bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

SummaryTwo‐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

SummaryRapid 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

SummaryWe 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.
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

AbstractPulsatile 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.
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

AbstractThe 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

AbstractSteroid 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

AbstractUltradian 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

AbstractThe 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

AbstractOrexin 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

AbstractPulsatile 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.
Photoresponsive Fischer 344 Rats are Reproductively Inhibited by Melatonin and Differ in 2‐[125I] Iodomelatonin Binding from Nonphotoresponsive Sprague‐Dawley Rats
Authors: Heideman P. D. , Bierl C. K. , Sylvester C. J. 
Published: March 2003

AbstractMany temperate‐zone species use photoperiod as an environmental cue to regulate reproductive timing. Strains of laboratory rats differ in their responsiveness to photoperiod, with the Fischer 344 (F344) strain being the most responsive known. F344 rats and closely related strains that differ in photoresponsiveness may be useful models to study the mechanisms and genetic basis for photoresponsiveness. We tested two hypotheses: (i) that melatonin mediates photoresponsiveness in F344 rats, as is the case in all other mammals tested, and (ii) that the location, abundance, or affinity of melatonin receptors, as estimated by the amount and location of binding of the radioligand 2‐[125I]‐iodomelatonin (IMEL) in the brain, might cause variation in photoresponsiveness among rat strains. Melatonin injections 1 h before lights off in a stimulatory photoperiod (L14 : D10) induced reproductive inhibition and reduced weight gain in a manner similar to short days of L8 : D16, while injections of ethanolic saline vehicle did not. Interestingly, melatonin injections administered during an inhibitory photoperiod (L10 : D14) caused greater inhibition of both reproduction and weight gain than short photoperiod alone. Pinealectomized F344 rats implanted subcutaneously with melatonin in a silastic capsule did not differ in testis size or body weight from controls with blank implants. The brains and pars tuberalis of the pituitary from photoresponsive F344 rats and nonphotoresponsive Harlan Sprague‐Dawley (HSD) rats were processed for autoradiography using IMEL. We found significantly higher specific IMEL binding in the anterior and posterior regions of the paraventricular nucleus of the thalamus (PVNt) and reuniens nucleus of the thalamus of F344 rats than in the same areas in HSD rats. There were no differences between strains in specific IMEL binding in the medial PVNt, anteroventral and anterodorsal nucleus of the thalamus, suprachiasmatic nucleus, or the pars tuberalis. These results indicate that melatonin mediates photoresponsiveness in F344 rats. In addition, they provide support for the hypothesis that F344 rats may be photoresponsive due to differences from other strains in the location, density, or affinity of melatonin receptors.
Evidence for Direct Involvement of β‐Catenin in Phorbol Ester‐Induced Neurite Outgrowth in GT1‐1 Hypothalamic Neurones
Authors: Sun W. , Lee H. , Choe Y. , Cho S. , Kim D. H. , Kim K. 
Published: March 2003

AbstractGonadotropin‐releasing hormone (GnRH) is a pivotal neuroendocrine regulator controlling reproductive functions. However, the scattered distribution of GnRH neurones in the mammalian brain has hindered studies on the development and differentiation of GnRH neurones. In the present study, we used the immortalized GnRH‐producing GT1‐1 cells to examine whether activation of protein kinase C (PKC) pathway with 12‐O‐tetradecanoyl‐13‐acetate (TPA) induces morphological and functional differentiation of GnRH neurones. TPA induced neurite outgrowth and inhibited proliferation of GT1‐1 cells that were specifically antagonized by cotreatment of PKC inhibitor, calphostin C. The functional significance of TPA‐induced differentiation of GT1‐1 cells was manifested in part by the changes in the effects of γ‐aminobutyric acid (GABA) on intracellular Ca2+ levels. In untreated GT1‐1 cells, activation of GABA‐A receptor with 10 µM muscimol increased intracellular Ca2+ levels, whereas such stimulatory effects disappeared in GT1‐1 cells bearing neurites. Accordingly, muscimol could not stimulate GnRH release in TPA‐treated GT1‐1 cells. To elucidate the molecular mechanism underlying TPA‐induced neurite outgrowth, we performed differential display reverse transcription‐polymerase chain reaction. Among several genes that are affected by TPA treatment, we found a significant induction of β‐catenin mRNA expression. Along with the rapid induction of β‐catenin protein levels, we observed that β‐catenin was reallocated from cell–cell adhesion sites to the growth cones within 3 h of TPA treatment. Transient transfection studies with green fluorescent protein as a reporter gene demonstrated that β‐catenin overexpression alone can promote neurite outgrowth in GT1‐1 cells. Moreover, TPA was found to increase the transcription‐activational roles of β‐catenin. Together, these data provide evidence that β‐catenin is involved in the TPA‐induced functional differentiation of immortalized GnRH neurones.
Regulation by Osmotic Stimuli of Galanin‐R1 Receptor Expression in Magnocellular Neurones of the Paraventricular and Supraoptic Nuclei of the Rat
Authors: Burazin T. C. D. , Larm J. A. , Gundlach A. L. 
Published: March 2003

AbstractNeurones of the supraoptic nucleus (SON) and the magnocellular and parvocellular divisions ofthe paraventricular nucleus (PVN) express galanin and [125I]galanin binding sites. Although the precise role(s) of galanin in these different cell populations is still unknown, it has been shown to regulate the electrophysiological, neurochemical and secretory activity of magnocellular neurones.In light of the well‐described effects of hyperosmotic stimuli, such as salt‐loading on magnocellular neurone activity and galanin synthesis and release, and the recent identification of multiple galanin receptors in brain, this study assessed the possible regulation of galanin receptor subtype expression in the PVN/SON of salt‐loaded, dehydrated and food‐deprived rats. Gal‐R1 mRNA was abundant in the SON (and magnocellular PVN) of control rats and levels were increased in these same cells after 4 days of salt‐loading (2% NaCl solution as drinking water) or water deprivation. The density of specific [125I]galanin(1–29) binding and the intensity of Gal‐R1‐like immunostaining were also increased in the characteristically enlarged, magnocellular neurones of the PVN and SON after these treatments. Gal‐R2 mRNA was detected in the parvocellular PVN, but levels were not altered by the hyperosmotic stimuli. In contrast, food deprivation (4 days), which has been shown to reduce levels of several neurochemical markers in magnocellular neurones, produced a significant reduction in Gal‐R1 (and galanin) mRNA levels in the SON, but no consistent change in neurone size, [125I]galanin binding levels, or Gal‐R1 immunostaining. Along with previous findings from this and other laboratories, these data suggest that the expression of galanin and Gal‐R1 receptors is regulated in parallel with functional and morphological changes in hypothalamic magnocellular neurones. Furthermore, Gal‐R1 immunoreactivity was primarily detected in somatodendritic areas and thus galanin may influence the activity of these cells, particularly vasopressin synthesis/release, via autocrine or paracrine activation of Gal‐R1 receptors, especially during long‐lasting stimulation.