bluuebunny/crossref_metadata_2025 huggingface dataset:
STM/SEM correlative study of facetted gold
Authors: ASENJO A. , GÓMEZ‐HERRERO J. , BARÓ A. M.
Published: November 2003

We have integrated an STM unit with a conventional scanning electron microscope in order to perform STM–SEM correlative microscopy. The method is applied to an electrochemically facetted gold sample, which provides a surface structure suitable for this study. We discuss the factors which are relevant in order to obtain a quantitative resolution of the topographic surface structure, by taking advantage of the performances of both techniques. In particular we suggest the use of the STM height distribution as the best parameter for STM/SEM correlation. Finally, from the STM data we deduce that the main process during electrochemical etching is the formation of (111) faces.
Effect of Cl on microstructure and mechanical properties of in situ Ti/TiB MMCs produced by a blended elemental powder metallurgy method
Authors: FAN Z. , NIU H. J. , CANTOR B. , MIODOWNIK A. P. , SAITO T.
Published: November 2003

A modified blended elemental powder metallurgy (MBEPM) method has been developed for the production of low‐cost Ti alloys and in situ Ti/TiB MMCs for automobile components such as connecting rods and inlet and exhaust valves. The MBEPM method uses Ti sponge fines as raw material, which contain a substantial amount of Cl. The Cl refines the microstructure of the as‐sintered Ti–6Al–4V alloys, with a reduced prior β‐grain size and a reduced α‐lath size and aspect ratio. However, the grain refining effect of Cl is much less pronounced in as‐sintered Ti–6Al–4V–10%TiB MMCs. The Cl is present in the as‐sintered microstructure in three forms: (1) shells consisting of fine NaCl particles in macropores; (2) cuboidal NaCl precipitates in the alloy matrix; and (3) Cl and Na segregated to prior β‐grain boundaries. Increasing the Cl content increases the tensile ductility of both Ti–6Al–4V alloys and Ti–6Al–4V–10%TiB MMCs, but has little effect on strength.
Chromatic shift in multicolour confocal microscopy
Authors: Manders E. M. M.
Published: November 2003

Multicolour confocal microscopy has proven to be a successful technique for the analysis of the spatial relationship between different biological structures in the same preparation. However, when the positions of objects are compared, e.g. co‐localization and distance measurements, any positional shift that arises between the colour components is clearly unacceptable. This paper presents a simple technique for measuring with high accuracy the positional shifts that occur between the colour components of an image. Multi‐labelled microbeads were scanned using two or three different detection channels. The position of each microbead was calculated separately for each detection channel. In general, the two calculated positions of the same microbead (one for each channel) are slightly different. This difference is a measure of the positional shift between the colours. This method enables the measurement of shift with a high accuracy (20 nm), and it has been applied to images from several experiments. The results of these experiments will give the reader an impression of typical contributions of different effects (such as chromatic aberration, misalignment of optical components and inaccuracy of the scanning unit) on the amount of positional shift.
Two‐photon fluorescence lifetime imaging microscopy of macrophage‐mediated antigen processing
Authors: French T. , So P. T. C. , Weaver, Jr D. J. , Coelho‐Sampaio T. , Gratton E. , Voss, Jr E. W. , Carrero J.
Published: November 2003

Two‐photon fluorescence lifetime imaging microscopy was used noninvasively to monitor a fluorescent antigen during macrophage‐mediated endocytosis, intracellular vacuolar encapsulation, and protease‐dependent processing. Fluorescein‐conjugated bovine serum albumin (FITC–BSA) served as the soluble exogenous antigen. As a relatively nonfluorescent probe in the native state, the antigen was designed to reflect sequential intracellular antigen processing events through time‐dependent changes in fluorescence properties. Using two‐photon lifetime imaging microscopy, antigen processing events were monitored continuously for several hours. During this time, the initial fluorescein fluorescence lifetime of 0.5 ns increased to α 3.0 ns. Control experiments using fluorescein conjugated poly‐l‐lysine and poly‐d‐lysine demonstrated that the increase in fluorescence parameters observed with FITC–BSA were due to intracellular proteolysis since addition of the inert d‐isomer did not promote an increase in fluorescence lifetime or intensity. Comparisons of intravacuolar and extracellular FITC–dextran concentration suggested active localization of dextran in the vacuoles by the macrophage. In addition, the kinetics of degradation observed using two‐photon microscopy were similar to results obtained on the flow cytometer, thus validating the use of flow cytometry for future studies.
Shear force imaging of DNA in a near‐field scanning optical microscope (NSOM)
Authors: Kirsch A. K. , Meyer C. K. , Jovin T. M.
Published: November 2003

A near‐field scanning optical module has been constructed as an accessory for a Nanoscope IIIa commercial scanning probe microscope. Distance feedback and topographic registration are accomplished with an uncoated optical fibre scanning tip by implementation of the shear force technique. The tip is driven by a piezoelectric actuator at a resonance frequency of 8–80 kHz. A laser diode beam is scattered by the tip and detected by a split photodiode, with lock‐in detection of the difference signal. The amplitude (r) and phase (τ) responses were characterized as a function of the calibrated tip–sample separation. Using an r cos τ feedback signal, imaging of pUC18 relaxed circular plasmid DNA spread on mica precoated with cetylpyridinium chloride was achieved. The apparent width (28 ± 5 nm) was approximately four times that achieved by scanning force measurements with the same instrument; the apparent height of the DNA (0.6 ± 0.3 nm) was similar with the two techniques. These results demonstrate the applicability of the shear force signal for imaging biological macromolecules according to topography and in conjunction with the optical signals of a near‐field scanning optical microscope (NSOM).
Serotonergic Modulation of Photically Induced Increase in Melatonin Receptor Density and Fos Immunoreactivity in the Suprachiasmatic Nuclei of the Rat
Authors: Recio Joaquín , Pévet Paul , Masson‐Pévet Mireille
Published: November 2003

The mammalian suprachiasmatic nuclei (SCN) contain a circadian clock which is regulated by neuronal photic and non‐photic afferences. Among these, the serotonergic input originating from the dorsal raphe nucleus (DRN) is extremely important. In rats, a light pulse administered during the dark period is known to induce the expression of the immediate early gene c‐fos and to increase melatonin receptor density in the SCN. The aim of this study was to assess whether, in rats, these two phenomena were regulated by serotonin, acting via 5‐HT1A receptors. Three days after pinealectomy, 4 groups of rats were injected i.p. 90 min before sacrifice with respectively: (1) vehicle, (2) the 5‐HT1A‐agonist 8‐OH‐DPAT (5 mg/kg), (3) the 5‐HT1A‐antagonist NAN‐190 (10 mg/kg) or (4) NAN‐190 and then 8‐OH‐DPAT. Half of the animals from each group were exposed to light for 60 min before sacrifice and the other half remained in darkness. Sacrifice took place 5 to 6 h after lights off. Our results show that the antagonist NAN‐190: (1) completely blocked the photically‐induced increase of melatonin receptor density in the SCN, with an IC50=0.352±0.103 mg/kg, and (2) partially blocked (30%) the photic induction of Fos (the protein product of c‐fos) in the ventrolateral subdivision of the SCN. The agonist 8‐OH‐DPAT enhanced the photically‐induced increase of melatonin receptors by 10% and decreased the photically‐induced increase in Fos by 18%. Both drugs were devoid of any effect in non‐light‐exposed animals. From these results we may suggest that, in rats, there is a serotonergic control of the neuronal path driving photic information to the SCN. This regulation seems to occur through 5‐HT1A or 5‐HT1A‐like receptors.
The Role of Oxytocin Release in the Paraventricular Nucleus in the Control of Maternal Behaviour in the Sheep
Authors: Da Costa Ana P. C. , Guevara‐Guzman Rosalinda G. , Ohkura Satoshi , Goode Jane A. , Kendrick Keith M.
Published: November 2003

Oxytocin (OT) release within the brain is thought to play a major role in inducing maternal behaviour in a number of mammalian species but little is known about the sites of release which are important in this respect. We have investigated whether the paraventricular nucleus of the hypothalamus (PVN) is a site of OT action on maternal behaviour in the sheep. In vivo microdialysis and retrodialysis was used to determine whether OT is released in the region of the PVN during the post‐partum induction of maternal behaviour and if its release at this site can stimulate maternal behaviour in non‐pregnant animals. In vivo sampling showed that OT concentrations increased significantly in the region of PVN at birth. When OT was retrodialysed bilaterally into the PVN (1 or 10 μM) of multiparous ewes treated with progesterone and oestradiol to stimulate lactation, maternal behaviour was induced in a significant number of animals (1 μM, 6/8 and 10 μM, 5/8) compared with controls (0/8 ewes). Similar infusions of the ring structure of OT, tocinoic acid (TOC—10 μM), also induced maternal behaviour in a significant proportion of animals (5/6 ewes) as did intracerebroventricular (ICV) OT (6/8 ewes) and artificial stimulation of the vagina and cervix (VCS, 8/9 ewes). On the other hand, vasopressin (AVP) 1 μM did not induce maternal behaviour in any ewes and a 10 μM dose only induced it in 2/8 animals. The neurochemical changes accompanying the above treatments were also investigated. Noradrenaline concentrations increased in the PVN after the retrodialysis administration of OT 1 μM and 10 μM, TOC 10 μM and AVP 1 μM, OT ICV and VCS. Dopamine concentrations were also increased by OT 10 μM, TOC 10 μM, AVP 1 μM and OT ICV. Aspartate and glutamate concentrations were significantly reduced by retrodialysis infusions of OT 1 μM and AVP 1 and 10 μM but not by any other treatment. Finally, the retrodialysis infusions of OT and TOC, as well as ICV OT, significantly increased plasma OT release whereas AVP infusions did not. These results provide evidence that OT is released in the PVN during parturition and is important for the induction of maternal behaviour. It seems probable that OT release at this site has a positive feedback effect on both parvocellular and magnocellular OT neurones to facilitate co‐ordinated OT release both in central OT terminal regions (to facilitate maternal behaviour) and peripherally into the blood (to facilitate uterine contractions/milk let down). The potential functional roles for the actions of OT on monoamine and amino acid transmitter release in the PVN are discussed.
Effects of Long‐Term Treatment with Antidepressant Drugs on Proopiomelanocortin and Neuropeptide Y mRNA Expression in the Hypothalamic Arcuate Nucleus of Rats
Authors: Baker Ross A. , Herkenham Miles , Brady Linda S.
Published: November 2003

Antidepressant drugs have in common a delayed onset of clinical efficacy. In rats, long‐term, daily administration of four different types of clinically effective antidepressant drugs results in decreased corticotropin releasing hormone (CRH) mRNA expression levels in the hypothalamic paraventricular nucleus (PVN). Because a subpopulation of neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus (Arc) projects to the PVN, we measured NPY and POMC mRNA expression in the Arc using in situ hybridization histochemistry at several time points following daily administration of four different antidepressant drugs. After 14 and 56 days of imipramine treatment, Arc NPY mRNA levels are decreased to 85% and 75% of control levels, but are unchanged compared to control after one or five days of treatment. Arc POMC mRNA levels are unchanged compared to controls at 1, 5, 14, or 56 days following imipramine treatment. Unlike after imipramine, Arc NPY and POMC mRNA levels are increased significantly to 134–172% of control following 56‐day treatment with the antidepressant drugs fluoxetine, phenelzine, or idazoxan. The divergent effects of imipramine vs the other 3 antidepressant drugs on Arc NPY mRNA expression are similar to the pattern of changes in tyrosine hydroxylase (TH) mRNA expression levels in the locus coeruleus (LC) using the same experimental paradigm, but are different from the unidirectional depressive effects of all four drugs on CRH mRNA expression in the PVN. Thus, the Arc NPY and LC noradrenergic systems may act coordinately in mediating antidepressant effects. The present data are consistent with the delayed onset of clinical efficacy for antidepressant drugs, and suggest that Arc NPY and POMC neurotransmitter systems play a role in the pathophysiology of depression.
Infusions of Lidocaine into the Amygdala, but not the Preoptic Area, Block Pseudopregnancy in the Rat
Authors: Coopersmith Carol , Gans Susan E. , Rowe David W. , Erskine Mary S.
Published: November 2003

In the rat, vaginocervical stimulation (VCS) received during mating is required for the subsequent expression of 10–12 days of twice‐daily prolactin surges that are necessary for pregnancy or pseudopregnancy (PSP). This temporal separation of sensory stimulus and neuroendocrine response suggests that a mnemonic of the vaginocervical stimulation is created in the brain that triggers and sustains the daily prolactin surges. We investigated the possible involvement of the medial preoptic area (mPOA) and the medial amygdala (mAMYG) as potential neural sites involved in the processing of this neuroendocrine arc. Cycling female rats were bilaterally implanted with intracerebral cannulae in either the mPOA or mAMYG. On proestrus, females were manually palpated to confirm sexual receptivity and then received bilateral infusions of either the local anesthetic lidocaine, the Ca++ channel blocker, verapamil, or phosphate‐buffered saline (PBS) into either brain site before or both before and after receipt of 15 intromissions from an experienced male. Unmated control females received comparable infusions of lidocaine or verapamil, and were placed in the empty test arena for 10 min. Infusions consisted of either a single bilateral infusion 15 min before mating (Expt. 1), bilateral infusions both 15 min before and after mating (Expt. 2) or eight bilateral infusions separated by 30 min intervals spanning a period beginning 45 min before and ending 2 h 45 min after mating (Expt. 3). None of the lidocaine infusions into the mPOA prevented the establishment of PSP, and neither verapamil infusions into the mAMYG nor the shorter‐term neural block (i.e. single or double lidocaine infusions) of the mAMYG prevented mating‐induced PSP. However, the longer‐term neural block (i.e. multiple lidocaine infusions) of the mAMYG significantly reduced the incidence of PSP. These data support previous findings that the mAMYG receives sensory input from VCS, and suggest that the mAMYG is a site at which a mnemonic of VCS is established.
Oxytocin Released within the Supraoptic Nucleus of the Rat Brain by Positive Feedback Action is Involved in Parturition‐Related Events
Authors: Neumann Inga , Douglas Alison J. , Pittman Quentin J. , Russell John A. , Landgraf Rainer
Published: November 2003

Oxytocin is released within the supraoptic nucleus during parturition and suckling. During suckling, such release is important in positive feedback stimulation of oxytocin neurons. We have investigated whether oxytocin released within this hypothalamic nucleus during parturition (1) acts on local receptors to further amplify its own release in a positive feedback manner and (2) is critically involved in the regulation of the delivery process.  To examine the effect of the oxytocin antagonist on oxytocin release within the supraoptic nucleus, microdialysates were sampled before and during parturition and either vehicle or the antagonist was infused adjacent to the microdialysis probe directly into the supraoptic nucleus after delivery of the second pup. Intranuclear infusion of an oxytocin receptor antagonist (des‐Gly‐NH2d(CH2)5[Tyr(Me)2Thr4]OVT; 50 ng/0.5 μl) significantly (P<0.01) diminished the parturition‐related rise in oxytocin release within the supraoptic nucleus and reduced the number of pups delivered during the first and second 30‐min dialysis period compared to vehicle‐treated controls.  Bilateral infusion of the oxytocin receptor antagonist into the supraoptic nucleus after delivery of the second pup significantly slowed parturition (P<0.05), although the parturition‐related rise in plasma oxytocin concentration was unchanged. In addition, the onset of suckling was significantly affected by the antagonist as indicated by fewer live pups and fewer surviving pups with milk in their stomachs 24 hours after parturition (P<0.05).  To seek other, periventricular sites of oxytocin action during parturition, oxytocin or the oxytocin antagonist was infused into the lateral cerebral ventricle from the birth of pup 2. Via this route, oxytocin speeded up parturition, but the antagonist was ineffective; thus it appears that periventricular oxytocin‐sensitive sites are not normally active in promoting parturition, but can do so.  The findings indicate a receptor‐mediated positive feedback action of oxytocin on its own release within the supraoptic nucleus during parturition, which seems to be involved in the progress of parturition without significantly affecting circulating oxytocin levels. Oxytocin released within the supraoptic nucleus might be important for the coordinated activation of oxytocin neurons and for the synergistic central and peripheral oxytocin effects involved in the regulation of parturition‐related events necessary for the survival of the newborn, including the onset of lactation.
Decrease of Endogenous Vasopressin Release Necessary for Expression of the Circadian Rise in Plasma Corticosterone: a Reverse Microdialysis Study
Authors: Kalsbeek Andries , van der Vliet Jan , Buijs Ruud M.
Published: November 2003

The mammalian suprachiasmatic nuclei (SCN) contain an endogenous pacemaker that generates daily rhythms in behavior and secretion of hormones. Previously we hypothesized that the SCN imposes its circadian rhythm on the rest of the brain through a rhythmic release of its transmitters in its target areas. In the present study we employed microdialysis‐mediated intracerebral administration of vasopressin (VP) and its V1 ‐antagonist to study the mechanisms underlying the circadian control of the release of the adrenal hormone corticosterone. Stress‐free application of the VP V1 ‐antagonist in the dorsomedial hypothalamus of freely moving, undisturbed animals during the middle of the light period (i.e. the trough of the corticosterone rhythm) caused an immediate increase of circulating plasma corticosterone levels. A similar administration of VP at the end of the light period completely prevented the diurnal rise in plasma corticosterone. These results indicate a pronounced inhibitory role for SCN‐derived VP at the level of the dorsomedial hypothalamus with respect to the activity of the hypothalamo‐pituitary‐adrenal axis during the day period. Thus, the daily decline in VP release sets a specific time window for the occurrence of the daily corticosterone peak. On the other hand, during the dark period corticosterone levels are decreasing together with basal VP levels. Therefore, in addition to the inhibitory VP signal from the SCN, there is also the need for an excitatory SCN signal in order to accomplish the complete circadian profile of plasma corticosterone.
Milk Ejection Bursts of Supraoptic Oxytocin Neurones during Bilateral and Unilateral Suckling in the Rat
Authors: Wang Yu‐Feng , Negoro Hideo , Honda Kazumasa
Published: November 2003

Extracellular recordings of the electrical activity of oxytocin neurones were made from the supraoptic nuclei (SON) of lactating rats, and the milk‐ejection bursts and the background activity of oxytocin neurones were investigated during unilateral and bilateral suckling. When application of pups was limited to the nipples on either the same side (ipsilateral suckling) or the side opposite (contralateral suckling) to the oxytocin neurone recorded, the burst amplitude and background firing rate were significantly (P<0.05) lower and the inter‐burst interval was significantly (P<0.05) longer than during bilateral suckling. Furthermore, the burst amplitude was significantly (P<0.05) lower during ipsilateral suckling than during contralateral suckling. The majority of the oxytocin neurones showed a gradual increase in the burst amplitude during bilateral (88.9%) and contralateral (77.3%) suckling, but during ipsilateral suckling only 40% of the neurones did. The inter‐burst interval became shorter with the progress of the milk ejection reflex during any mode of suckling. Three pairs of oxytocin neurones recorded simultaneously from both SON were successfully tested for the effect of bilateral and unilateral suckling on the electrical activity, and the results showed the same direction of change in the burst amplitude, background activity and burst interval as shown in single side recordings. These findings indicate that the burst amplitude mainly depends on the amount of afferent suckling signals arising from the nipples on the side opposite to the recording side, and that there may exist bilateral summation centres coordinating with the synchronization mechanism of milk‐ejection bursts of oxytocin neurones.
Estradiol Regulation of α1b‐Adrenoceptor mRNA in Female Rat Hypothalamus‐Preoptic Area
Authors: Karkanias G. B. , Ansonoff M. A. , Etgen A. M.
Published: November 2003

Estradiol treatment for 48 h increases the density of α1B‐adrenoceptors in the hypothalamus‐preoptic area of ovariectomized female rats by five‐ to six‐fold. Present studies tested the hypothesis that estradiol elevation of hypothalamus‐preoptic area α1B‐adrenoceptor density is correlated with increased levels of mRNA for this receptor. We developed a semiquantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) protocol for measuring brain α1b‐adrenoceptor mRNA. The primers chosen yielded the predicted 409 base pair PCR product when used to amplify authentic α1b‐adrenoceptor cDNA. The identity of the RT‐PCR products from rat brain was confirmed by restriction digest analysis and sequencing. Moreover, there was a good correlation between the levels of α1b‐adrenoceptor mRNA measured by RT‐PCR in liver, whole brain and cerebellum with previous measurements using Northern blots and RNAse protection assays. We then performed RT‐PCR on total RNA from hypothalamic‐preoptic area tissue taken from ovariectomized control rats and from ovariectomized rats injected once or twice with 2 μg of estradiol benzoate at 24 or 24 and 48 h before sacrifice. Exposure to estradiol for either 24 or 48 h significantly increased levels of α1b‐adrenoceptor mRNA by 86–110% in the hypothalamus‐preoptic area of ovariectomized female rats when compared to oil‐treated controls. We also examined whether estradiol regulates α1b‐adrenoceptor mRNA in the cortex. Cortical α1b‐adrenoceptor mRNA levels were reduced to approximately 20% of control levels when measured 24 h after hormone injection. A similar decrease in cortical α1b‐adrenoceptor mRNA was observed 48 h after estrogen administration. In summary, estradiol treatment significantly increases the level of α1b‐adrenoceptor mRNA in the hypothalamus‐preoptic area, a brain region involved in the control of reproductive function. In the cortex, a brain region with relatively few estrogen receptors, the same estrogen treatment reducesα1b‐adrenoceptor mRNA levels.
Modulation of 5HT1A Responsiveness in CA1 Pyramidal Neurons by in vivo Activation of Corticosteroid Receptors
Authors: Hesen W. , Joëls M.
Published: November 2003

In this study we describe modulatory effects exerted by in vivo activation of corticosteroid receptors on 5HT responsiveness of rat CA1 pyramidal neurons. In the first series of experiments, adrenalectomized (ADX) rats were injected with corticosterone one hour prior to decapitation (1–1000 μg/100 g body weight) after which 5HT1A induced hyperpolarizations were determined in vitro by means of intracellular recordings. It appeared that 5HT responsiveness was dose‐dependently affected by corticosterone injections: 5HT responses were relatively large when no corticosteroid receptors were activated (ADX); similar 5HT responses were observed or when both mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) were occupied by injection of high doses of corticosterone (100–1000 μg/100 g body weight). However, compared to the latter group, 5HT hyperpolarizations were significantly suppressed in slices from rats that received moderate amounts of corticosterone (10–30 μg/100g). Next, we investigated whether physiological variations of plasma corticosterone levels as occurring in intact rats correlated with the transmitter responsiveness. It was found that high plasma levels of corticosterone due to either stress or exogenous application of high doses of corticosterone correlated with large 5HT‐responses in vitro. Interestingly, the large 5HT responses recorded after stress were clearly suppressed by pretreatment with RU38486, a GR antagonist. Altogether, this study presents further evidence that 5HT transmission in hippocampal CA1 area is modulated by differential steroid receptor activation as may occur under physiological circumstances due to different plasma concentrations of corticosterone.
Dexamethasone Increases Growth Hormone (GH)‐Releasing Hormone (GRH) Receptor mRNA Levels in Cultured Rat Anterior Pituitary Cells
Authors: Tamaki M. , Sato M. , Matsubara S. , Wada Y. , Takahara J.
Published: November 2003

To examine the effects of glucocorticoid (GC) on growth hormone (GH)‐releasing hormone (GRH) receptor gene expression, a highly‐sensitive and quantitative reverse‐transcribed polymerase chain reaction (RT‐PCR) method was used in this study. Rat anterior pituitary cells were isolated and cultured for 4 days. The cultured cells were treated with dexamethasone for 2, 6, and 24 h. GRH receptor mRNA levels were determined by competitive RT‐PCR using a recombinant RNA as the competitor. Dexamethasone significantly increased GRH receptor mRNA levels at 5 nM after 6‐ and 24 h‐incubations, and the maximal effect was found at 25 nM. The GC receptor‐specific antagonist, RU 38486 completely eliminated the dexamethasone‐induced enhancement of GRH receptor mRNA levels. Dexamethasone did not alter the mRNA levels of β‐actin and prolactin at 5 nM for 24 h, whereas GH mRNA levels were significantly increased by the same treatment. The GH response to GRH was significantly enhanced by the 24‐h incubation with 5 nM dexamethasone. These findings suggest that GC stimulates GRH receptor gene expression through the ligand‐activated GC receptors in the rat somatotrophs. The direct effects of GC on the GRH receptor gene could explain the enhancement of GRH‐induced GH secretion.
Long‐Term Selective Down‐Regulation of Brain Glucocorticoid Receptors after Neonatal Dexamethasone Treatment in Rats
Authors: Felszeghy Klára , Gáspár Erzsébet , Nyakas Csaba
Published: November 2003

The development of corticosteroid receptors may be permanently modified by perinatal hormonal influences. Changes in binding characteristics of corticosteroid receptors were examined in rats treated sc. with 1 μg/g dexamethasone (DEX) on postnatal day (PND) 1 and subsequently two more times on PND 3 and 5 in several brain areas. [3H]Corticosterone (CORT) binding capacity (Bmax) and affinity (Kd) were determined at 3 weeks old and adult ages by using saturation analysis. The mineralocorticoid type receptor (MR) and the glucocorticoid receptor (GR) sites were measured separately with single point analysis applying a selective glucocorticoid ligand RU 28362 saturating GR. The decrease in CORT binding was due to a selective GR decrement in all structures and the MR concentration was not changed considerably. The basal levels of plasma CORT were not permanently influenced by neonatal DEX. In conclusion, DEX given during the first week of life resulted in long‐term and selective down‐regulation of GR and this decrement was independent of the actual circulating CORT level.
Corticotrophin‐Releasing Hormone Receptor Type 1: Generation and Characterization of Polyclonal Antipeptide Antibodies and their Localization in Pituitary Cells and Cortical Neurones in vitro
Authors: Castro M. G. , Morrison E. , Perone M. J. , Brown O. A. , Murray C. A. , Ahmed I. , Perkins A. V. , Europe‐Finner G. , Lowenstein P. R. , Linton E. A.
Published: November 2003

Corticotrophin‐releasing hormone (CRH) is a 41 amino acid neuropeptide which plays a major role in regulating the endocrine response to stress. CRH acts by first binding to specific receptors on the plasma membrane of target cells. A CRH receptor from a human corticotroph adenoma and rat brain has recently been cloned (CRH‐R1). In this paper, we have chosen three different peptide sequences within the CRH‐R1 molecule which bear no similarity to other members of this receptor subfamily (or indeed any known protein) and which are likely to be exposed on the surface of the native protein, for antibody production. Some of these fragments produced anti‐peptide antibodies of good titre which cross‐reacted with the CRH‐R1 receptor expressed in transiently transfected COS‐7 cells and in tissue extracts from rat cerebellum, cortex, pituitary gland and human myometrium, both in Western blots and in liquid‐phase radioimmunoassay. We used immunofluorescence techniques to localize the CRH receptor in transiently transfected COS‐7 cells, primary cultures of rat anterior pituitary (AP) cells, the corticotroph‐tumour cells AtT20 D16–16 and cortical neurons in primary culture. Our results indicate IR‐CRH‐R1 receptors have a punctate distribution on the plasma membrane of AP cells and AtT20 D16–16 cells. Whilst in AP cells their appearance is a fine punctate pattern, in AtT20 cells, they appear as large patches which could account for receptor clusters. Within primary cortical neurons, their distribution does not appear to be polarized. Our results suggest that distribution of CRH‐R1 receptors within the different cell‐types investigated depends not only on the amino acid sequence but also on cellular factors.
Species Differences in Central Oxytocin Receptor Gene Expression: Comparative Analysis of Promoter Sequences
Authors: Young Larry J. , Huot Becky , Nilsen Roger , Wang Zuoxin , Insel Thomas R.
Published: November 2003

The distribution of oxytocin binding sites in the brain is highly variable among mammals. Using two species of microtine rodents (voles) with strikingly different patterns of oxytocin binding sites in the brain, we demonstrate that these differences are due to differences in region specific gene expression and not post‐translational processing. The distribution of oxytocin receptor mRNA closely resembles the distribution of oxytocin receptor binding sites in both species. Analysis of the 5′ flanking region of the oxytocin receptor gene from both species reveals few differences in potential regulatory elements which could explain the differences in gene expression. These data suggest that species differences in oxytocin receptor binding are due to species differences in: i) distant DNA sequences further upstream or downstream which may influence expression; ii) the distribution of regulatory proteins such as transcription factors in the brain or iii) epigenetic factors, such as prenatal and perinatal environment which may affect gene expression in the adult.
Multiple Members of the Leucokinin Neuropeptide Family are Present in Cerebral and Abdominal Neurohemal Organs in the Cockroach Leucophaea maderae
Authors: Winther Åsa M. E. , Lundquist C. Tomas , Nässel Dick R.
Published: November 2003

Two neurohemal organs of the cockroach Leucophaea maderae, the corpora cardiaca and the lateral heart nerve are known to contain leucokinin immunoreactive material. We examined the corpora cardiaca and the lateral heart nerve to establish whether these neurohemal organs store all 8 known leucokinin isoforms or if the leucokinins have a differential distribution. Extracts of corpora cardiaca and abdominal hearts with attached lateral heart nerve were separated on reversed phase high performance liquid chromatography (rpHPLC), then tested for leucokinin immunoreactivity by a radioimmunoassay (RIA) able to detect all 8 leucokinin isoforms. Extracts from brain and optic lobes were also separated and assayed in the RIA. Synthetic leucokinin 1–8 were subjected to rpHPLC and their different retention times established by RIA for reference. Leucokinin immunoreactive material originating from the corpora cardiaca and lateral heart nerves eluted in fractions corresponding to those of the synthetic leucokinin 1–8. In this study we have thus demonstrated that probably all 8 leucokinin isoforms are stored in the corpora cardiaca and the lateral heart nerve. These observations suggest that all 8 leucokinins are likely to be released as neurohormones into the circulation.
Effect of Melatonin on the Release of Gonadotropin‐Releasing Hormone and Cyclic AMP from the Rat Hypothalamus: an in vitro Study
Authors: Messager S. , Caillol M. , Rossano B. , Martinet L.
Published: November 2003

Using an in vitro static incubation system of adult male rat hypothalami, we have studied the effect of melatonin on the release of gonadotropin‐releasing hormone (GnRH) and cyclic adenosine monophosphate (cAMP). Mediobasal hypothalamus (MBH) and preoptic area (POA) were incubated separately in Minimum Essential Medium (MEM) for 6 h. The release of GnRH was measured by radioimmunoassay in the incubation medium sampled every 7.5 min. In the MBH and POA incubation medium, the mean amount of GnRH released was 8.9±1.1 and 3.4±0.6 pg GnRH/7.5 min, respectively (P<0.01). The mean number of GnRH pulses under basal conditions was 2±0.3 per 2 h in the MBH and 1.6±0.3 per 2 h in the POA (P>0.05). Melatonin (10−8 M) did not alter the release of GnRH in the presence or absence of forskolin (10−4 M). Melatonin, which was without effect on basal cAMP, inhibited forskolin‐stimulated cAMP accumulation in the medium by 50% in the MBH and 40% in the POA. These results suggest that in our incubation system, melatonin does not modify GnRH release, but probably acts through the melatonin binding sites located in the hypothalamus to inhibit forskolin‐stimulated cAMP.
GnRH Secretion into CSF in Rams Treated With a GnRH Antagonist
Authors: Blache D. , Chagas L. M. , Caraty A. , Deghenghi R. , Delaleu B. , Blackberry M. A. , Martin G. B.
Published: November 2003

The equilibrium of the brain‐pituitary‐testicular axis is controlled by negative feedback exerted primarily through changes in the circulating concentrations of gonadal steroids. This is usually studied in gonadectomised animals treated with single large doses or constant low levels of exogenous steroid. However, the feedback system probably also contains dynamic components, perhaps expressed as delays to changes in GnRH secretion following a change in steroid concentration. These delays must be measured without interference from surgical procedures, including anaesthesia, bias associated with changes in pituitary responsiveness (which affect the efficiency of pulse detection), and chronic side‐effects of gonadectomy. We used a GnRH antagonist [‘Antarelix’: Ac‐d‐Nal, d‐Cpa, d‐Pal, Ser, Tyr, d‐Hci, Leu, Lys‐(iPr), Pro, d‐Ala‐NH2] to transiently block LH and steroid secretion (in effect, inducing and reversing castration) in mature male sheep, and measured GnRH secretion into cerebrospinal fluid (CSF) in the third cerebral ventricle. The CSF was withdrawn with a peristaltic pump at a rate of 2 ml/h and pooled every 20 min. Jugular plasma was sampled every 20 min and analysed for testosterone and LH pulses. The antagonist (500 μg i.v.) was injected after 6 h of baseline sampling and the study continued for a further 24 h. The pulses of LH and testosterone disappeared shortly after antagonist injection, with delays of 20±12 min for LH and 80±29 min for testosterone. This led to an increase in GnRH pulse frequency, starting 300±54 min after antagonist injection. Secretion of LH and testosterone pulses resumed at 553±38 and 530±30 min (after antagonist injection), and GnRH pulse frequency returned to baseline values after 170±42 min (relative to LH) and 117±35 min (relative to testosterone). The consistent nature of these responses across the group of animals suggests that this can be used to test the effects of exteroceptive factors on the dynamics of negative feedback.
Fos Expression by Naloxone in LHRH Neurons ofthe Mediobasal Hypothalamus and Effects of Pentobarbital Sodium in the Proestrous Rat
Authors: Funabashi Toshiya , Jinnai Kayoko , Kimura Fukuko
Published: November 2003

Because Fos is thought to be induced in neurons that are activated, we examined whether luteinizing hormone‐releasing hormone (LHRH) neurons expressed Fos protein when they were stimulated by an opioid receptor antagonist naloxone (NAL), expecting to identify LHRH neurons which are regulated by opioid neurons directly or indirectly. Further, we examined whether an ovulation‐blocking dosage of pentobarbital sodium (PB) would affect the NAL‐induced Fos expression. Female rats were infused with naloxone (5 mg/kg/h) for 90 min (10.00–11.30) in the morning of proestrus, during which infusion blood sampling was done, and were killed by i.v. injection with an overdose of PB at 11.30–12.00. Dual immunoperoxidase/immunofluorescence staining for both Fos and LHRH revealed that some LHRH immunoreactive (ir) neurons in the forebrain expressed Fos‐ir, associated with an increase in serum LH concentrations, but little co‐localization was found in rats in proestrus which were infused with saline as the control. The proportion of LHRH‐ir neurons which expressed Fos‐ir was about 35–62% in the caudal part of the forebrain including the mediobasal hypothalamus, and this was larger than that (10%) in the rostral part of the forebrain including the preoptic area. PB injection (32 mg/kg bw, i.p.) 15 min prior to the beginning of NAL infusion significantly enhanced the increase in LH secretion due to NAL, and also enhanced Fos‐ir expression in LHRH‐ir neurons. Together with the well‐established fact that PB blocks the LHRH surge generator and our previous findings that NAL stimulates the LHRH pulse generator even in the PB‐blocked proestrous rat, these results strongly suggest that the LHRH pulse generator exists in the mediobasal hypothalamus which contains LHRH neurons that are responsive to NAL and express Fos protein.
Decline in Basic Fibroblast Growth Factor (FGF‐2) mRNA Expression in Female Rat Hypothalamus at Puberty
Authors: Smyth Catherine E. , Knee Rai , Wilkinson Michael , Murphy Paul R.
Published: November 2003

There is a growing acceptance of the importance of hypothalamic growth factors in the control of sexual maturation. Basic fibroblast growth factor (bFGF, FGF‐2), a potent mitogen and neurotropic factor for brain cells in vitro, including hypothalamic cells, is widely expressed in the post‐natal CNS but its physiological functions there are largely unknown. Previously, studies of FGF‐2 mRNA regulation in vivo have been hampered by the low levels of FGF‐2 mRNA present in post‐natal tissues. We have applied a sensitive semi‐quantitative procedure based on reverse transcription followed by polymerase chain reaction amplification (RT‐PCR) to detect and estimate relative amounts of mRNAs encoding FGF‐2 and its receptor in the hypothalamic‐hypophyseal axis in individual female rats undergoing sexual maturation.
Changes in Sleep‐Endocrine Activity after Growth Hormone‐Releasing Hormone Depend on Time of Administration
Authors: Schier Thomas , Guldner Jürgen , Colla Michael , Holsboer Florian , Steiger Axel
Published: November 2003

When administered intravenously (i.v.) in a pulsatile mode during the first half of the night to young normal controls, growth hormone‐releasing hormone (GHRH) results in increased growth hormone (GH) plasma levels and slow wave sleep (SWS) and blunted cortisol release. In the present study we investigated whether GHRH has the same effects when administered in the early morning. Seven normal young male volunteers had 2 sessions each in the sleep laboratory (23.00 to 10.00 h) during which the secretion of GH, cortisol and corticotropin (ACTH) and polygraphic recordings were monitored. Verum (4 bolus injections of 50 μg GHRH) or placebo were injected i.v. at 04.00, 05.00, 06.00 and 07.00 h. GHRH stimulated GH plasma levels significantly whereas cortisol and ACTH were not altered. In the sleep‐electroencephalogram, only rapid‐eye‐movement density was decreased significantly during the period of active medication; all other sleep parameters were unaffected. We suggest that the physiological occurring high activity of the hypothalamic‐pituitary‐adrenocortical(HPA) system in the early morning prevents the effects of GHRH on cortisol plasma levels and SWS. Thus GHRH administered to healthy young men in the early morning hours has the same effect as GHRH administered during the first half of the night to patients with major depression who have HPA hyperactivity throughout the day.
Neuronal Activation of the Hypothalamic Magnocellular System in Response to Oropharyngeal Stimuli in the Rat
Authors: Naïmi Naceur , Rivest Serge , Racotta Ilie , Richard Denis
Published: November 2003

The present study was designed to delineate the neuronal site, the nature, and the gastrointestinal origin of the stimulation of the hypothalamic magnocellular system induced by the ingestion of sweetened condensed milk. Concomitant localization of the c‐fos protein (Fos) with either arginine‐vasopressin (AVP) mRNA or oxytocin (OT) mRNA in the paraventricular nucleus of the hypothalamus (PVH) and the supraoptic nucleus (SON) revealed that the hypothalamic neurons containing AVP and OT were activated following ingestion of sweetened condensed milk. Expression of c‐fos mRNA was also determined in rats implanted with a gastric cannula that allowed for real, sham, and gastric feeding of sweetened condensed milk. The results provide evidence that the stimulation of the PVH and SON induced by sweetened condensed milk originate from oropharyngeal stimuli. Indeed, in real‐and sham‐fed rats, the postprandial levels of c‐fos mRNA in the PVH and SON were significantly higher than the preprandial values, whereas there was no early postprandial rise in c‐fos mRNA levels within the magnocellular division of the PVH and SON after gastric feeding. The results of this study also suggested that the stimulation of the PVH and SON induced by sweetened condensed milk was related to the hypertonicity of the milk. Indeed, ingestion of an hypertonic solution of sucrose with a carbohydrate content close to that of sweetened condensed milk led to a stimulation of the PVH and SON that was comparable to that induced by the milk, whereas ingestion of an isotonic solution of sucrose did not trigger any significant activation of the PVH and SON. Taken together, the present results indicate that magnocellular neurosecretory neurons are sensitive to oropharyngeal stimuli and further support the view of the existence of oropharyngeal osmoreceptors.
Endogenous Neurotensin Regulates Hypothalamic‐Pituitary‐Adrenal Axis Activity and Peptidergic Neurons in the Rat Hypothalamic Paraventricular Nucleus
Authors: Nicot Arnaud , Rowe Wayne B. , De Kloet E. Ronald , Betancur Catalina , Jessop David S. , Lightman Stafford L. , Quirion Rémi , Rostène William , Bérod Anne
Published: November 2003

Adrenocorticotropin (ACTH) secretion depends primarily on hypophysiotrophic factors released from neurons of the paraventricular nucleus of the hypothalamus. However, the neurochemical factors controlling these neurons, in particular neuropeptides, have had little investigation. In this study, we have investigated the role of neurotensin in the regulation of the different components of the hypothalamo‐pituitary‐adrenal (HPA) axis under basal and stress conditions in rats. For this purpose, animals were implanted with bilateral cannulae filled with crystals of the neurotensin antagonist, SR 48692, and which were located above the paraventricular nucleus. Five days after surgery, the effects of SR 48692 implants were studied on basal and stress‐induced secretion of ACTH and corticosterone. Such treatment did not modify plasma levels of ACTH and corticosterone in basal conditions but reduced ACTH but not corticosterone levels after tail cut procedure. After an exposure to a novel environment for 30 min, both ACTH and corticosterone plasma levels were reduced in the SR 48692‐treated group. In situ hybridization studies revealed that chronic administration of SR 48692 induced a significant reduction of CRF mRNA levels in the parvocellular division of the paraventricular nucleus of the hypothalamus. In addition, a 2‐fold increase in basal levels of plasma vasopressin associated with an increase in vasopressin mRNA levels in the magnocellular neurons of the paraventricular nucleus was also detected. Finally, the basal plasma levels of oxytocin were not affected by the same treatment.
Differential Effects of Neuroactive Steroids on Somatostatin and Dopamine Secretion from Primary Hypothalamic Cell Cultures
Authors: Murray H. E. , Gillies G. E.
Published: November 2003

This study investigated the effects of neuroactive steroids, which have been reported to modulate GABA‐ergic transmission, on the secretion of somatostatin (SRIH) and also dopamine (DA) from primary rat hypothalamic cell cultures, where the release of both substances is regulated by a GABAA receptor‐mediated inhibitory tone. Pregnenolone sulphate (PS), a negative allosteric modulator at the GABAA receptor, enhanced SRIH secretion in a time and dose‐dependent manner (10−12–10−8 M). This effect was reversed by muscimol (10−8 M) and enhanced by bicuculline (10−6 M), thus supporting an action of PS at the GABAA receptor. The release of endogenously synthesized dopamine (DA) was, however, unaffected by PS. A number of other steroids were also tested for their potential actions on SRIH and DA secretion. Allopregnanolone had slight but significant stimulatory actions on SRIH secretion, whereas tetrahydro‐deoxycorticosterone (TH‐DOC) markedly stimulated SRIH secretion with a bell‐shaped dose response curve resembling that found for PS. The release of DA was unaffected by these neuroactive steroids but, unlike SRIH, DA release was stimulated by dehydroepiandrosterone sulphate (DHEAS). The results support the view that neuroactive steroids may play an important role in regulating some aspects of neuroendocrine function and they also provide the first demonstration of differential activities of neuroactive steroids within the hypothalamus at low, physiologically relevant concentrations. The results also raise the possibility that certain hypothalamic neuronal populations may posses uniquely different GABAA receptors and that such mechanisms may contribute to the functional development of the neuroendocrine system.