bluuebunny/crossref_metadata_2025 huggingface dataset:
Deconvolution improves colocalization analysis of multiple fluorochromes in 3D confocal data sets more than filtering techniques
Authors: Landmann L.
Published: March 2003

Background and noise impair image quality by affecting resolution and obscuring image detail in the low intensity range. Because background levels in unprocessed confocal images are frequently at about 30% maximum intensity, colocalization analysis, a typical segmentation process, is limited to high intensity signal and prone to noise‐induced, false‐positive events. This makes suppression or removal of background crucial for this kind of image analysis. This paper examines the effects of median filtering and deconvolution, two image‐processing techniques enhancing the signal‐to‐noise ratio (SNR), on the results of colocalization analysis in confocal data sets of biological specimens.

The data show that median filtering can improve the SNR by a factor of 2. The technique eliminates noise‐induced colocalization events successfully. However, because filtering recovers voxel values from the local neighbourhood false‐negative (‘dissipation’ of signal intensity below threshold value) as well as false‐positive (‘fusion’ of noise with low intensity signal resulting in above threshold intensities), results can be generated. In addition, filtering involves the convolution of an image with a kernel, a procedure that inherently impairs resolution.

Image restoration by deconvolution avoids both of these disadvantages. Such routines calculate a model of the object considering various parameters that impair image formation and are able to suppress background down to very low levels (< 10% maximum intensity, resulting in a SNR improved by a factor 3 as compared to raw images). This makes additional objects in the low intensity but high frequency range available to analysis. In addition, removal of noise and distortions induced by the optical system results in improved resolution, which is of critical importance in cases involving objects of near resolution size. The technique is, however, sensitive to overestimation of the background level. In conclusion, colocalization analysis will be improved by deconvolution more than by filtering. This applies especially to specimens characterized by small object size and/or low intensities.
Two‐photon fluorescence absorption and emission spectra of dyes relevant for cell imaging
Authors: Bestvater F. , Spiess E. , Stobrawa G. , Hacker M. , Feurer T. , Porwol T. , Berchner‐Pfannschmidt U. , Wotzlaw C. , Acker H.
Published: March 2003

Two‐photon absorption and emission spectra for fluorophores relevant in cell imaging were measured using a 45 fs Ti:sapphire laser, a continuously tuneable optical parametric amplifier for the excitation range 580–1150 nm and an optical multichannel analyser. The measurements included DNA stains, fluorescent dyes coupled to antibodies as well as organelle trackers, e.g. Alexa and Bodipy dyes, Cy2, Cy3, DAPI, Hoechst 33342, propidium iodide, FITC and rhodamine. In accordance with the two‐photon excitation theory, the majority of the investigated fluorochromes did not reveal significant discrepancies between the two‐photon and the one‐photon emission spectra. However, a blue‐shift of the absorption maxima ranging from a few nanometres up to considerably differing courses of the spectrum was found for most fluorochromes. The potential of non‐linear laser scanning fluorescence microscopy is demonstrated here by visualizing multiple intracellular structures in living cells. Combined with 3D reconstruction techniques, this approach gives a deeper insight into the spatial relationships of subcellular organelles.
Cryofixation of epithelial cells grown on sapphire coverslips by impact freezing
Authors: Reipert S. , Fischer I. , Wiche G.
Published: March 2003

Rapid cryofixation of cells cultured on coverslips without the use of chemical fixatives has proved advantageous for the immunolocalization of antigens by electron microscopy. Here, we demonstrate the application of sapphire‐attached tissue culture cells (PtK2 epithelial cells and mouse myoblasts) to metal‐mirror impact freezing. The potential of the Leica EM‐CPC cryoworkstation for routine freezing and for safe transfer of the cryofrozen samples into a sapphire disc magazine for freeze‐substitution (SD‐FS unit) has been exploited. Subsequently, the SD‐FS unit has been tested for its use in methanol freeze‐substitution and low temperature embedding for immunoelectron microscopy. The structural preservation of Lowicryl HM20‐embedded cells has been assessed as being free of damage by large ice crystals.
A novel light source for SICM–SNOM of living cells
Authors: Rothery A. M. , Gorelik J. , Bruckbauer A. , Yu W. , Korchev Y. E. , Klenerman D.
Published: March 2003

We have developed a novel light source for use in a scanning near‐field optical microscope (SNOM or NSOM) based on a nanopipette whose distance from the sample surface is controlled using scanning ion conductance microscopy. The light source is based on the general principle of the chemical reaction between a fluorophore in the pipette and ligand in the bath, to produce a highly fluorescent complex that is continually renewed at the pipette tip. In these experiments we used fluo‐3 and calcium, respectively. This complex is then excited with an Ar+ laser, focused on the pipette tip, to produce the light source. This method overcomes the transmission problem of more traditional SNOM probes and has been used to acquire simultaneous high‐resolution topographic and optical images of biological samples in physiological buffer. A resolution of ∼220 nm topographic and ∼190 nm optical was determined through imaging fixed sea‐urchin sperm flagella. Live A6 cells were also imaged, demonstrating the potential of this system for SNOM imaging of living cells.
Neuropeptide Y (NDY) Y1 Receptoe mRNA is Upregulated in Association with Transient Hyperphagia and Body Weight Gain: Evidence for a Hypothalamic Site for Concurrent Development of Leptin Resistance
Authors: Kalra P. S. , Dube M. G. , Xu B. , Farmerie W. G. , Kalra S. P.
Published: March 2003

Microinjection of colchicine (COL), a neurotoxin that blocks axoplasmic flow in the neurons, bilaterally into the ventromedial nucleus (VMN) evokes transient hyperphagia and body weight gain. These shifts in energy balance occurred in conjunction with development of increased sensitivity to neuropeptide Y (NPY), the endogenous orexigenic signal. In order to trace the aetiology of NPY supersensitivity, we have evaluated (1) NPY Y1 and Y5 receptor (R) gene expression in the hypothalamus and (2) the possibility of alterations in the inhibitory action of leptin, a hormone produced by lipocytes. Adult male rats were rendered hyperphagic with bilateral microinjections of COL (4 μg/side) into the VMN. We observed that hypothalamic NPY Y1 mRNA levels, as measured by RNAase protection assay, were significantly increased on day 2 and returned to the control level on day 4 in COL‐injected rats. The effects on NPY Y5R mRNA were not as clear cut. Interestingly, serum leptin levels increased in association with the hyperphagia and body weight gain, thereby raising the likelihood of development of resistance to the suppressive effect of endogenous leptin on food intake. Indeed, intracerebroventricular injection of 7 μg human recombinant leptin, a dose that attenuated daily food intake in normal and fasted rats, was completely ineffective in attenuating hyperphagia in COL‐treated rats. These results show that transient hyperphagia induced by interruption of signalling in the VMN may be caused by increased sensitivity to NPY, which may be caused, in part, by increased expression of NPY Y1R in hypothalamic sites involved in regulation of ingestive behaviour. Additionally, the observation of increased leptin release and concurrent development of leptin resistance suggest that a normally functioning VMN may be necessary for the central inhibitory effects of leptin on food intake.
Possible Involvement of Norepinephrine Transporter Activity in he pulsatility of Hypothalamic Gonadotropin‐Releasing Hormone Release: Influence of the Gonad
Authors: Pau Francis , Lee Cyrus , Cowles Angilene , Yang Shu‐Ping , Hess David L. , Spies Harold
Published: March 2003

Pulsatile secretion of hypothalamic gonadotropin‐releasing hormone (GnRH) is suppressed by α‐adrenergic antagonists in ovariectomized (OVX) rabbits, thus suggesting that initiation of GnRH pulses requires the presence of norepinephrine (NE) stimulation. Terminals of NE neurons are located in proximity with GnRH cells in the hypothalamus, including the arcuate nucleus‐median eminence (AME) region. Synaptic NE molecules may be catabolized or transported back to NE terminals (i.e. reuptake) via specific NE transporter proteins (NET). Thus, the amount of synaptic NE acting on GnRH cells is a function of the rate of NE release, metabolism and reuptake. Hypothetically, the rise and fall of a GnRH pulse may be associated with the similar fluctuations of synaptic NE release and/or NET activity. To test this hypothesis, we examined the effects of AME administration of desipramine (DMI, a specific NET blocking drug) on GnRH release. First, we delivered 0.2–10 mM doses of DMI continuously for 1 h via an AME microdialysis (μD) system into intact male rabbits. We found that each AME‐DMI infusion, between dosages of 1 mM and 10 mM, stimulated a GnRH pulse, and that the size of these GnRH pulses were proportional to the dosage of DMI. To confirm the specificity of DMI on NET, we measured catecholamine content in μD samples by HPLC. The temporal (60 min) DMI induced a pattern of NE release that included a rising limb within the first 20–30 min; although NE returned to baseline values within the period of DMI treatment. Neither epinephrine nor dopamine levels were changed by DMI. Second, a median dose of DMI (5 mM) was given by μD for 60 min in four separate rabbit models: gonadal intact females (F‐INT), intact males (M‐INT), gonadectomized females (F‐GDX) and castrated males (M‐GDX). Individual μD samples were measured for NE and GnRH. Regardless of gender or gonadal status, 5 mM of DMI concomitantly induced a pulse‐like release of NE and GnRH. Furthermore, the response of GnRH to DMI was greater in GDX rabbits than in INT animals of both genders. Third, we administered DMI (5 mM) for 30 min via a push‐pull perfusion (PPP) system during four repeated 90 min intervals, in either F‐INT or ovariectomized (F‐GDX) females, and measured GnRH in PPP samples. In both F‐INT and F‐GDX, each DMI challenge induced a GnRH pulse. In F‐INT, all sequential DMI‐induced GnRH pulses were nearly equal in size. In contrast, in F‐GDX, the first DMI‐induced GnRH pulse was greater than subsequent ones. Collectively, these observations are consistent with the concept of noradrenergic regulation of pulsatile GnRH release, and we conclude that the temporal activity of NET may be an integral part of the mechanism by which GnRH pulses operate.
Oxytocin Release from the Neurohypophysis after the Taste Stimuli Previously Paired with Intravenous Cholecystokinin in Anaesthetized Rats
Authors: Onaka Tatsushi , Yagi Kinji
Published: March 2003

Intravenously administered cholecystokinin octapeptide (CCK) induces oxytocin release from the neurohypophysis in anaesthetised rats. Memory of conditioned taste aversion can be acquired under anaesthesia. The present experiments aimed at investigating whether taste stimuli previously paired with iv CCK evoke oxytocin release from the neurohypophysis in urethane‐anaesthetised male rats. Sucrose solution (0.75–2.0 M) paired with iv CCK or the vehicle was applied to the tongue. After 3 h, sucrose solution was applied again. The second sucrose slightly increased plasma oxytocin concentration in rats that had received the first sucrose solution paired with the vehicle. Plasma oxytocin concentration after the second sucrose application, however, was significantly higher in CCK‐injected than in vehicle‐injected rats. In rats that received CCK 1 h before the first sucrose application, a second sucrose application did not produce the oxytocin response. The magnitude of the oxytocin response to the second sucrose solution was increased in a manner related to CCK doses. In separate experiments, NaCl solution (0.75 M) paired with CCK or the vehicle was applied to the tongue. The second NaCl solution applied 3 h after the first one facilitated oxytocin release both in the rats that had received CCK or the vehicle. The increase in plasma oxytocin, however, was significantly larger in CCK than in vehicle‐injected rats. In rats that had received the first sucrose solution paired with CCK, a second sucrose solution evoked a significantly larger increase in plasma oxytocin concentrations than a testing NaCl solution did. In rats that had received NaCl solution paired with CCK, a testing sucrose solution did not significantly change plasma oxytocin concentrations. These data suggest that the taste stimulus previously paired with iv CCK induces oxytocin release from the neurohypophysis in urethane‐anaesthetised rats.
Does Prolactin Mediate Induced Nest‐Building Behaviour in Pseudopregnant Gilts Treated with PGF2α?
Authors: Boulton  , Wickens  , Goode  , Lawrence  , Gilbert
Published: March 2003

Nest‐building behaviour occurs 6–24 h before parturition in pigs (gestation=116 days). Pseudopregnancy in pigs (induced with oestradiol valerate injections) lasts 50–80 days. We have shown that prostaglandin F2α (PG) administration on day 47 of pseudopregnancy induces nest‐building and changes to plasma prolactin, oxytocin, cortisol and progesterone similar to those seen before normal parturition. Peripheral prolactin has been proposed as a modulator of nest‐building. This study assessed nest‐building behaviour in prolactin‐deprived gilts. Jugular vein catheters were inserted on day 39 of pseudopregnancy and blood samples collected daily from days 40–48. Animals were injected im with either 40 mg bromocriptine in 2 ml 70% ethanol (n=8) or vehicle (n=7) at 17.00 h on day 46 and 09.00 h on day 47 of pseudopregnancy. PG (15 mg Lutalyse: Upjohn) was injected im at 11.00 h on day 47. Blood and behavioural samples were taken from 90 min before PG to 6 h post‐PG. Plasma prolactin increased in control but not bromocriptine treated animals following PG (P<0.05). Elevations in oxytocin, cortisol and progesterone (P<0.05) above pre‐PG concentrations were also seen, but of these only progesterone showed between group differences [greater (P<0.05) in control gilts on both days 47 and 48]. PG significantly (P<0.05) increased both the rate and proportion of total time spent performing straw/floor‐directed behaviours not including foraging (an index of nesting behaviour) in both treatment groups with no significant differences between groups. There were also no significant differences between groups in time spent performing pen fixture directed activities before or after PG. Bromocriptine suppressed the rise in prolactin concentrations after PG without suppressing nest‐building behaviour. We conclude that peripheral prolactin is not an essential component of the nest‐building complex in pigs.
Chronic Administration of Leptin into the Lateral Ventricle Induces Sexual Maturation in Severely Food‐restricted Female Rats
Authors: Gruaz Nadine M. , Lalaoui Meriem , Pierroz Dominique D. , Englaro Piera , Sizoneko Pierre C. , Blum Werner F. , Aubert Michel L.
Published: March 2003

In many species, delayed sexual maturation occurs when metabolic conditions are not satisfactory. Recently, leptin was shown to be involved in the regulation of food intake and body mass. Furthermore, leptin administration was shown to advance sexual maturation in mice and to rescue sexual function in adverse metabolic conditions. We examined plasma leptin levels in female rats during development and evaluated the role of leptin on sexual maturation in rats subjected to food restriction. In normal rats, plasma leptin levels were low at day 24 of life, then steadily increased during the juvenile period, reaching 740 ±56 pg/ml at 40 days at time of vaginal opening (VO) and further increasing by day 60 (957±73 pg/ml). Food restriction initiated at day 25 strongly impaired this increase, in proportion to the severity of the restriction. With a daily food intake reduced to 7–8 g/day, that permanently prevented VO, plasma leptin levels were very low at day 53 (169 ±67 pg/ml). Following switch to ad libitum feeding, plasma leptin reached high levels within 2 days (1577±123 pg/ml), and VO occurred 4 days later. If the severe food restriction was maintained and a central infusion of leptin (10 μg/day) was initiated, a significant decrease in body weight compared with vehicle‐infused controls was observed. In these conditions, VO occurred in eight out of the nine leptin‐treated rats, representing induction of the process of sexual maturation confirmed by increases in ovarian and uterine weights. This induction of sexual maturation exclusively results from a central effect of leptin because no leak of the icv administered leptin to the general circulation was observed. These data suggest that the rising plasma levels of leptin in the prepubertal period represent a signal to the brain indicating that the young animal is metabolically ready to go through the process of sexual maturation.
Stress‐Induced Changes of Gene Expression in the Paraventricular Nucleus are Enhanced in Spontaneously Hypertensive Rats
Authors: Imaki Toshihiro , Naruse Mitsuhide , Harada Shoko , Chikada Naoko , Nakajima Kishiko , Yoshimoto Takanobu , Demura Hiroshi
Published: March 2003

Heightened hypothalamic‐pituitary‐adrenal (HPA) axis responses have been implicated in hypertension in the spontaneously hypertensive rat (SHR), but the exact mechanisms involved are poorly understood. To determine changes in gene expression in SHR in the paraventricular nucleus (PVN), stress‐induced accumulation of CRF, CRF type 1 receptor (CRFR‐1) genes, and immediate‐early genes were examined using in situ hybridization in young (5 weeks old) and adult (12 weeks old) stroke‐prone SHR (SHRSP), compared with normotensive Wistar Kyoto (WKY) rats. Restraint stress‐induced accumulation of c‐fos, jun B, and NGFI‐B mRNA, and CRF hnRNA in the PVN was significantly higher in young and adult SHRSP than in WKY rats at 30 min, except for c‐fos in young rats. CRFR‐1 mRNA expression in the PVN was also significantly higher in adult SHRSP than in WKY rats at 120 min after stress onset. CRF mRNA was increased in response to stress in young SHRSP. The basal CRF mRNA level in the PVN was significantly lower in adult SHRSP than in WKY rats. Young SHRSP exhibit greater ACTH responses to stress without significant changes in plasma corticosterone concentrations. The adult SHRSP exhibited lower baseline concentrations of corticosterone and similar corticosterone response to stress with enhanced secretion of ACTH. Overall, these results demonstrated that stress‐induced activation of immediate early genes and CRF gene transcription in the PVN, and ACTH secretion is enhanced in early hypertensive, young, and adult SHRSP, suggesting that they are probably not the result of chronic alterations in blood pressure. The abnormal hypothalamic‐pituitary response to stress thus appears to be related to the development of hypertension.
Seasonal Neuroendocrine Rhythms in the Male Siberian Hamster Persist After Monosodium Glutamate‐Induced Lesions of the Arcuate Nucleus in the Neonatal Period
Authors: Ebling Francis P. , Arthurs Owen J. , Turney Ben W. , Cronin Anna S.
Published: March 2003

The aim of these experiments was to examine the role of the arcuate nucleus in the control of seasonal cycles of body weight, feed intake, moulting and reproduction in the Siberian hamster. The arcuate nucleus has previously been implicated as a central site where systemic feedback signals (e.g. leptin) might act to regulate feed intake and body weight, so it was predicted that hamsters with lesions of this structure would be unable to display the inhibitory effects of short days on these parameters. In the first series of studies, lesions that destroyed approximately 80% of the cells in the arcuate nucleus were produced by treating hamsters neonatally with monosodium glutamate (MSG; 4 mg/g body weight sc), and vehicle‐ and MSG‐treated males were raised from birth in long days (LD) or short days (SD). In hamsters raised in LD, the initial gain in body weight and testicular growth were significantly reduced by MSG treatment, however, growth rate and testis weight were still significantly greater than in vehicle‐ or MSG‐treated hamsters raised in SD. In the second study, hamsters treated neonatally with vehicle or MSG were raised in LD for 8 weeks and, subsequently, approximately half in each group were transferred to SD for 18 weeks. As expected, vehicle‐treated hamsters showed a characteristic decline in body weight when exposed to SD, while those remaining in LD continued to increase body weight. Feed intake decreased in parallel with the decline in body weight in SD, a complete moult to the white winter pelage occurred by 16 weeks in SD, and testicular regression occurred. Responses to SD also occurred in the MSG‐treated hamsters: body weight decreased in SD but increased in their lesioned litter mates remaining in LD, and feed intake paralleled body weight changes in these groups. The moult to winter pelage was significantly retarded in MSG‐treated hamsters transferred to SD. The testes were completely regressed in sham‐ and MSG‐treated hamsters exposed to SD, whereas testes weights in MSG‐treated hamsters maintained in LD were intermediate between those in vehicle‐treated hamsters in SD and LD. Thus, despite initial effects on growth, the MSG‐treated hamsters bearing substantial lesions of the arcuate nucleus were able to show appropriate responses to photoperiod, although not always of the same magnitude as the unlesioned controls. We conclude that feedback mechanisms operating via the arcuate nucleus are not the major regulators of seasonal cycles of body weight, feed intake, pelage and reproduction.
Interrelations between Hypothalamic Somatostatin and Proopiomelanocortin Neurons
Authors: Fodor Mariann , Csaba Zsolt , Epelbaum Jacques , Vaudry Hubert , Jegou Sylvie
Published: March 2003

Somatostatin receptors were visualized by [125I]‐Tyr0‐DTrp8‐somatostatin radioautography on 35% of arcuate neurons containing proopiomelanocortin (POMC) mRNA, as identified by in situ hybridization using a [35S] labelled riboprobe on 5 μm‐thick consecutive sections. Furthermore, double immunohistochemical staining revealed contacts of β‐endorphin or α‐MSH containing fibres with a majority of somatostatin perikarya in the anterior hypothalamic periventricular nucleus. Taken together, these data indicate that hypothalamic somatostatin and POMC neurons are interconnected. The results are discussed in term of intrahypothalamic control of GH secretion.
Ontogeny and Sexual Differentiation of Somatostatin Biosynthesis and Secretion in the Hypothalamic Periventricular‐Median Eminence Pathway
Authors: Murray  , Simonian  , Herbison  , Gillies
Published: March 2003

The biosynthesis of somatostatin (SRIH) in the hypothalamic periventricular nucleus (PeN) is sexually differentiated in neonatal and adult rats by virtue of the organizational and activational actions, respectively, of sex steroid hormones. Little information exists, however, on the normal pattern of maturation of these neurones or on how the sexually differentiated biosynthesis may relate to ontogenetic changes in somatostatin secretion during the neonatal and pubertal periods of development. Hence in the present study we determined the postnatal developmental profile of SRIH mRNA and peptide levels in the PeN–median eminence (ME) pathway as well as SRIH secretion, using an acute explant preparation, from the day of birth, through puberty and into adulthood in male and female rats. The results demonstrate that: (1) developmental sex differences in SRIH biosynthesis in PeN neurones occurred in an orderly cascade with differences observed for mRNA expression at postnatal day 5, for peptide content in the perikarya at postnatal day 10 and for peptide content in the nerve terminal (ME) by postnatal day 25; (2) sex differences in SRIH release were not evident prior to postnatal day 40; and (3) the developmental profile of SRIH biosynthesis in PeN neurones is unique compared with other hypothalamic (ventromedial nucleus) and extrahypothalamic (parietal cortex) populations. Specific developmental changes in the biosynthetic and secretory activity of the hypothalamic SRIH PeN–ME pathway may have a functional importance in the maturation of hypothalamic SRIH pathways involved in the regulation of GH secretion.
The Effect of Stressor Controllability on Stress‐Induced Neuropeptide mRNA Expression within the Paraventricular Nucleus of the Hypothalamus
Authors: Helmreich  , Watkins  , Deak  , Maier  , Akil  , Watson
Published: March 2003

Many stressors elicit changes in corticotrophin (CRH), enkephalin (ENK), and neurotensin (NT) mRNA levels within the medial parvocellular region of the paraventricular nucleus of the hypothalamus (mpPVN), and the pattern of changes in mRNA levels appears to depend on the physical characteristics of the stressor. We questioned whether psychologically distinct stressors would cause different patterns of neuropeptide mRNA expression within the PVN. Psychologically distinct stressors were created by employing a paradigm of escapable (controllable) vs non‐escapable (yoked) tail shock. An adult male rats could terminate the stress stimulus by performing wheel‐turning behaviour; his behaviour also terminated the stress for his yoked partner, who had no control over the termination of the shock. Four h post‐stress, brains were collected and processed for in‐situ hybridization histochemistry. Tail‐shock stress stimulated a significant increase in CRH, ENK, and NT mRNA levels within the mpPVN. The number of CRH identified neurones coexpressing AVP mRNA was also significantly elevated in both stress groups. Moreover, the pattern and magnitude of the stress‐induced increases in mRNA was similar in both stress groups. Additionally, no stress‐induced changes in CRH mRNA levels were observed in the central nucleus of the amygdala. In sum, two psychologically distinct stressors, escapable vs yoked tail shock stress, stimulated similar increases in CRH, NT, ENK, and AVP mRNA levels within the mpPVN. These results suggest that physical attributes of a stress, rather than psychological, may be the more important factors in determining the PVN mRNA response.
Evidence That Corticotropin‐Releasing Hormone Acts as a Growth Hormone‐Releasing Factor in a Primitive Teleost, the European Eel (Anguilla anguilla)
Authors: Rousseau  , Le Belle  , Marchelidon  , Dufour
Published: March 2003

The inhibitory control of growth hormone (GH) release by somatostatin (SRIH) has been conserved throughout vertebrate evolution. In contrast, the neuropeptides involved in the stimulatory control of GH vary according to species and/or physiological situations. We investigated the direct pituitary regulation of GH release in a primitive teleost, the European eel (Anguilla anguilla L.) at the juvenile stage. Short‐term serum‐free primary cultures of dispersed pituitary cells were used, and GH release was measured by an homologous radioimmunoassay. Whereas growth hormone‐releasing hormone (GHRH), gonadotropin‐releasing hormone (GnRH), thyrotropin‐releasing hormone (TRH), neuropeptide Y (NPY) and cholecystokinin (CCK) failed to induce any change in GH release, corticotropin‐releasing hormone (CRH) dose‐dependently stimulated GH release with a significant effect at 1 nM and a maximal effect (≥400% of controls at 24 h) at 100 nM. In agreement with our previous studies, PACAP also stimulated GH release but its maximal effect was lower than that ofCRH. Proopiomelanocortin (POMC)‐peptides, corticotropin (ACTH), melanotropin (α‐MSH), β‐endorphin) had no effect on GH release, at any dose tested (0.1–1000 nM), indicating that the stimulatory effect of CRH on GH release by somatotrophs was not mediated by CRH‐induced release of POMC‐peptides from corticotrophs and melanotrophs. The CRH antagonist, α‐helical CRH(9–41), significantly inhibited the stimulatory effect of CRH on GH release, suggesting the implication of specific CRH receptors related to mammalian ones. The stimulatory effect of CRH on GH release was reduced after 24 h of incubation, indicating a desensitization. In contrast, no desensitization to the inhibitory effect of SRIH was observed. SRIH inhibited CRH action in a dose‐dependent manner. The effect of SRIH was overriding, 1 nM SRIH being able to abolish the effect of 1000 nM CRH. In conclusion, in the eel, CRH stimulates GH release directly at the pituitary cell level. GH and cortisol secretions could interact in controlling several physiological functions such as metabolism and ion exchange. This study suggests that CRH may have played an important early role in vertebrates co‐ordinating the activation of various endocrine axes involved in metamorphosis, osmoregulation, stress and fasting. The stimulatory role of CRH on GH release may have been partially conserved during evolution, as it is found in some human physio‐pathological situations such as stress, fasting and depression.
Hyper‐Reactive Hypothalamo‐Pituitary‐Adrenocortical Axis in Rats Bred for High Anxiety‐Related Behaviour
Authors: Landgraf  , Wigger  , Holsboer  , Neumann
Published: March 2003

Psychiatric patients suffering from anxiety disorders or endogenous depression exhibit increased activity in their hypothalamo‐pituitary‐adrenocortical (HPA) axis. Recently, two Wistar rat lines, bred for high (HAB) and low (LAB) anxiety‐related behaviour on the elevated plus‐maze, were described as a unique psychopathological animal model (1). The present study focused on the HPA axis reactivity of HAB and LAB animals to an emotional stressor. Thus, adult male HAB and LAB animals, fitted with jugular vein catheters 5 days prior to the experiment, were exposed to an open arm of the elevated plus‐maze for 5 min. Whereas basal levels of ACTH and corticosterone were similar in both lines, HAB rats showed higher plasma concentrations at 5 and 15 min following stressor exposure (both hormones and both time points: P<0.01 vs LAB). Furthermore, increased basal (P<0.05 vs LAB) and stimulated (P<0.01 vs LAB) prolactin concentrations in HAB rats were found. In contrast to ACTH, corticosterone and prolactin, plasma oxytocin and vasopressin levels did not differ between HAB and LAB animals; oxytocin, but not vasopressin, responding to open arm exposure with a significant increase in both lines (P<0.05). In conclusion, particularly due to the association between inborn anxiety and HPA axis hyper‐reactivity, the HAB rat represents a promising animal model for further investigation of the relationship between emotional disturbance and neuroendocrine activity.
Expression Of Functional Growth Hormone Secretagogue Receptors In Human Pituitary Adenomas: Polymerase Chain Reaction, Triple In‐situ Hybridization And Cell Culture Studies1
Authors: Barlier  , Zamora  , Grino  , Gunz  , Pellegrini‐Bouiller  , Morange‐Ramos  , Figarella‐Branger  , Dufour  , Jaquet  , Enjalbert
Published: March 2003

We examined the expression of functional growth hormone secretagogue receptors (GHS‐R) in a series of 30 human pituitary adenomas–six secreting GH, three GH‐PRL, six prolactin (PRL), five adrenocorticotrophic hormone (ACTH), one thyroid stimulating hormone (TSH), four gonadotroph and five non‐secreting adenomas. By reverse transcriptase polymerase chain reaction (RT‐PCR), the coexpression of the two GHS‐R isoforms (Ia and Ib) was found in all the GH‐, GH‐PRL‐ and PRL‐secreting adenomas, and only in two out of three corticotroph, two out of four gonadotroph and one out of five non‐secreting tumours. They were absent in the TSH‐secreting adenoma. The PCR products of GHS‐R Ia and Ib were identical in size to those from two normal pituitaries. PCR cloning and sequencing of isoforms performed in two somatotroph adenomas revealed only two single, silent base mutations. Triple in‐situ hybridization showed colocalization of GHS‐R mRNA with messengers of GH and PRL, conjointly or separately, in individual cells of somatotroph, mammosomatotroph, and lactotroph adenomas. The presence of GHS‐R mRNA in cells expressing PRL mRNA is emphasized. In cultured cells from six somatotroph and two mammosomatotroph adenomas, the powerful GHS MK‐0677 stimulated GH release in a dose‐dependent manner, with maximal effect at 6 h. Contrarily, when GHRH was applied, only three somatotrophs and two mamosomatotrophs were stimulated. In the two mammosomatotrophs, the PRL response to MK‐0677 and to GHRH was similar to the GH response. An homologous desensitization of the GHS‐R and the GHRH receptor was observed 24 h after a first stimulation by a single dose of the corresponding agonist. Heterologous desensitization was not observed. Interestingly, MK‐0677 also stimulated, in a dose‐dependent way, the hormone release of cells from all tested lactotroph and corticotroph adenomas. The existence of a functional expression of GHS‐R in somatotroph, mammosomatotroph, lactotroph and corticotroph adenomas rises the question of the role played by GHS‐R in pituitary adenomas, particularly those not engaged in GH secretion.
Prolactin‐Releasing Peptide is Expressed in Afferents to the Endocrine Hypothalamus, but not in Neurosecretory Neurones
Authors: Morales T. , Hinuma S. , Sawchenko P. E.
Published: March 2003

Prolactin release from the anterior pituitary is regulated principally by inhibitory influences imparted by the tuberoinfundibular dopamine system. Stimulatory control is provided by several hypothalamic, peripheral and local factors. Recently a new peptide, prolactin releasing peptide (PrRP), showing prolactin‐secretagogue effects was discovered, synthesized and found to be expressed in brain. We have used histochemical and axonal transport methods to characterize the distribution of PrRP mRNA in the rat brain, and to identify possible pathways through which this factor might be delivered to the anterior lobe of the pituitary and thereby participate in the regulation of prolactin secretion. Analysis of histochemical preparations indicated that apart from a small population of cells in a non‐neurosecretory portion of the hypothalamus, PrRP mRNA is expressed exclusively in the caudal part of the nucleus of the solitary tract and in the caudal ventrolateral medulla. All medullary PrRP expressing cells could be immunolabeled for tyrosine hydroxylase, and none were found to stain for glucagon‐like peptide‐1, identifying them as comprising subsets of A2 and A1 noradrenergic neurones, respectively. Numerous PrRP‐expressing cells were retrogradely labelled following tracer injections in the paraventricular nucleus, while only a handful were backfilled following intravenous injections of tracer, indicating that this population issues substantial projections to the endocrine hypothalamus and meager ones to the median eminence and/or posterior pituitary. This conclusion was supported by the results of experiments in which the anterograde tracer, biotinylated dextran‐amine, was injected into the PrRP cell group in the nucleus of the solitary tract. These findings suggest that PrRP expressing neurones display a highly restricted distribution, and are in a position to regulate the output of particular cell types in the endocrine hypothalamus. Whether and how PrRP might be delivered to the anterior pituitary remains to be determined.
Melanocortin‐3 Receptor mRNA Expression in Pro‐Opiomelanocortin Neurones of the Rat Arcuate Nucleus
Authors: Jégou S. , Boutelet I. , Vaudry H.
Published: March 2003

The melanocortins α‐ and γ‐melanocyte‐stimulating hormones (α‐ and γ‐MSH) derive from the pro‐opiomelanocortin (POMC) precursor. Melanocortins exert a wide range of biological activities in the brain through activation of at least three distinct melanocortin receptor (MC‐R) subtypes. In order to determine whether POMC neurones can modulate their own activity, we looked for the possible expression of the MC3‐R gene in POMC‐positive cell bodies in the rat hypothalamus. In situ hybridization experiments revealed that the density of MC3‐R mRNA is particularly high in the arcuate nucleus which contains the main population of POMC neurones in the brain. The occurrence of MC3‐R mRNA in POMC‐positive cell bodies was demonstrated using a double‐labelling in situ hybridization technique. The proportion of POMC neurones expressing MC3‐R mRNA was significantly higher in the most rostral (43.5%) than in the most posterior part of the arcuate nucleus (8.2%). These results indicate that melanocortins likely exert a direct regulatory feedback on POMC neurones through activation of MC3‐R receptors. Our data also suggest that MC3‐R may be involved in the neuroendocrine responses induced by centrally administered melanocortins.
Gonadotropin‐Releasing Hormone Exhibits Circadian Rhythm in Phase with Arginine‐Vasopressin in Co‐Cultures of the Female Rat Preoptic Area and Suprachiasmatic Nucleus
Authors: Funabashi T. , Shinohara K. , Mitsushima D. , Kimura F.
Published: March 2003

To determine whether the suprachiasmatic nucleus can drive a circadian release of gonadotropin‐releasing hormone (GnRH) in the preoptic area, we measured the release of GnRH, arginine‐vasopressin and vasoactive intestinal polypeptide (VIP) in cocultures of the preoptic area and the suprachiasmatic nucleus at 2‐h intervals over a period of 120 h. The release of GnRH in cocultures exhibited a significant circadian rhythm in the presence of oestrogen but not in the absence of oestrogen. The period of the GnRH circadian rhythm was the same as that of the arginine‐vasopressin circadian rhythm, and different from the VIP circadian rhythm in each coculture. Furthermore, the peak phase of the GnRH rhythm occurred at the time same as that of the arginine‐vasopressin rhythm in each coculture. However, the peak phase of the GnRH rhythm was not always the same as that of the VIP rhythm. Administration of arginine‐vasopressin significantly increased GnRH release in single preoptic area cultures in the presence of oestrogen, but VIP did not. The result suggests that, in cocultures of the suprachiasmatic nucleus and the preoptic area, arginine‐vasopressin neurones drive the circadian release of GnRH in the presence of oestrogen. We suggest that arginine‐vasopressin neurones in the suprachiasmatic nucleus mediate the clock information to GnRH neurones in vivo as well.
Effects of Gonadal Steroids on Brain Serotonergic and Aromatase Activity During the Critical Period of Sexual Differentiation in Tilapia, Oreochromis mossambicus
Authors: Tsai C.‐L. , Wang L.‐H. , Chang C.‐F. , Kao C.‐C.
Published: March 2003

The effects of gonadal steroids on brain serotonin (5‐HT) and aromatase activity during the critical period of sexual differentiation were investigated in tilapia, Oreochromis mossambicus. Treatment of tilapia with 17β‐oestradiol (E2) between days 7 and 10 posthatching resulted in a significant increase in the female : male ratio as determined at day 90, and a significant reduction in brain 5‐HT content. Treatment between days 10 and 20, or between days 20 and 30, had no significant effect. Since the 5‐HT system may influence sexual differentiation, we examined the effects of para‐chlorophenylalanine (p‐CPA), a 5‐HT synthesis inhibitor. As with E2, treatment of tilapia with p‐CPA between days 7 and 10 posthatching resulted in a significant increase in the female : male ratio. Again, treatment between days 10 and 20, or between days 20 and 30, had no significant effect. Both p‐CPA and E2 significantly depressed brain aromatase activity when administrated between days 7 and 10, but not subsequently. In tilapia treated between days 7 and 10, the brain 5‐HT content was lowered by E2 to an extent similar to that seen with p‐CPA, which is consistent with the suggestion that the effect of E2 on sexual differentiation may be mediated by the 5‐HT system. Treatment of tilapia with 17α‐methyltestosterone (MT), by contrast, resulted in a reduction in the female : male ratio, and treatment was most effective when given between days 10 and 20. The period of maximal effect of MT upon sex ratio appears to coincide with the ability of MT to induce an increase in brain aromatase activity.
Nuclear Receptor Coactivators in Neuroendocrine Function
Authors: Tetel M. J.
Published: March 2003

Steroid hormones influence a variety of neuroendocrine events, including brain development, sexual differentiation and reproduction. Hormones elicit many of these effects by binding to neuronal steroid receptors, which are members of a nuclear receptor superfamily of transcriptional activators. However, the mechanisms by which activated steroid receptors regulate gene expression in brain are not well understood. Recently, a new class of proteins, known as nuclear receptor coactivators, have been found to dramatically enhance steroid receptor mediated transactivation of genes in vitro. Here, the proposed molecular mechanisms of how these coactivators enhance the transcriptional activity of steroid receptors are summarized. While much is known about the mechanisms of these coactivators in vitro, it is unclear how these cofactors function in hormone action in vivo or in the brain. This paper discusses some of the initial and enticing investigations into the role of these important coregulatory proteins in neuroendocrine events. Finally, some of the critical issues and future directions in nuclear receptor coactivator function in neuroendocrinology are highlighted.
Ultradian Corticosterone Rhythm and the Propensity to Behave Aggressively in Male Rats
Authors: Haller J. , Halasz J. , Mikics É. , Kruk M. R. , Makara G. B.
Published: March 2003

Ultradian fluctuations in plasma glucocorticoids have been demonstrated in a variety of species including humans. The significance of such rhythms is poorly known, although disorganized ultradian glucocorticoid rhythms have been associated with behavioural disorders. Here we report that ultradian glucocorticoid rhythms may establish the propensity to behave aggressively in male rats. Male rats were significantly more aggressive in the increasing phase of their corticosterone fluctuation when confronting a male intruder than counterparts in the decreasing phase of their corticosterone fluctuations facing such opponents. Corticosterone fluctuations were mimicked by a combination of treatments with the corticosterone synthesis inhibitor metyrapone and corticosterone. Again, males with increased plasma corticosterone levels were more aggressive than counterparts with a decreased plasma corticosterone concentration. These data suggest that the behavioural response to an aggressive challenge may vary in the same animal across the day due to the pulsating nature of corticosterone secretion. Aggressive behaviour is also episodic in humans; moreover, intermittent explosive behaviour is recognized as a psychological disorder. It can be hypothesized that a temporal coincidence between the occurrence of a challenge and a surge in plasma corticosterone concentration may be one of the factors that promote episodic aggressive outbursts.
Antagonistic Oxytocin/α2‐Adrenoreceptor Interactions in the Nucleus Tractus Solitarii: Relevance for Central Cardiovascular Control
Authors: Dı´az‐Cabiale Z. , Narváez J. A. , Garrido R. , Petersson M. , Uvnäs‐Moberg K. , Fuxe K.
Published: March 2003

The modulation of the central cardiovascular effects of α2‐adrenoceptor activation by oxytocin in the nucleus tractus solitarii has been evaluated by cardiovascular analysis and by quantitative receptor autoradiography. Microinjections in the nucleus tractus solitarii of a threshold dose of oxytocin effectively and significantly counteracted the vasodepressor and bradycardic actions of an ED50 dose of the α2−adrenoceptor agonist clonidine. The coinjection of a threshold dose of oxytocin with a threshold dose of clonidine did not produce any changes in the mean arterial pressure but a tachycardic response was observed. Receptor autoradiographical experiments showed that oxytocin (3 nM) significantly increased the Kd and Bmax values of [3H]p‐aminoclonidine binding sites in the nucleus tractus solitarii compatible with a possible antagonistic interaction with the α2‐adrenoceptors, and this effect was blocked by the presence of the specific oxytocin receptor antagonist 1‐deamino‐2‐D‐Tyr‐(OEt)‐4‐Thr‐8‐Orn‐oxytocin. These findings suggest the existence of an antagonistic oxytocin/α2‐adrenoceptor interaction in nucleus tractus solitarii that may be of relevance for the demonstrated modulation of α2−adrenoceptor induced cardiovascular responses by oxytocin.
Central Administration of Orexin A Suppresses Basal and Domperidone Stimulated Plasma Prolactin
Authors: Russell S. H. , Kim M. S. , Small C. J. , Abbott C. R. , Morgan D. G. A. , Taheri S. , Murphy K. G. , Todd J. F. , Ghatei M. A. , Bloom S. R.
Published: March 2003

Orexin immunoreactive fibres are abundant in the hypothalamus suggesting a neuroendocrine regulatory role. Intracerebroventricular (ICV) administration of orexin A suppressed plasma prolactin in male rats by 71% at 20 min post‐injection and 83% at 90 min post‐injection (P < 0.005 vs saline at both time points). To investigate whether this effect was through the tuberoinfundibular dopaminergic (TIDA) system, a supra‐maximal dose of domperidone, a dopamine receptor antagonist, was injected intraperitoneally (i.p.) prior to ICV injection of orexin A. ICV orexin A significantly suppressed domperidone (9 mg/kg)‐stimulated plasma prolactin levels, by up to 40% (i.p. domperidone + ICV orexin A 3 nmol 34.5 ± 7.4 ng/ml and i.p. domperidone + ICV orexin A 20 nmol 43.5 ± 4.3 ng/ml, both P < 0.005 vs i.p. domperidone + ICV saline 57.9 ± 2.7 ng/ml). Orexin A, 100 nM, significantly stimulated release of neurotensin, vasoactive intestinal polypeptide, somatostatin, corticotropin releasing factor and luteinizing hormone releasing hormone, but had no effect on release of dopamine, thyrotropin releasing hormone (TRH), vasopressin or melanin‐concentrating hormone from hypothalamic explants in vitro. Orexin A did not alter basal or TRH stimulated prolactin release in dispersed pituitary cells harvested from male rats. The data suggest that ICV administration of orexin A suppresses plasma prolactin in part through a pathway independent of the dopaminergic system.
Intracerebroventricular Administration of the Rat Growth Hormone (GH) Receptor Antagonist G118R Stimulates GH Secretion: Evidence for the Existence of Short Loop Negative Feedback of GH
Authors: Nass R. , Toogood A. A. , Hellmann P. , Bissonette E. , Gaylinn B. , Clark R. , Thorner M. O.
Published: March 2003

Pulsatile growth hormone (GH) secretion is regulated by three hypothalamic factors, growth hormone‐releasing hormone (GHRH), somatostatin and the natural ligand for the GH secretagogue receptor (Ghrelin). These factors and their effects are, in turn, affected by short loop feedback of GH itself. To test the hypothesis that hypothalamic GH receptors are involved in the ultradian rhythmicity of pituitary GH secretion, the rat GH receptor antagonist (G118R) was administered to adult male rats by intracerebroventricular (i.c.v.) injection and the effects on spontaneous GH secretion were studied. Normal saline was administered i.c.v. to eight control rats. Mean GH concentrations increased significantly in the rat treated with G118R compared to rats that received normal saline. The pulse amplitude rose by a mean of 33.3 ng/ml and the total area under the curve increased by a mean of 15 061 ng/ml × min. The number of GH peaks did not change significantly following G118R. These data suggest that GH regulates its own secretion by acting directly on hypothalamic GH receptors.