bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct measurement of glutathione in epidermal cells of intact Arabidopsis roots by two‐photon laser scanning microscopy
Authors: Meyer A. J. , Fricker M. D.
Published: March 2003

Two‐photon laser scanning microscopy (TPLSM) was used to directly measure glutathione (GSH) as its fluorescent glutathione S‐bimane conjugate (GSB) in developing root hair cells (trichoblasts) and non‐root hair cells (atrichoblasts) of intact Arabidopsis roots. In comparison to confocal microscopy, TPLSM showed more detail deep within the tissue with less signal attenuation. The total level of GSB labelling reached a plateau after 60 min in both trichoblasts and atrichoblasts, reflecting depletion of GSH. GSB was formed initially in the cytoplasm and was subsequently transported into the vacuole. The volume ratio of vacuole to cytoplasm was determined using the Cavalieri estimator of volume and used to calculate the amount of GSB per volume of cytoplasm in each cell type. At the end of the time‐course the cytoplasmic concentration of GSB was 2.7 ± 0.5 mm (n = 5) in trichoblasts and 5.5 ± 0.8 mm (n = 5) in atrichoblasts. In trichoblasts this value represents the initial concentration of GSH in the cytoplasm. Labelling of roots with monochlorobimane (MCB) on ice led to the formation of GSB in the cytoplasm, but prevented vacuolar sequestration. After washing prelabelled roots and transfer to room temperature, vacuolar transport resumed. Although no free MCB was present the total amount of GSB in atrichoblasts increased further, indicating that the higher values recorded in the atrichoblasts might reflect additional symplastic transport and sequestration of GSB from neighbouring cells.
Cathodoluminescence imaging and spectroscopy by near‐field detection
Authors: Bubendorff J. L. , Pastré D. , Troyon M.
Published: March 2003

Using near‐field techniques, we have developed an experimental set‐up for spatially resolved cathodoluminescence (CL) spectroscopy and monochromatic imaging. It combines a scanning near‐field optical/force microscope with a scanning electron microscope equipped with a field emission gun. The potentialities of this scanning near‐field cathodoluminescence microscope are demonstrated on two kinds of sample: an indented MgO crystal and AlGaN/GaN quantum wells grown on GaN/sapphire. Monochromatic CL imaging allows a clear distinction between the emission of quantum wells and the GaN substrate, and for the MgO crystal, the localization on the slip bands, near the indentation, of luminescent centres emitting at 450 nm.
Off‐axis electron holography of patterned magnetic nanostructures
Authors: Dunin‐Borkowski R. E. , Mccartney M. R. , Kardynal B. , Parkin S. S. P. , Scheinfein M. R. , Smith D. J.
Published: March 2003

Magnetization reversal processes in lithographically patterned magnetic elements that have lateral dimensions of 70–500 nm, thicknesses of 3–30 nm and a wide range of shapes and layer sequences have been followed in situ using off‐axis electron holography in the transmission electron microscope. This technique allows domain structures within individual elements and the magnetic interactions between them to be quantified at close to the nanometre scale. The behaviour of 30 nm‐thick Co elements was compared with that of 10 nm‐thick Ni and Co elements, as well as with Co/Au/Ni trilayers. The hysteresis loops of individual elements were determined directly from the measured holographic phase images. The reproducibility of an element's domain structure in successive cycles was found to be affected by the out‐of‐plane component of the applied magnetic field and by the exact details of its initial magnetic state. Close proximity to adjacent elements led to strong intercell coupling, and remanent states with the in‐plane magnetic field removed included domain structures such as solenoidal (vortex) states that were never observed during hysteresis cycling. Narrow rectangular bars reversed without the formation of end domains, whereas closely separated magnetic layers within individual elements were observed to couple to each other during field reversal.
A comparison of optical geometries for combined flash photolysis and total internal reflection fluorescence microscopy
Authors: Conibear P. B. , Bagshaw C. R.
Published: March 2003

Total internal reflection fluorescence (TIRF) microscopy, used in conjunction with flash photolysis, provides a useful way of investigating the kinetics of macromolecular interactions. We compare different TIRF optical geometries to establish an optimal combination. Excitation light was introduced via four different arrangements: (1) a prism positioned on the microscope optical axis, (2) an offset prism with propagation through a silica slide trans to the objective lens, (3) an offset prism with propagation through a silica coverslip cis to a water‐immersion objective lens and (4) a prismless arrangement using a high NA oil‐immersion objective lens. Photolysis was achieved using a Xe flash lamp and a customised silica condenser lens. Single myosin molecules labelled with a Cy3 fluorophore were used as a test sample. Although the offset trans prism gave the best signal‐to‐background ratio, a customised thin rhombic prism incorporated, on axis, into the flash condenser assembly was almost as good and was more practical for scanning multiple fields. An oil‐immersion lens gave the brightest image for sample depths < 30 µm but above this limit, a water‐immersion lens was better. The prismless arrangement may offer advantages in other situations but it is important to check the actual numerical aperture of the objective lens.
Microstructural analysis of a FeAl/quasicrystal‐based composite prepared using a focused ion beam miller
Authors: Cairney J. M. , Munroe P. R. , Sordelet D. J.
Published: March 2003

A composite consisting of a brittle multiphase matrix containing both an Al‐based quasicrystalline phase (ψ) and an ordered body centred cubic phase (β) and a relatively ductile ordered body centred cubic intermetallic FeAl phase has been developed as an abrasive wear‐resistant coating material. It is applied as a 500 μm thick layer onto stainless steel substrates through plasma spray processing. The microstructure of such materials can be readily examined by optical and scanning electron microscopy, but the inherent difficulty of preparing transmission electron microscope (TEM) samples has inhibited higher resolution studies. However, the relatively recent development of the focused ion beam (FIB) miller as a tool in materials science provides a method ideal for the preparation of TEM specimens of these materials. In this study a coating consisting of a mixture of an Al–Cu–Fe based quasicrystal and FeAl+Cr was deposited on to a 304 stainless steel substrate. TEM specimens were prepared using a FIB and subjected to detailed microstructural characterization. The structure consisted of elongated bands of a FeAl phase about 100 nm in width and several micrometres in length, which enclosed more equiaxed regions about 1 μm in diameter that consisted of fine mixtures of quasicrystal and two Al‐Fe‐Cu phases isostructurally related to FeAl.
Microcracking mechanism in a SiCf–SiBC composite creep‐tested in argon
Authors: Darzens S. , Chermant J. L. , Vicens J.
Published: March 2003

This paper addresses the creep behaviour of a woven SiCf–SiBC composite, tested in tension under a partial pressure of argon, between 1273 K and 1473 K. It appears that the creep strain begins from 1273 K and becomes larger at higher temperatures. Moreover, the shapes of the creep curves led to the assumption of the existence of two competing deformation mechanisms depending on the temperature domain. The creep mechanism involved is microcrack damage‐creep. From higher resolution studies at higher scales (scanning electron microscopy, transmission electron microscopy (TEM) and high resolution electron microscopy (HREM)), many types of damage were observed, for example matrix microcracking, fibre/matrix debonding and fibre/matrix sliding. The observations via TEM and HREM enabled us to specify the existence or not of the classical creep mechanism of the constituents of the composite, and also to characterize the behaviour and the role of the different interfaces and especially of the pyrocarbon interphase. These multiscale observations will be discussed in order to highlight the creep‐damage mechanism as a function of temperature of the SiCf‐SiBC composites.
A simple contrast enhancement by potassium permanganate oxidation for Lowicryl K4M ultrathin sections prepared by high pressure freezing/freeze substitution
Authors: Sawaguchi A. , Ide S. , Goto Y. , Kawano J.‐I. , Oinuma T. , Suganuma T.
Published: March 2003

A simple contrast enhancement method is presented for Lowicryl K4M ultrathin sections prepared by high pressure freezing/freeze substitution. The sections were treated with an acidified potassium permanganate oxidizing solution followed by uranyl acetate and lead citrate staining. The method, designated KMnO4–UA/Pb staining, provided a much greater contrast in electron microscopy than conventional UA/Pb staining. In detail, the visibility of plasma membrane was especially improved and the nuclear heterochromatin, mitochondria and cytoplasmic ribosomes showed an adequate increase in electron density. In the mucous cells of rat Brunner's glands, the Golgi cisternae were well defined with the KMnO4–UA/Pb staining. Interestingly, the membranes of the intermediate compartments were moderately reactive to the KMnO4–UA/Pb staining, whereas the cis and the trans compartments were only faintly stained. It should be emphasized that the KMnO4 oxidation following colloidal gold labelling did not cause a remarkable reduction of immunogold labelling and the enhanced contrast helped us to examine the gold particles with high accuracy. This contrast enhancement method is highly promising, with the potential to become a useful tool for histochemical investigation, including immunocytochemistry with the Lowicryl K4M ultrathin sections prepared by high pressure freezing/freeze substitution techniques.
Microstructural analysis of silicon carbide monofilaments
Authors: Shatwell R. A. , Dyos K. L. , Prentice C. , Ward Y. , Young R. J.
Published: March 2003

In the development of monofilaments, a good understanding of the process/property relationships is essential. Transmission electron microscopy (TEM) is a powerful tool but too slow and expensive to be used routinely. Alternative, cheaper techniques have therefore been investigated. The microstructures of three SiC monofilaments (DERA Sigma SM1140+, Textron SCS‐6 and Ultra‐SCS) and some experimental samples were studied using a combination of TEM, electron microprobe analysis, Raman microprobe analysis, thermo‐gravimetric analysis (TGA) and differential scanning calorimetry (DSC). It was found that the Raman technique was complementary to TEM and easily identified the presence of amorphous C and Si. These could not be seen by electron or X‐ray diffraction techniques. DSC indicated the presence of free Si in the DERA Sigma SM1140+ monofilament by a distinctive peak at ∼1400 °C. TGA showed the reaction of monofilament components with gaseous species. The Textron SCS‐6 and Ultra species lost weight as C was oxidized to gaseous CO. By contrast, the Sigma monofilament gained weight from formation of SiO2 from the free Si. The separations of the transverse optical phonon peaks in the Raman spectra were correlated with the density of stacking faults in the SiC crystallites. This was similar in all monofilaments. Analysis of the polarization of the Raman scattering gave information on the orientation of crystallites. The crystallites in SM1140+ and SCS‐6 were orientated predominantly with the <111> parallel to the radius. Preliminary interpretation of the polarized Raman scattering from Ultra‐SCS indicated more than one orientation of crystallite. One possibility was a mixture of <111> and <110> directions parallel to the radius.
The role of tip plasmons in near‐field Raman microscopy
Authors: Milner R. G. , Richards D.
Published: March 2003

The enhancement in electric field strength in the vicinity of a metal tip, through the excitation of plasma modes in the tip, is investigated using the finite difference time domain method; such tip enhancement has significant potential for application in scanning near‐field Raman microscopy. To represent an experimentally realistic geometry the near‐field probe is described by a conical metal tip with a spherical apex, with radii 20 nm and 200 nm considered, in close proximity to a glass substrate. Illumination through the substrate is considered, both at normal incidence and close to the critical angle, with the polarization in the plane of incidence. By modelling the frequency dependent dielectric response of the metal tip we are able to highlight the dependence on the scattering geometry of the nature of the electromagnetic excitations in the tip. In particular, the strongest electric field enhancement with the greatest confinement occurs for the excitation of modes localized at the tip apex, excited only for off‐normal incidence. Bulk modes excited in the tip also produce enhancement, although over a larger area and with significantly less enhancement than that of the localized modes; however, the excitation of bulk modes is independent of the angle of incidence.
Non‐regularly shaped plasmon resonant nanoparticle as localized light source for near‐field microscopy
Authors: Kottmann J. P. , Martin O. J. F. , Smith D. R. , Schultz S.
Published: March 2003

We study numerically two‐dimensional nanoparticles with a non‐regular shape and demonstrate that these particles can support many more plasmon resonances than a particle with a regular shape (e.g. an ellipse). The electric field distributions associated with these different resonances are investigated in detail in the context of near‐field microscopy. Depending on the particle shape, extremely strong and localized near‐fields, with intensity larger than 105 that of the illumination wave, can be generated. We also discuss the spectral dependence of these near‐fields and show that different spatial distributions are observed, depending which plasmon resonance is excited in the particle.
Dislocation mobility and electronic effects in semiconductor compounds
Authors: Vanderschaeve G. , Levade C. , Caillard D.
Published: March 2003

In situ transmission electron microscopy experiments provide a unique way to investigate in real time the dislocation behaviour at a microscopic scale and to decide which elementary process controls the dislocation glide in semiconductors. In this review the experimental results obtained on different semiconductors are presented and discussed. Particular attention is devoted to the radiation‐enhanced glide process.
A new approach for high‐pressure freezing of primary culture cells: the fine structure and stimulation‐associated transformation of cultured rabbit gastric parietal cells
Authors: Sawaguchi A. , Mcdonald K. L. , Karvar S. , Forte J. G.
Published: March 2003

A newly designated procedure for high‐pressure freezing of primary culture cells provided excellent ultrastructure of rabbit gastric parietal cells. The isolated parietal cells were cultivated on Matrigel‐coated aluminium plates for conventional subsequential cryoimmobilization by high‐pressure freezing. The ultrastructure of different organelles (Golgi apparatus, mitochondria, multivesicular bodies, etc.) was well preserved compared to conventional chemical fixation. In detail, actin filaments were clearly shown within the microvilli and the subapical cytoplasm. Another striking finding on the cytoskeleton system is the abundance of microtubules among the tubulovesicles. Interestingly, some microtubules appeared to be associating with tubulovesicles. A large number of electron‐dense coated pits and vesicles were observed around the apical membrane vacuoles in cimetidine‐treated resting parietal cells, consistent with an active membrane uptake in the resting state. Immunogold labelling of H+/K+‐ATPase was seen on the tubulovesicular membranes. When stimulated with histamine, the cultured parietal cells undergo morphological transformation, resulting in great expansion of apical membrane vacuoles. Immunogold labelling of H+/K+‐ATPase was present not only on the microvilli of expanded apical plasma membrane vacuoles but also in the electron‐dense coated pits. The present findings provide a clue to vesicular membrane trafficking in cultured gastric parietal cells, and assure the utility of the new procedure for high‐pressure freezing of primary culture cells.
CRFergic Innervation Of the Paraventricular Nucleus of the Rat Hypothalamus: A Tract‐Tracing Study
Authors: Champagne Danielle , Beaulieu Johanne , Drolet Guy
Published: March 2003

It has been reported that corticotropin‐releasing factor (CRF) may regulate its own biosynthesis in the paraventricular nucleus of the hypothalamus (PVH). Whether this CRF autoregulation is mediated by local circuitry or from extra‐PVH CRF neuronal fibers terminating on CRF perikarya within the PVH is unknown. In the present study, we sought to determine the origin(s) of this CRF innervation using retrograde transport of wheat germ‐conjugated‐gold particles (WGA‐apoHRP‐Au) combined with immunohistochemistry for CRF. The rats also received colchicine (100 μg, icv) 5–7 days after tracer injection and were perfused 24 h later. Results of retrograde labeling with pressure injections of WGA‐apoHRP‐Au centered to PVH and subsequent immunohistochemical staining for CRF demonstrated numerous retrogradely labeled CRF neurons in the perifornical hypothalamic nucleus (PeF), the dorsolateral hypothalamic area (DA) (medial and lateral portions) and the dorsomedial nucleus of the hypothalamus (DMH). Smaller groups of CRF‐ir neurons that were retrogradely labeled were found in the bed nuclei of the stria terminalis (BnST), the Barrington’s nucleus (Bar) and the dorsal raphé (DR). These CRFergic pathways to the PVH may represent an anatomical substrate underlying the function of the stress‐integrative PVH neurons in the autonomic, behavioral and neuroendocrine regulation during the stress response, including CRF autoregulation.
Effect of Growth Hormone‐Releasing Peptide‐2 (GHRP‐2) and GH‐Releasing Hormone (GHRH) on the cAMP Levels and GH Release from Cultured Acromegalic Tumours
Authors: Chen Chen , Pullar Michael , Loneragan Kylie , Zhang Jin , Clarke Iain J
Published: March 2003

There is a difference between the sheep and rat somatotrophs in the response to growth hormone‐releasing peptide‐2 (GHRP‐2), which raises the question of what the response may be in human somatotrophs. In the present study, cells were obtained from seven human acromegalic tumours and the effects of GHRP‐2 were studied. Cells were dissociated and kept in primary culture for 1–3 weeks before experimentation. Application of GHRP‐2 for 30 min induced a significant increase in GH secretion from the cultured cells from all seven tumours whereas human GH‐releasing hormone (hGHRH) at a dose of 10 nM induced a significant GH release in only four of seven tumours. The intracellular levels of cAMP in all seven tumours were significantly increased by both 10 nM GHRP‐2 and GHRH, but the response to GHRH was significantly higher than the response to GHRP‐2. The adenylyl cyclase inhibitor, MDL 12330A, blocked the effect of GHRH and GHRP‐2 on intracellular cAMP levels, whereas the Ca2+ channel blocker Co2+ (0.5 mM) did not attenuate the cAMP response. For the tumours in which GH secretion was increased by GHRH and GHRP‐2, the cAMP antagonist Rp‐cAMP blocked the GH response to GHRH but not to GHRP‐2. When a protein kinase A (PKA) inhibitor (H89) was applied, GHRH stimulated GH release was blocked, but cAMP accumulation was not affected. The response to GHRP‐2 was not altered by H89. Calphostin C [a protein kinase C (PKC) inhibitor] reduced the effect of GHRP‐2 on the secretion of GH but did not affect the response to GHRH. Both GHRH and GHRP‐2 increased the intracellular Ca2+ concentration in a concentration‐dependent manner. We conclude that (1) GHRH increases GH secretion from human GH tumours via the cAMP pathway whereas GHRP‐2 increases GH secretion mainly via the PKC pathway; (2) GHRH increases cAMP (without GH release) in a subset of tumours whereas GHRP‐2 increases cAMP levels (slightly) and GH secretion in all tumours; and (3) GHRP‐2 and GHRH do not act on the same receptor on human somatotrophs derived from acromegalic tumours.
Dendritic Release of Vasopressin and Oxytocin
Authors: Ludwig
Published: March 2003

In addition to the release of neurotransmitters from their axon terminals, several neuronal populations are able to release their products from their dendrites. The cell bodies and dendrites of vasopressin‐ and oxytocin‐producing neurones are mainly located within the hypothalamic supraoptic and paraventricular nuclei and neuropeptide release within the magnocellular nuclei has been shown in vitro and in vivo. Local release is induced by a range of physiological and pharmacological stimuli, and is regulated by a number of brain areas; locally released peptides are mainly involved in pre‐ and postsynaptic modulation of the electrical activity of magnocellular neurones. Spatial and temporal differences between peptide release within the nuclei and that from the distant axonal varicosities indicate that the release mechanisms are at least partially independent, supporting the hypothesis of locally regulated dendritic release of vasopressin and oxytocin. In this respect, magnocellular neurones show similarities to other neuronal populations and thus autoregulation of neuronal activity by dendritic neuromodulator release may be a general phenomenon within the brain.
A Possible Role of the Pineal Gland in Acute Immobilization‐Related Suppression of Naloxone‐Induced LH Release in Ovariectomized Estrogen‐Primed Rats
Authors: Chiba Atsuhiko , Akema Tatsuo , Iigo Masayuki , Nagami Yuta , Kimura Fukuko , Toyoda Jun‐ichi
Published: March 2003

It has been recently reported that acute immobilization stress almost completely suppresses the luteinizing hormone (LH) release induced by naloxone, a μ‐opioid antagonist, in ovariectomized estrogen‐primed rats. The present study examined the possible involvement of the pineal gland in the acute immobilization‐related suppression of the naloxone‐induced LH release. An intraventricular (ICV) injection of 15 μg naloxone produced an abrupt increase in circulating LH concentrations in non‐stressed rats. The naloxone‐induced LH release was completely eliminated when tested 60 min after the end of a 30 min session of acute immobilization. The same stress conditions did not affect LH‐releasing hormone (LHRH)‐induced LH release, suggesting that the stress‐related suppression of the naloxone‐induced LH release was a suprapituitary event. In chronically‐pinealectomized rats, but not in sham‐pinealectomized rats, naloxone injected 60 min after the end of the stress session evoked a significant increase in serum LH concentrations. However, naloxone injected ICV during the acute immobilization did not elicit LH release in either pinealectomized or sham‐operated rats. Under non‐stressed conditions, the LH secretory response to naloxone was similar in pinealectomized and sham‐operated animals. The same stress (30 min immobilization) significantly increased pineal melatonin content as well as plasma melatonin concentrations in rats bearing intact pineal glands, indicating that stress actually affected the pineal function. These results provide evidence for a role of the pineal in the suppression of the LH response to naloxone after stress, but not during stress.
Influence of Neonatal Rearing Conditions on Stress‐Induced Adrenocorticotropin Responses and Norepinepherine Release in the Hypothalamic Paraventricular Nucleus
Authors: Liu  , Caldji  , Sharma  , Plotsky  , Meaney
Published: March 2003

Postnatal rearing conditions influence the development of hypothalamic‐pituitary‐adrenal (HPA) responses to stress in the rat. Thus, postnatal handling dampens HPA responsivity to stress, while prolonged periods of maternal separation have the opposite effect. HPA responses to stress are initiated by the release of corticotropin‐releasing factor and/or arginine vasopressin from the neurones of the paraventricular nucleus of the hypothalamus (PVNh). A major source of input to the PVNh arises from brainstem noradrenergic neurones with signalling occurring via α1 adrenoreceptors. We examined the noradrenergic response to stress in the PVNh in adult animals exposed to daily periods of handling or maternal separation over the first 2 weeks of life using microdialysis in conscious animals. Maternal separation increased, while handling greatly decreased and norepinepherine responses to restraint stress in the PVNh as compared to non‐handled controls; the same pattern was observed for plasma adrenocorticotropic hormone (ACTH) responses to stress. Rearing condition did not affect either α1 or α2 receptor levels in the PVNh. However, α2 receptor binding levels in the noradrenergic cell body regions of the locus coeruleus and the n. tractus solitarius were significantly increased in handled animals. These α2 receptors are principally located on noradrenergic neurones (i.e. autoreceptors) and inhibit noradrenaline release at terminal sites. The effects on α2 receptor levels could serve as a mechanism for the differences in stress‐induced noradrenaline levels in the PVNh and in HPA activity among handled vs non‐handled and maternal separation animals. Thus, early life events may serve to influence the differentiation of noradrenergic neurones and thus alter HPA responses stress in adulthood.
Interleukin‐3 and Interleukin‐6 Stimulate Bovine Adrenal Cortisol Secretion Through Different Pathways
Authors: Michl  , Beikler  , Engelhardt  , Weber
Published: March 2003

Several interleukins have been reported to play a major role in the regulation of steroid secretion at all three levels of the hypothalamic‐pituitary‐adrenal axis. The objective of this study was to investigate the effect of interleukin‐3 (IL‐3) and interleukin‐6 (IL‐6) on cortisol secretion of bovine adrenocortical cells in primary culture under serum‐free conditions. Both IL‐3 and IL‐6 stimulated basal cortisol secretion dose‐dependently to a similar extent at a similar time course. After incubation with IL‐3 or IL‐6 at concentrations of 100 μg/l, a maximum 4.1‐fold increase of the cortisol secretion was reached after 12 h (P<0.01). Coincubation of IL‐3 and IL‐6 (100 μg/l) revealed no significant synergism. To elucidate a possible involvement of arachidonic acid metabolites in the signal transduction, we coincubated IL‐3 or IL‐6 together with the cyclo‐oxygenase inhibitor indometacin or the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA). Coincubation with indometacin completely abolished the stimulatory effect of IL‐6 but had no effect on IL‐3 stimulated cortisol secretion. In contrast, specific inhibition of the lipoxygenase system by nordihydroguaiaretic acid blocked IL‐3 stimulated steroidogenesis while the effect of IL‐6 was not affected. Neither IL‐3 nor IL‐6 altered cAMP levels significantly, whereas ACTH significantly induced cAMP levels in parallel to its steroidogenic effect. In conclusion, our data indicate that IL‐3 and IL‐6 stimulate the steroid secretion of bovine adrenocortical cells to a similar extent and with a similar time course. However, the effects of IL‐3 and IL‐6 are mediated through different, cAMP‐independent pathways. While the stimulatory effect of IL‐3 seems to be dependent on the lipoxygenase pathway, the effect of IL‐6 on adrenocortical cortisol secretion is mediated through the cyclo‐oxygenase pathway.
Triggering of Neuroendocrine Refractoriness to Short‐Day Patterns of Melatonin in Siberian Hamsters
Authors: Prendergast  , Flynn  , Zucker
Published: March 2003

Short day lengths induce gonadal regression within 6 weeks in Siberian hamsters, but this inhibitory effect eventually wanes and reproductive competence is regained after 4–6 months in short days. These experiments were undertaken to determine whether continuous exposure to short days for several months is necessary to induce neuroendocrine refractoriness, or if a few weeks of short days are sufficient to trigger processes that culminate in refractoriness several months later. Adult male Siberian hamsters transferred from their natal long‐day photoperiod of 15L (15 h light/day) to a short‐day photoperiod of 10L were pinealectomized (PINx) after 0, 6, 12, 21 or 40 weeks of exposure to 10L. Intact hamsters kept in 10L manifested gonadal regression by week 6 and completed gonadal recrudescence by week 40, at which time they too were PINx. Beginning at week 40 all hamsters were infused s.c. with melatonin for 10 h/day for 6 consecutive weeks. This treatment induces gonadal regression in photosensitive hamsters. At the end of melatonin treatment, maximal gonadal regression was recorded for hamsters PINx at week 0 and those PINx after 40 weeks in long days. Hamsters PINx after 40 week of short day treatment were completely unresponsive to melatonin whereas those PINx after 6 and 12 weeks had intermediate responses. The percentage of hamsters whose reproductive apparatus was refractory to melatonin at week 40 increased with increasing duration of exposure to short days prior to PINx. Refractoriness was induced by relatively few weeks of short days in some hamsters, whereas others required much more extensive exposure. Induction of refractoriness is triggered by a fraction of the short days that hamsters experience in nature but may not be manifested until many weeks later when it coincides with gonadal recrudescence. In nature all hamsters are exposed to short days for at least 25 weeks, ensuring refractoriness in most individuals.
Norepinephrine Regulation of Growth Hormone Release from Goldfish Pituitary Cells. II. Intracellular Sites of Action
Authors: Yunker  , Lee  , Wong  , Chang
Published: March 2003

Previous results suggest that norepinephrine decreases growth hormone (GH) release in goldfish by means of α‐2 adrenoceptor activation. The intracellular mechanisms by which norepinephrine inhibits GH release were examined in the present study using dispersed goldfish pituitary cells. In 2‐h static incubation experiments, norepinephrine and the α‐2 agonist clonidine decreased basal GH release and the GH responses to stimulation by the dopamine D1 agonist SKF38393 and two native gonadotropin‐releasing hormones (GnRH). Norepinephrine also reduced GH responses to the adenylate cyclase activator forskolin, two protein kinase C (PKC) activators (phorbol ester and synthetic diacylglycerol), and two Ca2+ ionophores (ionomycin and A23187). Similarly, norepinephrine applied as a 1‐h pulse in cell column perifusion experiments reduced basal GH release and abolished the GH response to a 5‐min pulse of arachidonic acid. In goldfish, D1‐stimulated GH release is mediated by AC‐, arachidonic acid‐and Ca2+‐dependent pathways, whereas GnRH action is coupled to PKC‐and Ca2+‐dependent mechanisms. These results suggest that norepinephrine activation of α‐2 receptors inhibits ligand‐induced GH secretion by actions subsequent to activation of these second messenger cascades. To further characterize norepinephrine mechanisms of action on unstimulated hormone release, the ability of norepinephrine and an α‐2 agonist to affect activation of two second messenger cascades under basal conditions was also investigated. Static incubation with clonidine reduced cAMP production in a time‐and dose‐dependent manner, suggesting that norepinephrine inhibitory action can also be expressed at the level of cAMP production. Resting intracellular free calcium levels in single, identified goldfish somatotropes was unaffected by norepinephrine. However, the inhibitory effects of norepinephrine on basal GH secretion was not observed in the presence of a voltage‐sensitive Ca2+ channel agonist. Whether these channels are targets for norepinephrine action on unstimulated GH release requires further investigation.
Masculinizing Effect of Dihydrotestosterone on Growth Hormone Secretion is Inhibited in Ovariectomized Rats with Anterolateral Deafferentation of the Medial Basal Hypothalamus or in Intact Female Rats
Authors: Tamura  , Sugihara  , Kamegai  , Minami  , Wakabayashi
Published: March 2003

There is a striking sex difference in the pattern of growth hormone (GH) secretion in rats. Our previous studies showed that short‐term administration of pharmacological doses of testosterone or dihydrotestosterone (DHT) masculinized the GH secretory pattern in ovariectomized (OVX) rats. The locus where testosterone or DHT interacts with the somatotropic axis is believed to be the hypothalamus. To obtain insights into this phenomenon, we administered a single dose of DHT s.c. to adult OVX rats at 0.01, 0.1 or 1 mg/rat. Blood GH concentrations were measured in unanaesthetized rats. Six to12 h after the s.c. administration of all three doses of DHT, the GH secretory pattern revealed a male‐like secretory pattern as shown by episodic bursts occurring at 2–3‐h intervals with low or undetectable trough levels. When anterolateral deafferentation of the medial basal hypothalamus (ALC) was performed, the blood concentrations revealed irregularly occurring small fluctuations, instead of the usual high bursts, but the basal GH concentration was significantly higher than that of OVX‐sham‐operated rats. DHT treatment did not elicit pulsatile GH secretion or alter GH concentrations in OVX rats with ALC. When intact adult female rats received DHT at a dose of 1 mg/rat, the male‐like GH secretory pattern was not induced. These results suggest that neural inputs from the anterolateral direction to the medial basal hypothalamus are necessary for the masculinizing effect of DHT on the GH secretory pattern in OVX rats, and that oestrogen in intact female rats prevents the masculinizing effect of DHT.
The Central Effects of Orexin‐A in the Hypothalamic‐Pituitary‐Adrenal Axis In Vivo and In Vitro in Male Rats
Authors: Russell S. H. , Small C. J. , Dakin C. L. , Abbott C. R. , Morgan D. G. A. , Ghatei M. A. , Bloom S. R.
Published: March 2003

Orexin‐A is synthesized in the posterolateral hypothalamus and immunoreactive fibres project to many central nervous system structures, including the paraventricular nucleus, which is rich in corticotropin releasing factor (CRF) neurones and neuropeptide Y (NPY) innervation. We investigated the central effects of orexin‐A on the hypothalamic‐pituitary‐adrenal (HPA) axis by measuring plasma concentrations of corticosterone and adrenocorticotropic hormone (ACTH) in vivo. We explored the potential neuropeptide pathways involved by investigating the effects of orexin‐A on CRF, NPY, arginine vasopressin (AVP) and noradrenaline release from hypothalamic explants in vitro. Intracerebroventricular (i.c.v.) injection of orexin‐A (3 nmol) in male rats stimulated increases in plasma concentrations of corticosterone between 10 and 40 min after injection, and of plasma ACTH at 20 and 90 min after injection. Orexin‐A significantly stimulated CRF and NPY release from hypothalamic explants in vitro. Orexin‐A did not stimulate CRF release in the presence of the selective NPY Y1 receptor antagonist, BIBP3226. BIBP3226 alone did not alter CRF release from hypothalamic explants. Orexin‐A had no effect in vitro on the release of other neuropeptides, AVP and noradrenaline, involved in the central regulation of the HPA axis. These results suggest that orexin‐A is involved in activation of the HPA axis, and that these effects could be mediated via the release of NPY.
Effects of Melanocortin Receptor Ligands on Thyrotropin‐Releasing Hormone Release: Evidence for the Differential Roles of Melanocortin 3 and 4 Receptors
Authors: Kim M. S. , Small C. J. , Russell S. H. , Morgan D. G. A. , Abbott C. R. , AlAhmed S. H. , Hay D. L. , Ghatei M. A. , Smith D. M. , Bloom S. R.
Published: March 2003

The hypothalamic melanocortin system is important in the central regulation of food intake and body weight. We have previously demonstrated that intracerebroventricular administration of α‐melanocyte stimulating hormone (α‐MSH), a nonselective MC3 and MC4 receptor agonist, stimulated plasma thyroid‐stimulating hormone, and agouti‐related protein (AgRP), an MC3 and MC4 receptor antagonist, suppressed it. In this study, we investigated the effects of MC3 and MC4 receptor (MC3‐R and MC4‐R) selective agonists and antagonists on the release of thyrotropin‐releasing hormone (TRH) from hypothalamic explants in vitro. α‐MSH stimulated TRH release from the rat hypothalamic explants (α‐MSH 100 nM 230 ± 22.9% basal, P < 0.005). In contrast, γ2‐MSH, a selective MC3‐R agonist, suppressed TRH release (γ2‐MSH 10 µM 76.2 ± 7.4% basal, P < 0.05). AgRP (83‐132), a nonselective MC3/4‐R antagonist, induced no change in TRH release whilst JKC‐363 (cyclic [Mpr11, D‐Nal14, Cys18, Asp22‐NH2]‐β‐MSH 11‐22), a selective MC4‐R antagonist, suppressed it (JKC‐363 10 nM 57.2 ± 11.5% basal, P < 0.05). Both AgRP (83‐132) and JKC‐363 blocked α‐MSH stimulated TRH release but only AgRP (83‐132) blocked the inhibitory effect of γ2‐MSH on TRH release. These data suggest differential roles for the MC3 and MC4 receptors in TRH release; MC3‐R agonism inhibiting and MC4‐R agonism stimulating TRH release.
Variation in Reproductive Behaviour within a Sex:Neural Systems and Endocrine Activation
Authors: Rhen T. , Crews D.
Published: March 2003

Intrasexual variation in reproductive behaviour, morphology and physiology is taxonomically widespread in vertebrates, and is as biologically and ecologically significant as the differences between the sexes. In this review, we examine the diverse patterns of intrasexual variation in reproductive behaviours within vertebrates. By illustrating the genetic, cellular, hormonal and/or neural mechanisms underlying behavioural variation in a number of species, another level of complexity is added to studies of brain organization and function. Such information increases our understanding of the unique and conserved mechanisms underlying sex and individual differences in behaviour in vertebrates as a whole. Here, we show that intrasexual variation in behaviour may be discrete or continuous in nature. Moreover, this variation may be due to polymorphism at a single genetic locus or many loci, or may even be the result of phenotypic plasticity. Phenotypic plasticity simply refers to cases where a single genotype (or individual) can produce (or display) different phenotypes. Defined in this way, plasticity subsumes many different types of behavioural variation. For example, some behavioural phenotypes are established by environmental factors during early ontogeny, others are the result of developmental transitions from one phenotype early in life to another later in life, and still other strategies are facultative with different behaviours displayed in different social contexts.
Physiological Concentrations of Melatonin and Corticosterone in Stress and their Relationship with Phagocytic Activity
Authors: Barriga C. , Martín M. I. , Ortega E. , Rodriguez A. B.
Published: March 2003

The aim of the present work was to investigate the possible connection between melatonin, corticosterone and the nonspecific immune response under both basal and stressed conditions. The concentrations used in the present study were as determined in an earlier work, and corresponded to the minimum and maximum concentrations of the hormones melatonin and corticosterone over one circadian cycle in mice subjected to stress and in a basal group of mice. The concentrations were 15 pg/ml and 90 pg/ml of melatonin and 50 ng/ml and 200 ng/ml of corticosterone for incubations of the basal group macrophages, and 15 pg/ml and 70 pg/ml and 200 ng/ml and 500 ng/ml of melatonin and corticosterone, respectively, for incubations of macrophages from the group of stressed mice. The results indicate that corticosterone stimulates antigen ingestion at all physiological doses tested in the macrophages from the unstressed mice. The greatest phagocytic stimulation was obtained in macrophages from mice in a basal situation, and which had been incubated with the diurnal concentrations of the two hormones (maximum corticosterone 200 ng/ml; minimum melatonin 15 pg/ml). When the macrophages were from stressed mice, neither corticosterone nor melatonin led to any change in their phagocytic activity.
Hypothalamic Interactions Between Neuropeptide Y, Agouti‐Related Protein, Cocaine‐ and Amphetamine‐Regulated Transcript and Alpha‐Melanocyte‐Stimulating Hormone In Vitro in Male Rats
Authors: Dhillo W. S. , Small C. J. , Stanley S. A. , Jethwa P. H. , Seal L. J. , Murphy K. G. , Ghatei M. A. , Bloom S. R.
Published: March 2003

A number of neuropeptides implicated in the hypothalamic regulation of appetite are synthesized in the arcuate nucleus (Arc). Neuropeptide Y (NPY) and agouti‐related protein (Agrp) are orexigenic. The pro‐opiomelanocortin (POMC) product alpha‐melanocyte‐stimulating hormone (α‐MSH) is anorectic. Intracerebroventricular administration of cocaine‐ and amphetamine‐regulated transcript (CART) decreases food intake. However, recent results show that CART is orexigenic when injected into discrete hypothalamic nuclei. There is almost complete coexpression of NPY and Agrp mRNA in Arc neurones, and the majority of CART‐containing neurones in the Arc also contain POMC mRNA. We investigated possible interactions between these neuropeptides in vitro using a rat hypothalamic explant system. Administration of 1, 10 and 100 nm of NPY to hypothalamic explants significantly increased release of Agrp(83‐132)‐immunoreactivity (IR). NPY (10 and 100 nm) significantly increased the release of CART(55‐102)‐IR and α‐MSH‐IR from hypothalamic explants. Agrp(83‐132) (10 nm) administered to hypothalamic explants significantly increased the release of NPY‐IR. Agrp(83‐132) (10 and 100 nm) significantly decreased the release of CART(55‐102)‐IR from hypothalamic explants. Administration of 1, 10 and 100 nm CART(55‐102) to hypothalamic explants resulted in a significant increase in NPY‐IR release. Administration of 10 nm CART(55‐102) to hypothalamic explants significantly increased the release of Agrp(83‐132)‐IR. NDP‐MSH (10 nm) administered to hypothalamic explants significantly increased the release of NPY‐IR. NDP‐MSH (10 and 100 nm) significantly increased the release of Agrp(83‐132)‐IR from hypothalamic explants. These data suggest that orexigenic neuropeptides in the arcuate nucleus stimulate the release of each other, perhaps reinforcing orexigenic behaviour via a positive‐feedback loop. Our results are also in keeping with the possibility that the melanocortin‐3 receptor in the arcuate nucleus may influence the release of arcuate neuropeptides.
Resistance to the Satiety Action of Leptin Following Chronic Central Leptin Infusion is Associated with the Development of Leptin Resistance in Neuropeptide Y Neurones
Authors: Sahu A.
Published: March 2003

Leptin regulates food intake and body weight by acting primarily in the hypothalamus. In humans and rodents, obesity is associated with hyperleptinaemia, suggesting a possible state of leptin resistance. Thus, to begin to examine the mechanisms of leptin resistance, we developed a rat model in which chronic central leptin infusion results in the development of resistance to leptin's satiety action. Adult male rats were infused chronically into the lateral cerebroventricle with leptin (160 ng/h) or phosphate‐buffered saline via Alzet pumps for 28 days, followed by artificial cerebrospinal fluid infusion for 3 weeks. After the initial decrease in food intake, rats developed resistance to the satiety action of leptin, and withdrawal of the chronic leptin infusion resulted in hyperphagia. During leptin infusion, body weight was gradually decreased to reach a nadir on day 12, and thereafter, body weight was sustained at a reduced level throughout the entire 28‐day infusion, despite normalization in food intake. Body weight was mostly normalized by day 22 postleptin. Since neuropeptide Y (NPY) neurones are one of the targets of leptin signalling in the hypothalamus, we next examined whether the development of resistance to the satiety action of leptin was due to altered NPY gene expression. On day 3–4 of infusion, hypothalamic NPY mRNA levels, as determined by RNAse protection assay (RPA), were significantly decreased in leptin treated rats compared to controls. By contrast, on day 16 of infusion, NPY mRNA levels in the leptin treated group had returned to control levels. In situ hybridization study confirmed the results obtained with RPA and showed further that the effect of chronic leptin infusion on NPY mRNA levels was restricted to the rostral and middle parts of the arcuate nucleus. Overall, the finding that the action of continuous leptin exposure on NPY neurones was not sustained suggests that NPY neurones may be involved in the development of leptin resistance to the satiety action of leptin in the hypothalamus.
Evidence for the Biosynthesis of a Prolactin‐Releasing Factor From the Ovine Pars Tuberalis, Which is Distinct from Thyrotropin‐Releasing Hormone
Authors: Graham E. Scott , Webster C. A. , Hazlerigg D. G. , Morgan P. J.
Published: March 2003

This study demonstrates the presence of two prolactin‐releasing (PR) factors in media conditioned by primary pars tuberalis cells prepared from dispersed pars tuberalis tissue. One factor was identified as thyrotropin‐releasing hormone (TRH) on the basis of immunoreactivity and following purification by high‐performance liquid chromatography and mass spectrometry. The origin of TRH in the pars tuberalis conditioned media was investigated by measuring the expression of glutaminyl‐cyclase (QC) by in situ hybridization. QC expression was not detected in pars tuberalis‐specific cells, but was relatively abundant in cells in the pars distalis and hypothalamic paraventricular nucleus. These data suggest that TRH is not synthesized by the ovine pars tuberalis and more likely originated from the hypothalamic neuronal processes from the paraventricular nucleus that terminate in the median eminence. The second component of the conditioned media PR bioactivity was insensitive to the TRH‐antiserum, less than 1 kDa and was not retained by the C18 reverse‐phase column. The biosynthesis of the PR bioactivity by pars tuberalis cells was investigated using cycloheximide, forskolin and melatonin. Cycloheximide reduced the level of PR bioactivity produced by the pars tuberalis cells. Melatonin inhibited the increased level of PR bioactivity stimulated by forskolin. Collectively, these data demonstrate the synthesis of at least one regulator of prolactin secretion by ovine pars tuberalis‐specific cells.