to 50 μM dopamine. In the VMH the D1 agonist SKF38393, but not dopamine, stimulated adenylate cyclase activity. In conclusion, these results demonstrate that D1 binding sites exist within the hypothalamus in the VMH and that these binding sites have the characteristics of D1 receptors. These receptors are a potential site of action for dopamine in facilitating prolactin release. In addition, the results show that at least for some dopamine analogues, receptor binding affinity does not always correlate with biological activity.
Activational Effects of Gonadal Steroids on Hypothalamo‐Pituitary‐Adrenal Regulation in the Rat Disclosed by Response to Dexamethasone Suppression
Authors: Almeida Osborne F. X. , Canoine Virginie , Ali Sinan , Holsboer Florian , Patchev Vladimir K.
Published: November 2003

Previous studies demonstrated that gonadal steroids secreted during perinatal life permanently ‘organize’ the mechanisms governing hypothalamo‐pituitary‐adrenal (HPA) function, leading to sexually differentiated patterns of pituitary‐adrenal activity under basal and stress conditions. In this paper, we show that gonadal steroids can also exert ‘activational’ effects upon the HPA system. Examination of the ability of different doses of dexamethasone to suppress the nocturnal increase in corticosterone secretion and to attenuate the gene expression of CRH in the hypothalamic paraventricular nucleus of intact and gonadectomized male and female rats revealed that ovarian steroids make an important contribution to the higher sensitivity of the pituitary‐adrenal axis in females to glucocorticoid suppression, whereas testicular steroids may be causal to the male’s moderate responsiveness to glucocorticoid feedback. These findings may be implicated in a number of psychiatric and neurological disease states commonly associated with impaired HPA regulation, but which may be primarily rooted in altered gonadal steroid secretion.
Synthesis of C‐Type Natriuretic Peptide (CNP) byImmortalized LHRH Cells
Authors: Middendorff R. , Paust H. J. , Davidoff M. S. , Olcese J.
Published: November 2003

Former studies have indicated an influence of natriuretic peptides on LHRH secretion. In this report we demonstrate local synthesis of CNP in immortalized LHRH neurons (GT1–7 cells). Using reverse transcription‐polymerase chain reaction and RNase protection assays a transcript for the CNP precursor was identified in these cells. Immunocytochemical data revealed the presence of the peptide CNP in GT1 cells, using a specific polyclonal antiserum against CNP. Electron microscopic immunohistochemical investigations also showed the strongest CNP‐immunoreactivity in some small vesicles, providing initial evidence for the potential secretion of this peptide by immortalized LHRH neurons. Subsequent experiments demonstrated also that CNP elevates LHRH production in static cultures of GT1 cells. These data show for the first time the co‐production of the functionally relevant natriuretic peptide, CNP, by immortalized LHRH neurons. Together with the recent demonstration of CNP receptor expression by these cells, we suggest that CNP may represent a novel autocrine regulator of LHRH neuronal activity. It remains to be elucidated, however, to what extent CNP expression in immortalized LHRH neurons reflects a co‐localization in situ of CNP and LHRH peptides.
Differential Expression of NGFI‐B and RNR‐1 Genes in Various Tissues and Developing Brain of the Rat: Comparative Study by Quantitative Reverse Transcription‐Polymerase Chain Reaction
Authors: Bandoh Shuji , Tsukada Toshihiko , Maruyama Kouji , Ohkura Naganari , Yamaguchi Ken
Published: November 2003

NGFI‐B and RNR‐1 are closely related transcription factors that constitute a distinct subclass within the steroid/thyroid hormone receptor superfamily. They have been implicated in neuronal differentiation, neuroendocrine regulation of adrenocortical function and T‐cell apoptosis. In this study, we measured and compared NGFI‐B and RNR‐1 mRNA levels in various adult rat tissues and in the developing rat brain by means of the quantitative reverse transcription‐polymerase chain reaction. The use of RNA standards synthesized in vitro allowed direct comparison of the amount of the transcripts of these two genes. We demonstrated that the transcripts of both genes were present in all tissues examined although the expression levels widely varied. We found the highest constitutive expression of both genes in the pituitary. High levels of NGFI‐B were also expressed in the cerebral cortex, muscle, ventral prostate, thymus and adrenal glands, whereas high levels of RNR‐1 expression were restricted to the pituitary and cerebral cortex. These findings were consistent with the notion that NGFI‐B and RNR‐1 are involved in various signal transduction systems in diverse cell types. The amount of NGFI‐B mRNA was greater than that of RNR‐1 mRNA in all adult rat tissues, with the highest ratio of NGFI‐B relative to RNR‐1 expression in the muscle and leukocytes. In contrast, fetal rat brain showed relatively high RNR‐1 gene expression. These findings suggested that the NGFI‐B and RNR‐1 genes are differentially expressed in a tissue‐specific and developmentally regulated manner.
Involvement of Neurosteroids in the Effect of the Endogenous Benzodiazepine Receptor Ligand Octadecaneuropeptide (ODN) on Gonadotropin‐Releasing Hormone Gene Expression in Rat Brain
Authors: Li S. , Givalois L. , Pelletier G.
Published: November 2003

We have recently demonstrated that different activators of the GABAA receptor complex including reduced progesterone metabolites and the endozepine octodecaneuropeptide (ODN) exert an inhibitory influence on GnRH gene expression. In order to investigate the possible involvement of neurosteroids, especially progesterone metabolites in the effect of ODN, we have evaluated in adrenalectomized and castrated male rats the influence of pretreatment with an inhibitor of 3β‐hydroxysteroid dehydrogenase (3β‐HSD) trilostane (TRIL) and an inhibitor of 5α‐reductase MK‐906 on GnRH mRNA levels in ODN‐treated rats. TRIL completely prevented the inhibitory influence of ODN on GnRH mRNA. It was also found that the inhibitor of 3β‐HSD as well as pregnenolone sulfate (PREG‐S), which has been shown to be increased following TRIL treatment, could induce an increase in GnRH mRNA. MK‐906 could also completely reverse the negative influence of ODN. When administered alone, this antagonist of 5α‐reductase induced an increase in GnRH mRNA. These results clearly indicate that the inhibition of two key enzymes for the synthesis of reduced progesterone metabolites can completely prevent the inhibitory influence of the endozepine ODN, suggesting that the effect of this endogenous ligand might be completely or partially mediated by an activation of the synthesis of active progesterone metabolites. On the other hand, it remains possible that, as a consequence of enzymatic inhibition, an increase in some precursors known as antagonists of GABAA may play a role in the prevention of the ODN effect by the two enzyme antagonists.
Endocrine and Behavioural Responses to Noise Stress:Comparison of Virgin and Lactating Female Ratsduring Non‐Disrupted Maternal Activity
Authors: Windle R. J. , Wood S. , Shanks N. , Perks P. , Conde G. L. , Da Costa A. P. C. , Ingram C. D. , Lightman S. L.
Published: November 2003

The behavioural and endocrine responses to a 10 min white noise stress have been characterized in female virgin and undisturbed lactating Sprague‐Dawley rats. Animals were continuously video‐taped and frequent blood samples were collected using an automated sampling system. Noise stress caused hypothalamo‐pituitary‐adrenal (HPA) activation, as indicated by a rapid increase in plasma corticosterone and ACTH in the virgins: corticosterone concentrations peaked 20 min after initiation of the stress before declining rapidly back to basal concentrations. In contrast, noise stress had no significant effect on either plasma corticosterone or ACTH concentrations in the lactating animals. However, 72 h after weaning the corticosterone response of the ex‐lactating rats was of comparable magnitude, but longer duration to that seen in the virgins. Plasma prolactin concentrations were significantly higher in the lactating animals and declined in response to the noise whereas, a transient but reproducible increase was seen in the virgin group. In situ hybridization revealed a significantly lower basal expression of CRF mRNA in the paraventricular nucleus of lactating rats as compared to the virgins, but noise stress had no further effect. Virgin animals showed behavioural responses to the stress, including an increase in the total activity, exploratory behaviours (rearing) and displacement behaviours (grooming). Lactating animals also showed behavioural responses to the noise, but their activities were principally directed towards the pups. These data show that although lactating rats showed normal behavioural reactivity to a psychological stress they showed no statisitically significant activation of the HPA axis, suggesting a dissociation of behavioural and neuroendocrine responses to this mild stress.
Ontogeny of Growth Hormone and LHβ‐, FSHβ‐ and α‐Subunit mRNA Levels in the Porcine Fetal and Neonatal Anterior Pituitary
Authors: Granz S. , Ellendorff F. , Grossmann R. , Kato Y. , Mühlbauer E. , Elsaesser F.
Published: November 2003

The mechanism underlying the ontogenetic increase in plasma growth hormone (GH), luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentration during fetal life in mammalian species and the prenatal sex difference in these hormones in some species is not fully understood. To this end anterior pituitaries were collected from German Landrace fetuses and piglets at day (d) 50, 65, 80, 95, 110 pc and d 6 pp and pituitary GH, LHβ, FSHβ and α‐subunit mRNA levels were determined by measuring Northern blot hybridization signals. GH mRNA was detected in both sexes as early as d 50 pc. The mRNA level markedly increased with age in both sexes (males>females, P≤0.05) reaching its maximum at d 95/110 pc. LHβ mRNA signals were first detected at d 50 pc in females and at d 65 pc in males increasing thereafter to a maximum at d 6 pp in both sexes (P≤0.05). In males the augmentation in LHβ mRNA was delayed compared to females (P≤0.01). Before d 80 pc no FSHβ mRNA hybridization signals were apparent. Thereafter the mRNA level continuously increased with age (P≤0.01) in both sexes reaching its maximum at d 6 pp. The FSHβ mRNA level in females was always higher than in males (P≤0.01). As early as d 50 pc the α‐subunit mRNA level was high in both sexes and further increased without sex difference to d 6 pp (P≤0.05). In conclusion, the mRNA levels of GH, LHβ and FSHβ are age and sex dependent during fetal development. We suggest that the fetal increase in plasma GH concentration can be accounted for by changes in GH mRNA levels, while the dramatic perinatal decrease in plasma GH concentration seems to be primarily controlled at the posttranslational and/or secretion level. The fetal sex difference and the increase in plasma LH and FSH concentrations seems to be primarily dependent on the cellular concentration of the gonadotropin β‐subunit mRNAs and/or number of gonadotrophs.