bluuebunny/crossref_metadata_2025 huggingface dataset:
Atomic force microscopy of the erythrocyte membrane skeleton
Authors: Swihart A. H. , Mikrut J. M. , Ketterson J. B. , Macdonald R. C.
Published: March 2003

The atomic force microscope was used to examine the cytoplasmic surface of untreated as well as fixed human erythrocyte membranes that had been continuously maintained under aqueous solutions. To assess the effects of drying, some membranes were examined in air. Erythrocytes attached to mica or glass were sheared open with a stream of isotonic buffer, which allowed access to the cytoplasmic membrane face without exposing cells to non‐physiological ionic strength solutions. Under these conditions of examination, the unfixed cytoplasmic membrane face revealed an irregular meshwork that appeared to be a mixture largely of triangular and rectilinear openings with mesh sizes that varied from 35 to 100 nm, although few were at the upper limit. Fixed ghosts were similar, but slightly more contracted. These features represent the membrane skeleton, as when the ghosts were treated to extract spectrin and actin, these meshworks were largely removed. Direct measurements of the thickness of the membrane skeleton and of the lateral dimensions of features in the images suggested that, especially when air dried, spectrin can cluster into large, quite regularly distributed aggregates. Aggregation of cytoskeletal components was also favoured when the cells were attached to a polylysine‐treated substrate. In contrast, the membrane skeletons of cells attached to substrates rendered positively charged by chemical derivatization with a cationic silane were much more resistant to aggregation. As steps were taken to reduce the possibility of change of the skeleton after opening the cells, the aggregates and voids were eliminated, and the observed structures became shorter and thinner. Ghosts treated with Triton X‐100 solutions to remove the bilayer revealed a meshwork having aggregated components resembling those seen in air. These findings support the proposition that the end‐to‐end distance of spectrin tetramers in the cell in the equilibrium state is much shorter than the contour length of the molecule and that substantial rearrangements of the spectrin‐actin network occur when it is expanded by low ionic strength extraction from the cell. This study demonstrates the applicability of AFM for imaging the erythrocyte membrane skeleton at a resolution that appears adequate to identify major components of the membrane skeleton under near‐physiological conditions.
A preliminary electron backscattered diffraction study of sintered NdFeB‐type magnets
Authors: Lillywhite S. J. , Williams A. J. , Davies B. E. , Harris I. R.
Published: March 2003

This paper reports, for the first time, the use of electron backscattered diffraction (EBSD) to study orientation in sintered NdFeB type magnets. The magnetic properties of NdFeB magnets are greatly improved if a strong crystallographic texture is firstly achieved, namely, the direction of the c‐axis is along the direction of magnetization. A systematic survey of sample preparation techniques showed that samples that were mechanically polished and then etched gave the most reliable EBSD data. Analyses were made using both fully automated EBSD scans and by EBSD measurements taken after manual movement of the beam. The EBSD results are presented as secondary electron SEM micrographs, orientation images and 001 pole figures. For the selection of grains investigated, the deviation of the c‐axis was shown to be between 10° and 30° from the ideal [001]//magnetization direction. It is demonstrated that EBSD is a valuable tool for characterizing the microstructure and texture relationships and for assessing the performance of the processing routes of NdFeB magnets.
Adaptive correction of depth‐induced aberrations in multiphoton scanning microscopy using a deformable mirror
Authors: Sherman L. , Ye J. Y. , Albert O. , Norris T. B.
Published: March 2003

We demonstrate adaptive aberration correction for depth‐induced spherical aberration in a multiphoton scanning microscope with a micromachined deformable mirror. Correction was made using a genetic learning algorithm with two‐photon fluorescence intensity feedback to determine the desired shape for an adaptive mirror. For a 40×/0.6 NA long working distance objective, the axial scanning range was increased from 150 mm to 600 mm.
The distribution of caveolin‐3 immunofluorescence in skeletal muscle fibre membrane defined by dual channel confocal laser scanning microscopy, fast Fourier transform and image modelling
Authors: Ockleford C. D. , Cairns H. , Rowe A. J. , Byrne S. , Scott J. J. A. , Willingale R.
Published: March 2003

Membrane domains rich in caveolin‐3 overlie sarcomeric actin in skeletal muscle. The membrane exhibits a regular array of caveolin‐3 immunofluorescence using confocal laser scanning microscopy (CLSM). Fourier analysis of tissue imaged by CLSM accurately defines a repeating intensity with a long‐axis spacing of 1.48 µm confirmed by measurement of direct images. Reverse fast Fourier transform (FFT) and image‐modelling allow reconstruction of the pattern. Mathematical modelling has allowed replication of several features of the FFT, including the second order maxima that confirm the relatively high information content of the original images. Measurements of membrane‐pattern primary long‐axis spacings are consistent with our measurements of the I‐band sarcomere repeat in similarly prepared specimens labelled with fluorescent phalloidin or imaged using differential interference contrast microscopy. Dual‐channel CLSM analysis of the sarcomeric banding pattern of actin and the repeating pattern of muscle fibre membrane caveolin showed that caveolae overlie the I‐band. The anti‐caveolin immunofluorescence is deficient over the Z‐disc and maximal toward each of the I‐band extremities. A mechanism of membrane shape change in which membrane–lipid molecules are interposed between more stable anchored rafts associated with caveolae can be envisaged. Thus, increasing girth and reducing length of the sarcolemma in rapid contraction may be explained.
A new high‐aperture glycerol immersion objective lens and its application to 3D‐fluorescence microscopy
Authors: Martini N. , Bewersdorf J. , Hell S. W.
Published: March 2003

High‐resolution light microscopy of glycerol‐mounted biological specimens is performed almost exclusively with oil immersion lenses. The reason is that the index of refraction of the oil and the cover slip of ~1.51 is close to that of ~1.45 of the glycerol mountant, so that refractive index mismatch‐induced spherical aberrations are tolerable to some extent. Here we report the application of novel cover glass‐corrected glycerol immersion lenses of high numerical aperture (NA) and the avoidance of these aberrations. The new lenses feature a semi‐aperture angle of 68.5°, which is slightly larger than that of the diffraction‐limited 1.4 NA oil immersion lenses. The glycerol lenses are corrected for a quartz cover glass of 220 µm thickness and for a 80% glycerol‐water immersion solution. Featuring an aberration correction collar, the lens can adapt to glycerol concentrations ranging between 72% and 88%, to slight variations of the temperature, and to the cover glass thickness. As the refractive index mismatch‐induced aberrations are particularly important to quantitative confocal fluorescence microscopy, we investigated the axial sectioning ability and the axial chromatic aberrations in such a microscope as well as the image brightness as a function of the penetration depth. Whereas there is a significant decrease in image brightness associated with oil immersion, this decrease is absent with the glycerol immersion system. In addition, we show directly the compression of the optic axis in the case of oil immersion and its absence in the glycerol system. The unique advantages of these new lenses in high‐resolution microscopy with two coherently used opposing lenses, such as 4 Pi‐microscopy, are discussed.
Structure of the (110) antiphase boundary in gallium phosphide
Authors: Cohen D. , Carter C. B.
Published: March 2003

The morphology of antiphase boundaries in GaP films grown by molecular beam epitaxy on Si (001) has been studied by transmission electron microscopy. The inversion of the crystal polarity between antiphase domains was confirmed by convergent‐beam electron diffraction. The APBs were often found to facet parallel to {110} planes. Strong‐beam α‐fringe contrast observed along the (110) facets indicates that adjacent antiphase domains are related by an additional rigid‐body lattice translation. Diffraction‐contrast analysis shows that this R corresponds to a shear parallel to the [001] direction and a small expansion. The magnitude of the translation was inferred, quantitatively, through a comparison between energy‐filtered zero‐loss images of the α‐fringe contrast with numerical calculations. The components of the rigid‐body lattice translation were determined to be 0.023 ± 0.0033 nm in the [001] direction and 0.005 ± 0.002 nm in the 0 direction. Based upon a geometric model of the {110} antiphase boundary, the lengths of the Ga and P antisite bonds were calculated to be 254 ± 2 pm and 227 ± 4 pm, respectively.
Finasteride Blocks the Reduction in Ictal Activity Produced by Exogenous Estrous Cyclicity
Authors: Frye Cheryl A , Scalise Trudy J , Bayon Laura E
Published: March 2003

The purpose of the present study was to examine seizure activity during reduced 5α‐pregnan‐3α‐ol‐20‐one (3α,5α‐THP) production. Ovariectomized Long‐Evans rats were stereotaxically implanted with bipolar electrodes above the perforant pathway; silastic implants filled with estradiol‐17‐benzoate (EB) and progesterone were inserted subcutaneously to mimic diestrus. Estrus was then induced in half of these animals by injection of EB (30 μg) and progesterone (2.5 mg), 48 and 4 h, respectively, prior to perforant pathway stimulation. Half of the estrous and diestrous rats also received a 5α‐reductase inhibitor, finasteride (50 mg/kg), 6 h prior to perforant pathway stimulation. The estrous condition was associated with reduced number and duration of partial seizures, improved performance on a Morris water maze recovery of function test, reduced neuronal loss in the hilar region of the hippocampus, and elevated central and plasma 3α,5α‐THP, compared to estrus+finasteride, diestrus+vehicle and diestrus+finasteride conditions, which did not differ from each another. These data suggest antiseizure effects of estrus may be caused, in part, by the action of 3α,5α‐THP and that the precipitous decline in 3α,5α‐THP may restore seizure threshold to control levels.
Modification of Nitric Oxide Synthase Activity and Neuronal Response in Rat Striatum by Melatonin and Kynurenine Derivatives
Authors: León Josefa , Vives Francisco , Crespo Elena , Camacho Encarnación , Espinosa Antonio , Gallo MigueL. A. , Escames Germaine , Acuña‐Castroviejo Darío
Published: March 2003

Tryptophan is mainly metabolized in the brain through methoxyindole and kynurenine pathways. The methoxyindole pathway produces (among other compounds) melatonin, which displays inhibitory effects on human and animal central nervous systems, including a significant attenuation of excitatory, glutamate‐mediated responses. The kynurenine pathway produces kynurenines that interact with brain glutamate‐mediated responses. Nitric oxide (NO) increases glutamate release, and melatonin and kynurenines may act via modification of NO synthesis. In the present study, the effects of melatonin and four synthetic kynurenines were studied on the activity of rat striatal nitric oxide synthase (NOS) and on the response of rat striatal neurons to sensorimotor cortex (SMCx) stimulation, a glutamate‐mediated response. Melatonin inhibited both NOS activity and the striatal glutamate response, and these effects were dose‐related. Compound A (2‐acetamide‐4‐(3‐methoxyphenyl)‐4‐oxobutyric acid) did not inhibit NOS activity but inhibited the striatal response similarly to melatonin. Compound B (2‐acetamide‐4‐(2‐amino‐5‐methoxyphenyl)‐

4‐oxobutyric acid) was more potent than melatonin in inhibiting both NOS activity and the striatal response. Compound C (2‐butyramide‐4‐(3‐methoxyphenyl)‐4‐oxobutyric acid) did not change NOS activity, but increased the striatal response. Compound D (2‐butyramide‐4‐(2‐amino‐5‐methoxyphenyl)‐4‐oxobutyric acid) showed potent inhibitory effects on both NOS activity and striatal glutamate‐mediated response. A structure‐related effect of the kynurenine derivatives was observed, and those with an amino group in position 2 of the benzenic ring had more potent effects than melatonin itself in inhibiting striatal NOS activity and the response of striatal neurons to SMCx.
Regulation of Melatonin Receptors in the Pars Tuberalis of Syrian Hamsters Transferred from Long to Short Photoperiod: Implication of Melatonin and Testosterone
Authors: Recio Joaquín , Pévet Paul , Masson‐Pévet Mireille
Published: March 2003

The exposure of long day seasonal breeders to a short photoperiod (SP) induces both sexual quiescence and a decrease in pars tuberalis (PT) melatonin receptor density. Therefore, we studied the respective roles of melatonin and testosterone on the regulation of PT melatonin receptors in Syrian hamsters transferred from long photoperiod (LP) to SP. Compared with intact sexually active animals in LP, the density of melatonin receptors was not affected by the absence of melatonin after removal of the pineal gland from animals kept in either SP or LP. In contrast, the presence of a long melatonin peak in the blood which induces gonadal atrophy induced a significant decrease in binding capacity. The SP‐induced decrease in PT melatonin receptor density was also observed in castrated animals showing that it was directly regulated by melatonin, independently of circulating testosterone concentrations. However, the absence of testosterone induced an increased binding in LP, while increasing blood testosterone concentration after implantation of one testosterone‐filled silastic tube resulted in a decrease in binding both in LP‐and in SP‐animals. These results indicate that testosterone induces a photoperiod‐independent decrease in PT melatonin receptor density. In summary, these results show that both melatonin and testosterone have negative regulatory effects on the density of PT melatonin receptors.
The Ovine Melatonin‐Related Receptor: Cloning and Preliminary Distribution and Binding Studies
Authors: Drew Janice E. , Barrett Perry , Williams Lynda M. , Conway Shaun , Morgan Peter J.
Published: March 2003

A melatonin‐related receptor was cloned from an ovine genomic library. The sequenced gene has a similar structure to that of the melatonin receptor gene family and consists of two exons separated by an intron of approximately 3 kb. Exon 1 and exon 2 of the ovine melatonin‐related receptor encode a protein of 575 amino acids which is 73.8% homologous to the human melatonin‐related receptor and shows 40.9% homology with the ovine Mel1a melatonin receptor. COS‐7 cells transiently expressing ovine melatonin‐related receptors did not bind 2‐[125I]iodomelatonin or 3H‐melatonin. Reverse transcription‐polymerase chain reaction (RT‐PCR) and in situ hybridization studies revealed expression of the ovine melatonin‐related receptor in the hypothalamus, pituitary, retina and retinal pigment epithelium. Furthermore, expression of the ovine melatonin‐related receptor is shown to be coincident with Mel1a and 2‐[125I]iodomelatonin binding in the pituitary and serotonin N‐acetyl transferase (arylalkylamine N‐acetyl transferase, AANAT) expression in the retina. Expression patterns and similarity with the melatonin receptor gene family suggest a role for this novel G protein‐coupled receptor in control and regulation of endocrine function and retinal physiology.
Hypothalamic Dopamine D1 Receptors are Involved in the Stimulation of Prolactin Secretion by High Environmental Temperature on the Female Sheep
Authors: Colthorpe  , Anderson  , Martin  , Curlewis
Published: March 2003

Recent evidence suggests that dopamine, acting via its D1 receptors, may function as a neurotransmitter in intrahypothalamic pathways involved in the stimulation of prolactin secretion. Functional dopamine D1 receptors are present in the ventromedial hypothalamic nucleus (VMH) and we hypothesized that they might be part of a prolactin‐stimulatory pathway activated by stress. We tested this hypothesis in a series of experiments on sheep involving two different forms of stressors, audiovisual (barking dog) and high environmental temperature. We attempted to block the stimulation of prolactin secretion by infusion into the VMH of an antagonist specific for the D1 receptor. Ovariectomised, oestradiol‐implanted merino ewes were surgically implanted with bilateral guide tubes directed at the VMH. After a 180 min pretreatment period, the ewes either were or were not exposed to a stressor (30 min of barking dog or 120 min at 35 °C, 65% relative humidity). D1 receptor antagonist, SCH23390 or vehicle (0.9% saline) was infused into the VMH (1.7 μl/h, 120 nmol/h) for 60 min prior to and during the stressor period. Blood was sampled every 15 min via jugular cannulae and the plasma was assayed for prolactin, cortisol and growth hormone (GH). Both stressors significantly increased prolactin concentrations over control levels. SCH23390 infusion significantly attenuated the prolactin response to high environmental temperature, but had no effect on the prolactin response to audiovisual stress. Cortisol concentrations were significantly increased by audiovisual stress only and were not affected by SCH23390. GH concentrations were not changed by either stressor or infusion. Drug infusion alone did not affect the concentration of the hormones. The data suggest that the VMH D1 receptors are involved in a prolactin stimulatory pathway in response to high environmental temperature. The inability of the D1 antagonist to affect the response to the barking dog indicates that this pathway is stress‐specific, implying that there is more than one mechanism or pathway involved in the prolactin response to different stressors.
Differential Regulation of Hippocampal Glucocorticoid Receptors mRNA and Fast Feedback: Relevance to Post‐Traumatic Stress Disorder
Authors: Liberzon  , López  , Flagel  , Vázquez  , Young
Published: March 2003

Hippocampal glucocorticoid receptors (GR and MR) play an important role in glucocorticoid negative feedback. Abnormalities in negative feedback are found in depression and in post‐traumatic stress disorder (PTSD), suggesting that GR and MR might be involved in the pathophysiology of these disorders. Enhanced negative feedback, the PTSD‐specific neuroendocrine abnormality, can be induced in animals using a single prolonged stress (SPS) paradigm (a number of different stressors in one prolonged session, ‘no stress’ interval and a testing session one week later). In the current study, we examined hippocampal GR and MR mRNA distribution in the same animals that exhibited altered negative feedback following the SPS. Seven groups of adult Sprague‐Dawley male rats (seven animals each) were used in two studies, comparing unstressed controls to acutely stressed animals (SPS: 24 h group), SPS animals (seven and 14 days), and SPS+chronic stress animals. GR and MR mRNA distribution across hippocampal subfields was studied using in‐situ hybridization with 35S‐labelled cRNA probes. Acute stress produced down‐regulation of GR and MR mRNA across all hippocampal subfields. Seven days later (SPS‐7 group), there was a differential recovery, with GR mRNA reaching higher than the prestress levels, and MR mRNA remaining down‐regulated. The same differential regulation was present in the 14‐day group. Chronically stressed animals that exhibited normal fast feedback also had normalization in their GR and MR mRNA levels. The MR/GR ratio was decreased only in animals that had enhanced fast feedback. These findings suggest that the increase in GR, in hippocampus is involved in the fast feedback hypersensitivity observed in the SPS animals, and might also underlie enhanced dexamethasone sensitivity found in PTSD. Since differential activation of GR and MR can modulate memory, behavioural responsivity, anxiety and fear, change in MR/GR ratio might also explain other PTSD‐related phenomena.
Corticotropin‐Releasing Factor But Not Urocortin Is Involved in Adrenalectomy‐Induced Adrenocorticotropin Release
Authors: Masuzawa  , Oki  , Ozawa  , Watanabe  , Yoshimi
Published: March 2003

Urocortin, a new corticotropin‐releasing factor (CRF)‐related peptide, has been reported to have the ability to bind to CRF receptors and to stimulate adrenocorticotropin (ACTH) secretion from the rat anterior pituitary in vivo and in vitro. In this study, we examined the effect of intravenous administration of urocortin‐antiserum to investigate the role of endogenous urocortin on ACTH secretion from rat anterior pituitary after adrenalectomy. Male Sprague‐Dawley rats, which were maintained in a conscious and undisturbed condition, were administered non‐immunized rabbit serum (NRS), CRF‐antiserum or urocortin‐antiserum at a volume of 1 ml/kg b.w. 15 min before the injection of secretagogues. Synthetic rat urocortin (2 μg/kg B.W.) increased plasma ACTH concentrations by about sixfold the basal concentration. The pretreatment with urocortin‐antiserum but not CRF‐antiserum abolished the urocortin‐induced increase in plasma ACTH concentrations. In adrenalectomized rats, plasma ACTH concentrations were markedly increased at basal conditions, and rapidly reduced after the administration of CRF‐antiserum. By contrast, administration of urocortin‐antiserum did not alter ACTH secretion induced by adrenalectomy. Our results suggest that endogenous urocortin is unlikely to be involved in ACTH release in adrenalectomized rats.
5HT1A‐Receptor Binding in the Brain of Cyclic and Ovariectomized Female Rats
Authors: Flügge  , Pfender  , Rudolph  , Jarry  , Fuchs
Published: March 2003

Although it has been reported that hypothalamic 5HT1A‐receptor functioning is modulated by oestrogen and that this modulation contributes to the regulation of female sexual behaviour, there have been no reports up to now showing changes in numbers of these receptors during the oestrus cycle and after oestrogen treatment. We therefore analysed 5HT1A‐receptors in eight brain areas of female rats at different stages of the oestrus cycle, and in ovariectomized (OVX) females without and with oestrogen replacement. In‐vitro receptor autoradiography with the agonist 3H‐8‐OH‐DPAT(3H‐8‐hydroxy‐2‐[di‐n‐propylamino]tetralin) was used to determine numbers and affinities of 5HT1A‐receptors. To evaluate the hormonal state of the animals, serum concentrations of oestradiol, progesterone, luteinizing hormone (LH), and prolactin were also measured. Hormone determinations confirmed the expected endocrine states of the animals. In the ventromedial hypothalamic nucleus, the number of 3H‐8‐OH‐DPAT binding sites (Bmax‐value) during oestrus was increased compared to dioestrus yielding significant differences when using ANOVA statistics. In OVX females, the number of binding sites was decreased compared to pro‐oestrus and oestrus, and after oestrogen replacement, it was as high as during oestrus. All other brain areas analysed (medial preoptic area, bed nucleus of the stria terminalis, lateral septum, cingulate cortex, amygdala, hippocampal region CA1, and layers V and VI of the occipital cortex) showed no significant changes in 3H‐8‐OH‐DPAT binding site numbers. Also the affinity of 3H‐8‐OH‐DPAT binding sites did not change during the oestrus cycle, but in the medial preoptic area, oestradiol‐treated OVX animals showed a tendency for increased affinity compared to untreated OVX females. This was indicated by a change in Kd which appeared to be significant when groups were compared with the t‐test. We conclude from our data, that in the ventromedial hypothalamic nucleus, which is involved in the regulation of sexual function, 5HT1A‐receptors are up‐regulated during oestrus, that ovariectomy reduces the receptor numbers, and that oestradiol replacement counteracts the effect of ovariectomy. Since the ventromedial hypothalamic nucleus contains a high number of oestrogen receptive cells, our data indicate that oestrogen up‐regulates 5HT1A‐receptor expression in this nucleus.
A Suprachiasmatic Nucleus Generated Rhythm In Basal Glucose Concentrations
Authors: La Fleur  , Kalsbeek  , Wortel  , Buijs
Published: March 2003

The daily rhythm in feeding activity in mammals, as driven by the biological clock, largely determines the daily fluctuations in basal concentrations of glucose and insulin. To investigate a possible direct impact of the suprachiasmatic nucleus (SCN) on these parameters, we subjected intact rats and SCN‐lesioned rats to a fasting regimen of 36 h, or to a scheduled feeding regimen of six identical meals equally distributed over the light:dark‐cycle. Plasma profiles of glucose and insulin in rats during the final 24 h of the 36 h of fasting, and in rats subjected to the scheduled feeding regimen were compared to profiles in rats fed ad libitum. In rats fed ad libitum, in fasted rats and in rats subjected to a scheduled feeding regimen basal glucose concentrations showed a pronounced 24‐h rhythm that was not found in rats that had been SCN‐lesioned. Basal insulin levels showed a 24‐h rhythm in 50% of the rats fed ad libitum and in 50% of the rats subjected to a scheduled feeding regimen; neither rhythms were present in SCN‐lesioned rats. However, none of the fasted rats showed a 24‐h rhythm in basal insulin concentrations. These data provide clear evidence that the SCN directly controls basal glucose concentrations independent of its influence on feeding activity. At the same time, we found no consistent evidence for a strong impact of the SCN on basal insulin concentrations.
Effect of Corticosteroid Treatment In Vitro on Adrenalectomy‐Induced Impairment of Synaptic Transmission in the Rat Dentate Gyrus
Authors: Stienstra  , Joëls
Published: March 2003

Removal of the rat adrenals results after 3 days in the appearance of apoptotic cells in the dentate gyrus. Apoptosis is accompanied by an impaired synaptic transmission in the dentate gyrus. Substitution in vivo with a low dose of corticosterone was found to prevent both the appearance of apoptotic cells and the functional impairment. In the present study we determined whether the functional normalisation after corticosterone treatment critically depends on prevention of apoptosis. To address this question, brain slices from rats showing apoptosis after adrenalectomy were treated in vitro with the mineralocorticoid aldosterone (3 nM) or with 30 nM corticosterone, which is assumed to activate both mineralo‐ and glucocorticoid receptors. Steroids were briefly applied either during recording (acute effects) or several hours before recording (long‐term effects). While the slope of the fEPSP recorded in the outer molecular layer of the dentate gyrus in response to perforant path stimulation was not affected up to 1 h after acute administration of the steroids, fEPSP slopes recorded 2.5–3 h after corticosterone or aldosterone treatment were significantly increased, to the level of the sham‐operated controls. The results indicate that delayed corticosteroid effects through in vitro activation of mineralocorticoid receptors (MRs) are sufficient to normalise synaptic transmission in the dentate gyrus of ADX rats, even in the presence of apoptotic cells. We tentatively conclude that the impaired synaptic transmission seen after ADX is probably not primarily caused by the appearance of apoptotic cells.
Thyroid Hormone Acts Centrally to Programme Photostimulated Male American Tree Sparrows (Spizella arborea) for Vernal and Autumnal Components of Seasonality1
Authors: Wilson  , Reinert
Published: March 2003

Thyroid hormone and long days interact to programme American tree sparrows (Spizella arborea) for seasonality (i.e. thyroid hormone‐dependent photoperiodic gonadal growth, photorefractoriness, and postnuptial moult). This study explored in radiothyroidectomized (THX) males given thyroid hormone replacement therapy whether thyroid hormone acts within the brain and, additionally, the identity of the putative tissue‐active thyroid hormone. The minimum dose (30 ng) of
l‐thyroxine (T4) that restored all components of seasonality when given i.c.v. daily during the first 21 days of photostimulation restored no component of seasonality when given s.c. The same dose of
l‐triiodothyronine (T3) also was ineffective when administered s.c., but restored photoperiodic testicular growth (though neither photorefractoriness nor postnuptial moult) when admiministered i.c.v. Three of seven birds given a 10‐fold lower dose of T4 (3 ng) exhibited thyroid hormone‐dependent photoperiodic testicular growth, albeit damped. The other four birds given 3 ng T4 and all birds given 3 ng T3 responded like THX controls, exhibiting only slight thyroid hormone‐independent photoperiodic testicular growth. The highest dose (300 ng) of T3 restored all components of seasonality only when administered i.c.v. daily during the first 49 days of photostimulation. This demonstration in American tree sparrows is the first in any species that the thyroid‐dependent transition from the breeding season to the non‐breeding season can be effected by T3. The same dose of reverse T3 administered daily over the same 49 days restored photoperiodic testicular growth in only half of 10 subjects and photorefractoriness and moult in none. Collectively, the data support the hypothesis that thyroid hormone acts centrally to programme photostimulated male American tree sparrows for all components of seasonality. The most parsimonious interpretation of the data, including the threshold‐like effect of 3 ng T4, favours T4 as the tissue‐active thyroid hormone for vernal as well as autumnal events, but does not entirely exclude T3.
Co‐Expression of Melatonin (MEL1a) Receptor and Arginine Vasopressin mRNAs in the Siberian Hamster Suprachiasmatic Nucleus
Authors: Song C. Kay , Bartness  , Petersen  , Bittman
Published: March 2003

Durational melatonin signals, cued by the photoperiod and generated by the pineal gland, are processed in the brain to induce seasonally appropriate physiological and behavioural adaptations. The melatonin receptor subtype MEL1a (also known as mt1) appears to regulate seasonal responses. Single label in situ hybridization for MEL1a receptor mRNA revealed labelled cells in several brain regions of Siberian hamsters, including the suprachiasmatic nucleus, the paraventricular nucleus of the thalamus, and the reuniens nucleus of the thalamus. To characterize suprachiasmatic nucleus cells containing MEL1a receptor mRNA, we used 35S‐labelled cRNA probes for MEL1a receptor mRNA in combination with digoxigenin‐labelled cRNA probes for vasopressin, somatostatin, or orphan retinoid Z receptor β (RZRβ; a putative nuclear melatonin receptor). Cells in the suprachiasmatic nucleus that contained MEL1a receptor mRNA also contained mRNAs for vasopressin and RZRβ, but not for somatostatin. These data suggest that suprachiasmatic nucleus vasopressin cells may respond to melatonin signals, raising the possibility that suprachiasmatic nucleus vasopressin output mediates some of the effects of melatonin on seasonal or circadian responses.
Corticosterone Facilitates Saccharin Intake in Adrenalectomized Rats: Does Corticosterone Increase Stimulus Salience?
Authors: Bhatnagar  , Bell  , Liang  , Soriano  , Nagy  , Dallman
Published: March 2003

Unlike normal rats, adrenalectomized rats do not voluntarily drink sweet saccharin solutions. To test whether this is a function of corticosterone in the circulation, and if corticosterone also increases the impetus for drinking saccharin after a period of withdrawal, we performed the following experiments. Young male rats were sham adrenalectomized (sham) or adrenalectomized (ADX); the ADX rats were provided with subcutaneous pellets containing (percent replacement of corticosterone, %B) 0%B, 15%B, 30%B or 100%B. Sham and ADX rats were immediately provided with saline (0.5%) and saccharin (2 mM) bottles in their home cages. Saccharin was allowed for 4 days on, 3 days off, 4 days on, 3 days off and a final day on, over the 15 days experiment. The dose of corticosterone determined both how much saccharin was voluntarily drunk by the ADX rats and the degree of overshoot after days off. Corticosterone also determined energy balance of the groups of ADX rats. The 30%B pellets restored food intake, body weight gain, insulin and caloric efficiency to the normal levels observed in sham rats. White fat depot weights and uncoupling protein concentration in brown adipose tissue were restored to sham levels by 100%B, suggesting that these variables which depend on activity in the sympathetic nervous system require considerable glucocorticoid receptor occupancy. We conclude that corticosterone increases the willingness to ingest sweetened water in a unimodal, dose‐related manner, while moderate doses of corticosterone restore energy balance.
Synchronized Clusters of Action Potentials can Increase or Decrease the Excitability of the Axons of Magnocellular Hypothalamic Neurosecretory Cells
Authors: Dyball R. E. J. , McKenzie D. N.
Published: March 2003

Extracellular recordings were made from supraoptic nucleus (SON) cells in urethane anaesthetized male rats in vivo. Two stimulating electrodes were positioned to activate the cells antidromically, one in the mid axon region of the cells and the other at the axon terminals. Trains of 5–20 just‐subthreshold stimuli at 5 s intervals decreased the threshold for antidromic activation from both sites. Whereas neither single stimuli, nor the stimuli at the beginning of a train of 20 stimuli evoked antidromic action potentials, later action potentials did so. Paradoxically, trains of 20 just‐suprathreshold stimuli increased the threshold for activation of both axons and terminals. In recordings from the same cells, stimuli were applied singly at 5 s intervals at an intensity which almost invariably evoked an antidromic action potential. Identical stimuli were then applied in trains of 20 stimuli at 50 Hz. After the first train, the initial stimulus pulses of the trains frequently fell below threshold. Following a conditioning train of five stimuli applied to one electrode, the period of decreased threshold (increased excitability) at the other electrode lasted less than 100 ms and the period of increased threshold (decreased excitability) after 12 trains of 20 stimuli lasted between 5 and 10 s. Both decreased and increased excitability were seen at axons and terminals of both putative oxytocin and vasopressin cells. Since the excitability changes were shown in vivo at frequencies encountered during recordings, it is likely that they influence the probability of spike propagation and hormone secretion under physiological conditions.
The Effects of Ionotropic Agonists of Excitatory Amino Acids on the Release of Arginine Vasopressin in Rat Hypothalamic Slices
Authors: Joanny P. , Steinberg J. , Guerrero F. , Sauze N. , Oliver C. , Grino M.
Published: March 2003

The effects of ionotropic excitatory amino acids agonists on the release of vasopressin from rat hypothalamic slices were studied. Incubation with increasing doses of NMDA, kainate or AMPA decreased the release of vasopressin in a dose‐dependent manner. The values of the IC50 were 1.0, 9.6, or 3.7 × 10−8 M, respectively. The inhibitory effect of the various excitatory amino acids tested was blocked by coincubation with their respective antagonists. Vasopressin secretion was stimulated to 140.3 ± 7.6% of controls when the slices were obtained from chronically (7 days) salt‐loaded rats. Addition of 1 × 10−7 M NMDA or 1 × 10−6 M kainate to the incubation medium antagonized the salt loading‐induced increase in vasopressin release. Incubation with 1 × 10−4 M tetrodotoxin did not change basal vasopressin release, but it blocked the decrease in vasopressin secretion induced by 1 × 10−7 M NMDA or 1 × 10−6 M kainate or 1 × 10−6 M AMPA. Incubation with 1 × 10−5 M phaclophen (a GABAB antagonist) and 1 × 10−5 M bicuculline (a GABAA antagonist) was without effect on basal vasopressin secretion while it reversed the inhibition of vasopressin release induced by 1 × 10−7 M NMDA. Incubation with 1 × 10−6 M GABA alone decreased vasopressin secretion to 64.6 ± 6.9% of control values. The inhibitory effect of GABA did not change when 1 × 10−7 M NMDA was added to the incubation medium. These findings demonstrate that ionotropic excitatory amino acids agonists inhibit vasopressin secretion from hypothalamic slices. They strongly suggest that this inhibitory effect is mediated through local GABAergic interneurones.
Calcium‐Independent, Tyrosine Phosphorylation‐Dependent Effects of Serum on the Morphology of Cultured Neurohypophysial Astrocytes
Authors: Ramsell K. D. , Cobbett P.
Published: March 2003

Activation of adenylate cyclase induces cultured neurohypophysial astrocytes (pituicytes) to change from a protoplasmic, nonstellate form to a stellate form. Stellation is inhibited and reversed (destellation) by serum. The objective of the present studies was to examine the roles of Ca2+ and tyrosine phosphorylation in mediating these morphological changes. The effects of forskolin (to induce stellation) and serum (to inhibit and reverse stellation) were not affected by replacement of Ca2+ with Co2+ in the medium or by treatment of cultures with thapsigargin. However, genistein, a specific inhibitor of tyrosine kinase(s), significantly reduced the effect of serum on forskolin‐induced stellation. Also, dephostatin, a specific inhibitor of tyrosine phosphatase, inhibited forskolin‐induced stellation. In contrast, genistein did not have a dramatic effect on serum‐induced destellation. The data demonstrate that morphological changes exhibited by cultured pituicytes are independent of Ca2+ but may be modulated by the activity of tyrosine kinase(s) and phosphatase(s).
Oestrogen Upregulates Noradrenaline Release in the Mediobasal Hypothalamus and Tyrosine Hydroxylase Gene Expression in the Brainstem of Ovariectomized Rhesus Macaques
Authors: Pau K.‐Y. F. , Hess D. L. , Kohama S. , Bao J. , Pau C. Y. , Spies H. G.
Published: March 2003

Noradrenaline plays a key role in the initiation of ovulation in nonprimate species. A similar noradrenaline role in the primate has not been established experimentally. We utilized the ovariectomized‐oestrogen‐supplemented (OVX + E) rhesus macaque to examine the effects of intravenous (i.v.) infusion of oestradiol‐17β (E2) on the activity of the brain noradrenaline system. Experiment 1 established the induction of a preovulatory surge‐like release of luteinizing hormone in OVX + E monkeys by i.v. infusion of E2 (OVX + E + E2). In experiment 2, a marked increase in hypothalamic microdialysate noradrenaline concentrations occurred after identical E2 infusion into the OVX + E monkeys that were used in experiment 1. In experiment 3, tyrosine hydroxylase (TH) mRNA expression in the locus coeruleus of the brainstem increased at various times after E2 infusion as determined by semiquantitative in situ hybridization. The amount of TH mRNA in OVX + E + E2 animals was higher (P < 0.05) than that in either the OVX + E or OVX monkeys; no difference was found in the latter two groups. Moreover, selected locus coeruleus sections from E2‐infused monkeys were examined for the localization of oestrogen receptors (ER) by in situ hybridization. Both ER‐α and ER‐β mRNAs were expressed in the locus coeruleus, although the expression was greater for ER‐α than for ER‐β. We conclude that i.v. infusion of E2, which induces a preovulatory surge‐like release of LH, stimulates brain noradrenaline activity; this enhanced activity likely involves an ER‐mediated process and is reflected by hypothalamic noradrenaline release and locus coeruleus TH mRNA expression. The results support the concept that noradrenaline can influence the E2‐stimulated ovulation in nonhuman primates and that the brainstem is one of the components in this neuroendocrine process.
Variations in Maternal Behaviour are Associated with Differences in Oxytocin Receptor Levels in the Rat
Authors: Francis D. D. , Champagne F. C. , Meaney M. J.
Published: March 2003

Female Long‐Evans rats exhibit stable individual differences in maternal behaviours such as pup licking/grooming and arched‐back nursing posture (LG‐ABN). These variations in maternal behaviour are accompanied by differences in lactation‐induced increases in oxytocin receptor levels in brain regions known to mediate the expression of maternal care in this species (i.e. the bed nucleus of the stria terminalis, the medial preoptic area and the lateral septum). Oxytocin receptor levels in the central nucleus of the amygdala were significantly higher in high compared to low LG‐ABN females regardless of reproductive status. These findings suggest that individual differences in maternal behaviour may be directly related to variations in oxytocin receptor expression.
Effects of Chronic Bromocriptine Treatment on Tyrosine Hydroxylase (TH) mRNA Expression, TH Activity and Median Eminence Dopamine Concentrations in Ageing Rats
Authors: MohanKumar P. S. , MohanKumar S. M. J. , Quadri S. K. , Voogt J. L.
Published: March 2003

The purpose of this study was to investigate the age‐related changes in the responsiveness of tuberoinfundibular dopamine (TIDA) neurones to chronic hypoprolactinemia induced by treatment with bromocriptine, a dopamine receptor agonist. In one experiment, TIDA neuronal activity after acute hypoprolactinemia or exogenous prolactin was monitored by measuring tyrosine hydroxylase (TH) activity in the stalk median eminence of middle‐aged cycling female rats (10–12 months), old constant oestrous rats (18–20 months) and old pseudopregnant rats (22–24 months). In another experiment, middle‐aged cycling (10–12 months) rats were treated with bromocriptine for 6 or 12 months. TH activity was measured in the stalk median eminence, TH mRNA levels were measured in the arcuate nucleus and dopamine concentrations were measured in the arcuate nucleus and median eminence. Responsiveness of TIDA neurones to exogenous prolactin and to the withdrawal of bromocriptine in these rats was also tested. While the TIDA neurones in all three age groups responded to acute hypoprolactinemia by showing a reduction in TH activity, older rats failed to respond to exogenous prolactin administration. In contrast, chronic hypoprolactinemia for 12 months enabled the rats to retain TIDA neuronal responsiveness to exogenous prolactin. It also decreased TIDA neuronal function as measured by dopamine concentrations in the median eminence, TH activity in the stalk median eminence and TH mRNA in the arcuate nucleus of ageing rats. The restoration of the responsiveness of these neurones to prolactin stimulation in older rats demonstrates for the first time that hypoprolactinemia produced by chronic bromocriptine treatment indeed provides a neuroprotective effect on TIDA neurones. These results indicate that maintaining a low level of neuronal activity by lowering prolactin levels may be a contributing factor in retaining the plasticity of TIDA neurones.
The Insulin Hypoglycemia Test: Hypoglycemic Criteria and Reproducibility
Authors: Nye E. J. , Grice J. E. , Hockings G. I. , Strakosch C. R. , Crosbie G. V. , Walters M. M. , Torpy D. J. , Jackson R. V.
Published: March 2003

The insulin hypoglycemia test (IHT) is widely regarded as the ‘gold standard’ for dynamic stimulation of the hypothalamic‐pituitary‐adrenal (HPA) axis. This study aimed to investigate the temporal relationship between a rapid decrease in plasma glucose and the corresponding rise in plasma adenocorticotropic hormone (ACTH), and to assess the reproducibility of hormone responses to hypoglycemia in normal humans. Ten normal subjects underwent IHTs, using an insulin dose of 0.15 U/kg. Of these, eight had a second IHT (IHT2) and three went on to a third test (IHT3). Plasma ACTH and cortisol were measured at 15‐min intervals and, additionally, in four IHT2s and the three IHT3s, ACTH was measured at 2.5‐ or 5‐min intervals. Mean glucose nadirs and mean ACTH and cortisol responses were not significantly different between IHT1, IHT2 and IHT3. Combined data from all 21 tests showed the magnitude of the cortisol responses, but not the ACTH responses, correlated significantly with the depth and duration of hypoglycemia. All subjects achieved glucose concentrations of of ≤ 1.6 mmol/l before any detectable rise in ACTH occurred. In the seven tests performed with frequent sampling, an ACTH rise never preceeded the glucose nadir, but occurred at the nadir, or up to 15 min after. On repeat testing, peak ACTH levels varied markedly within individuals, whereas peak cortisol levels were more reproducible (mean coefficient of variation 7%). In conclusion, hypoglycemia of ≤ 1.6 mmol/l was sufficient to cause stimulation of the HPA axis in all 21 IHTs conducted in normal subjects. Nonetheless, our data cannot reveal whether higher glucose nadirs would stimulate increased HPA axis activity in all subjects. Overall, the cortisol response to hypoglycemia is more reproducible than the ACTH response but, in an individual subject, the difference in peak cortisol between two IHTs may exceed 100 nmol/l.
PACAP Stimulation of Gonadotropin‐II Secretion in Goldfish Pituitary Cells: Mechanisms of Action and Interaction with Gonadotropin Releasing Hormone Signalling
Authors: Chang J. P. , Wirachowsky N. R. , Kwong P. , Johnson J. D.
Published: March 2003

Pituitary adenylate cyclase‐activating polypeptide (PACAP) has recently been shown to be a hypophysiotropic factor in the goldfish. In this study, we examined the mechanisms of PACAP action on goldfish maturational gonadotropin (GTH‐II) release using primary cultures of pituitary cells. The GTH‐II response to mammalian PACAP1−38 (mPACAP) was inhibited by a PACAP receptor antagonist suggesting a receptor‐mediated action. Addition of either an adenylate cyclase inhibitor or a protein kinase A (PKA) inhibitor reduced the mPACAP‐induced GTH‐II release. In addition, when GTH‐II release was already stimulated by either forskolin or 8‐bromo‐cAMP (8Br‐cAMP), mPACAP did not further increase GTH‐II secretion. These results strongly implicated the involvement of an adenylate cyclase/cAMP/PKA pathway in PACAP‐stimulated GTH‐II release. Although mPACAP induced a rise in intracellular Ca2+ level in identified gonadotropes, results with voltage‐sensitive Ca2+ channel inhibitors indicated that the GTH‐II responses to mPACAP, forskolin and 8Br‐cAMP did not depend upon Ca2+ entry through these channels. Two protein kinase C (PKC) inhibitors did not affect mPACAP‐elicited GTH‐II release, and mPACAP further increased GTH‐II secretion in the presence of PKC activators. These results indicate that PKC‐dependent elements are not essential for the stimulatory action of mPACAP in gonadotropes. Interestingly, while GTH‐II responses to a stimulatory concentration of mPACAP were additive to responses elicited by maximal effective concentrations of two endogenous gonadotropin releasing hormones (GnRHs), a subthreshold concentration of mPACAP potentiated GnRH and PKC activator stimulation of GTH‐II secretion. Similarly, submaximal concentrations of forskolin potentiated the GTH‐II response to the PKC activator, tetradecanoyl phorbol acetate. These data suggest that PACAP and its cAMP‐dependent signalling mechanisms provide an alternate stimulatory input to goldfish gonadotropes and may influence the effectiveness of the major neuroendocrine control exerted by the PKC‐dependent GnRH signalling pathway.