bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative phase‐amplitude microscopy II: differential interference contrast imaging for biological TEM
Authors: McMahon P. J. , Barone‐Nugent E. D. , Allman B. E. , Nugent K. A.
Published: March 2003

Although phase contrast microscopy is widespread in optical microscopy, it has not been as widely adopted in transmission electron microscopy (TEM), which has therefore to a large extent relied on staining techniques to yield sufficient contrast. Those methods of phase contrast that are used in biological electron microscopy have been limited by factors such as the need for small phase shifts in very thin samples, the requirement for difficult experimental conditions, or the use of complex data analysis methods. We here demonstrate a simple method for quantitative TEM phase microscopy that is suitable for large phase shifts and requires only two images. We present a TEM phase image of unstained Radula sp. (liverwort spore). We show how the image may be transformed into the differential interference contrast image format familiar from optical microscopy. The phase images contain features not visible with the other imaging modalities. The resulting technique should permit phase contrast TEM to be performed almost as readily as phase contrast optical microscopy.
Measurement of nanosecond time‐resolved fluorescence with a directly gated interline CCD camera
Authors: Mitchell A. C. , Wall J. E. , Murray J. G. , Morgan C. G.
Published: March 2003

CCD cameras coupled optically to gated image intensifiers have been used for fast time‐resolved measurements for some years. Image intensifiers have disadvantages, however, and for some applications it would be better if the image sensor could be gated directly at high speed. Control of the ‘charge drain’ function on an interline‐transfer CCD allows the sensor to be switched rapidly from an insensitive state. The temporal and spatial properties of the charge drain are explored in the present paper and it is shown that nanosecond time resolution with acceptable spatial uniformity can be achieved for a small commercial sensor. A fluorescence lifetime imaging system is demonstrated, based on a repetitively pulsed laser excitation source synchronized to the CCD control circuitry via a programmable delay unit.
Freeze substitution of high‐pressure frozen samples: the visibility of biological membranes is improved when the substitution medium contains water
Authors: Walther P. , Ziegler A.
Published: March 2003

Biological membranes are often poorly visible with the electron microscope after high‐pressure freezing and freeze‐substitution. The water content of the sample and of the substitution medium is one factor among others that strongly influences membrane visibility. In order to investigate this effect, high‐pressure frozen yeast cells, rat‐pancreas tissue and arthropod tissue were freeze‐substituted with and without adding water to the substitution medium. The visibility of the biological membranes was generally improved if the substitution medium contained 1–5% water. The effect was especially pronounced in yeast cells, where membrane visibility was poor after freeze‐substitution with water‐free medium but good after addition of 5% water to the substitution medium.
High‐resolution scanning electron microscopy of immunogold‐labelled cells by the use of thin plasma coating of osmium
Authors: Suzuki E.
Published: March 2003

The feasibility of plasma coating of a thin osmium layer for high‐resolution immuno‐scanning electron microscopy of cell surfaces was tested, using Drosophila embryonic motor neurones as a model system. The neuro‐muscular preparations were fixed with formaldehyde and labelled with a neurone‐specific antibody and 10 or 5 nm colloidal gold‐conjugated secondary antibodies. The specimens were post‐fixed with osmium tetroxide and freeze‐dried. Then they were coated with a 1–2 nm thick layer of osmium using a hollow cathode plasma coater. The thin and continuous coating of amorphous osmium gave good signals of gold particles and fine surface structures of neurites in backscattered electron images simultaneously. This method makes it possible to visualize the antigen distribution and the three‐dimensionally complex surface structures of cellular processes with a resolution of several nanometres.
Quantitative polarized light microscopy
Authors: Massoumian F. , Juškaitis R. , Neil M. A. A. , Wilson T.
Published: March 2003

We describe a simple modification to a confocal microscope, which analyses the state of polarization of light emerging from the specimen so as to permit quantitative polarized light microscopy to be performed. The system uses a novel form of rotating analyser which, together with lock‐in detection, permits images to be obtained where the image contrast corresponds to both specimen retardance and orientation (e.g. in the case of a birefringent specimen). Images are presented from a wide range of specimens and the origin of the contrast observed from simple point scatterers is investigated both theoretically and experimentally.
PACAP Increases the Cytosolic Ca2−/Concentration and Stimulates Somatodendritic Vasopresson Release in Rat Supraoptic Neurons
Authors: Shibuya Izumi , Noguchi Jun , Tanaka Keiko , Harayama Nobuya , Inoue Yoshitaka , Kabashima Narutoshi , Ueta Yoichi , Hattori Yukio , Yamashita Hiroshi
Published: March 2003

Pituitary adenylate cyclase activating polypeptide (PACAP)‐like immunoreactivity and its receptor mRNA have been reported in the supraoptic and the paraventricular nucleus (SON and PVN, respectively) and PACAP has been implicated in the regulation of magnocellular neurosecretory cell function. To examine the site and the mechanism of the action of PACAP in the neurosecretory cells, we measured AVP release from SON slice preparations and the cytosolic Ca2+concentration ([Ca2+]i) from single dissociated SON neurons. PACAP at concentrations from 10−12to 10−7M increased [Ca2+]iin dissociated SON neurons in a dose‐dependent manner. The patterns of the PACAP‐induced [Ca2+]iincrease were either sustained increase or cytosolic Ca2+oscillations. PACAP (10−7M) increased [Ca2+]iin 27 of 27 neurons and glutamate (10−4M) increased [Ca2+]iin 19 of 19 SON neurons examined, whereas angiotensin II (10−7M) increased [Ca2+]iin only 15 of 60 SON neurons examined. PACAP at lower concentrations (10−10to 10−8M) increased [Ca2+]iin 70–80% of neurons examined. Although the onset and recovery of the PACAP‐induced [Ca2+]iincrease were slower than those observed with glutamate, the spatial distribution of the [Ca2+]iincreases in response to the two ligands were similar: [Ca2+]iincrease at the proximal dendrites was larger and faster and that at the center of the soma was smaller and slower. The PACAP‐induced [Ca2+]iresponseswere abolished by extracellular Ca2+removal, thel‐type Ca2+‐channel blocker, nicardipine, or by replacement of extracellular Na+with N‐methyld‐glucamine, and were partially inhibited by the Na+‐channel blocker, tetrodotoxin. The N‐type Ca2+‐channel blocker,ω‐conotoxin GVIA did not significantly inhibit the PACAP‐induced [Ca2+]iresponses. Furthermore, PACAP (10−7M) as well as glutamate (10−4M) increased AVP release from SON slice preparations, and extracellular Ca2+removal or nicardipine inhibited the AVP release in response to PACAP.

These results indicate that PACAP enhances Ca2+entry via voltage‐gated Ca2+channels and increases [Ca2+]i, which, in turn, stimulates somatodendritic vasopressin release by directly activating PACAP receptors on SON neurons. The results also suggest that PACAP in the SON may play a pivotal role in the control of the neurohypophyseal function at the level of the soma or the dendrites.
Prolactin Receptor Gene Expression and Foetal Adipose Tissue
Authors: Symonds  , Phillips  , Anthony  , Owens  , McMillen
Published: March 2003

We have investigated the effects of increasing gestational age, maternal undernutrition or restricted placental growth on prolactin receptor (PRLR) gene expression in perirenal adipose tissue collected from foetal sheep during late gestation (term=147 d±3 d of gestation). Foetal nutrient supply was reduced by either restriction of placental growth following removal of endometrial caruncles before mating or by reducing maternal feed intake by 50% from 115 d of gestation. Total RNA was extracted from adipose tissue taken from foetal sheep between 90 and 145 d of gestation, and only at 141–145 d in placentally restricted, nutrient restricted and control foetuses. Messenger RNAs encoding the long (PRLR1) and short (PRLR2) forms of the PRLR and glyceraldehyde‐phosphate‐dehydrogenase (GAPDH) were detected and quantified in a ribonuclease protection assay using an antisense RNA probe complementary to ovine PRLR2 and GAPDH. There was a 7.5‐fold increase in the amount of perirenal adipose tissue between 90 and 125 d of gestation, compared with a 1.3‐fold increase between 125 and 145 d of gestation. The abundance of mRNA encoding PRLR1 and PRLR2 in perirenal adipose tissue increased 10‐ and sixfold, respectively, between 90 and 125 d of gestation, and then declined by 145 d of gestation. Both placental restriction and maternal undernutrition significantly reduced foetal adipose tissue deposition. The abundance of PRLR1 but not PRLR2 mRNA was reduced in adipose tissue from the placentally restricted group, where as GAPDH mRNA was three times higher than in controls. In contrast, maternal undernutrition from 115 d of gestation did not affect PRLR1, PRLR2 or GAPDH mRNA expression in foetal adipose tissue. It is concluded that during the period of rapid deposition of perirenal adipose tissue, there is a concomitant increase in PRLR gene expression. This indicates that prolactin may play an important role in the growth and maturation of foetal adipose tissue which occurs before birth.
The Effects of Progesterone on Oxytocin mRNA Levels in the Paraventricular Nucleus of the Female Rat Can Be Altered by the Administration of Diazepam or RU4861
Authors: Thomas  , Shughrue  , Merchenthaler  , Amico
Published: March 2003

Oxytocin (OT) facilitates the onset of maternal behaviour in the late pregnant rat, enhances uterine contractility at parturition, and elicits milk ejection during lactation. If the rising estradiol (E2) and declining progesterone (P) of late pregnancy is reproduced in a virgin ovariectomized rat by implanting E2‐ and P‐filled capsules for 2 weeks followed by removal of P‐containing implants 36–48 h prior to death, OT messenger ribonucleic acid (mRNA) levels increase in the paraventricular and supraoptic nuclei (PVN and SON) of the rat. Both E2 administration and P withdrawal are necessary to increase OT mRNA, but the mechanisms of these effects are not understood. P may work within the PVN although P receptors are reported to be sparse or non‐existent in the PVN or outside the PVN on PR‐containing neurones that project to OT‐containing neurones or via membrane bound receptors that are known to bind neurosteroids and gamma aminobutyric acid (GABA). To determine the mechanism through which P may inhibit or P withdrawal may increase OT mRNA levels, virgin ovariectomized (OVX) rats received sequential E2 and P via Silastic implants for 14 days. On day 13, prior to removal of P capsules on day 14, the rats were given the benzodiazepine agonist, diazepam, or saline injections subcutaneously (s.c.) twice daily until death on day 16. OT mRNA levels were increased in the steroid‐treated group that received saline but not diazepam. In experiment 2, P capsules were removed on day 14 or pharmacological P withdrawal was induced by injecting RU486 injections s.c. twice daily until death 48 h later. OT mRNA levels were increased in the steroid‐treated group that received RU486. Subsequent studies demonstrated the expression of PR mRNA within the rat PVN. The data suggest that gonadal steroids may influence PVN OT mRNA levels by modulating the GABAA receptor or by directly altering gene transcription via the PR.
Hypothalamic Involvement in the Regulation of Maternal Behaviour in the Rat: Inhibitory Roles for the Ventromedial Hypothalamus and the Dorsal/Anterior Hypothalamic Areas
Authors: Bridges  , Mann  , Coppeta
Published: March 2003

The present report examines the possible involvement of the ventromedial hypothalamus (VMH), the dorsal hypothalamus (DH), and the anterior hypothalamic area (AHA) in the regulation of maternal behaviour in the female rat. In a series of experiments it was found that either infusions of saline or lowering cannulas into the VMH stimulated a rapid onset of maternal behaviour in progesterone plus oestrogen‐primed, nulliparous rats. The stimulatory effect of cannula lowering into the VMH on maternal behaviour was shown to be steroid‐dependent. Next, the involvement of cell bodies located in the DH/AHA in maternal behaviour was examined after bilateral lesions of these regions with the neurotoxin, N‐methyl‐ d‐aspartic acid (NMA). NMA lesions of the DH/AHA stimulated a rapid onset of maternal behaviour in oestrogen‐treated, nulliparous rats, while NMA lesions in non‐steroid‐treated animals or vehicle infusions in steroid or non‐steroid‐treated rats failed to induce a rapid onset of behaviour. In a final study the effects of NMA lesions of the VMH were evaluated. As in AHA lesioned rats, NMA lesions of the VMH stimulated a fast onset of maternal behaviour in steroid‐primed females. These findings indicate that the VMH as well as the DH/AHA exert chronic steroid‐dependent inhibitory influences on the induction of maternal behaviour.
An Increase in Glutamate Release Follows a Decrease in Gamma Aminobutyric Acid and the Pubertal Increase in Luteinizing Hormone Releasing Hormone Release in Female Rhesus Monkeys
Authors: Terasawa  , Luchansky  , Kasuya  , Nyberg‡
Published: March 2003

Previously we have shown that release of γ‐aminobutyric acid (GABA) in the stalk‐median eminence (S‐ME) is high in prepubertal monkeys and that a decrease in GABA release triggers the onset of puberty. However, it is still unclear how disinhibition of the luteinizing hormone releasing hormone (LHRH) neuronal system from GABA input is followed (or accompanied) by an increase in stimulatory signals, such as glutamatergic input to LHRH neurons. To clarify the temporal relationship between the reduction of the GABAergic inhibitory signal and the enhancement of the glutamatergic stimulatory signal in the control of LHRH release at the onset of puberty, we conducted two experiments using a push–pull perfusion method. In the first experiment, we measured developmental changes in release of LHRH, GABA, and glutamate in the S‐ME. LHRH levels were very low in prepubertal monkeys, increased to higher levels in early pubertal monkeys, with the highest LHRH levels occurring in mid‐pubertal monkeys. As we previously observed, GABA levels were high in prepubertal monkeys and then decreased in early‐ and mid‐pubertal monkeys. In contrast, glutamate levels were very low in prepubertal monkeys, increased dramatically in early pubertal monkeys, and then slightly decreased in mid‐pubertal monkeys, although mid‐pubertal levels remained much higher than prepubertal levels. In the second experiment, we measured GABA, glutamate and LHRH in the same samples obtained from prepubertal monkeys which were infused with an antisense oligodeoxynucleotide (AS) for glutamic acid decarboxylase (GAD) 67 mRNA into the S‐ME. GAD67 is a catalytic enzyme for GABA synthesis from glutamate, and AS GAD67 mRNA interferes with GAD67 synthesis. Infusion of the AS GAD67 induced a decrease in GABA release, which subsequently resulted in an increase in LHRH release. Surprisingly, glutamate release also increased several hours after the decrease in GABA release, and the increased LHRH release continued. These data are interpreted to mean that a decrease in GABA synthesis by interference with GAD67 synthesis and the reduction of GABA release in the S‐ME trigger an increase in LHRH release, but that a subsequent increase in glutamate release in the S‐ME further contributes to the pubertal increase in LHRH release at the onset of puberty. The data further support our hypothesis that GAD plays an important role in the mechanism of the onset of puberty.
GnRH mRNA Increases with Puberty in the Male Syrian Hamster Brain
Authors: Parfitt  , Thompson  , Richardson  , Romeo  , Sisk
Published: March 2003

Puberty in the male Syrian hamster (Mesocricetus auratus) is characterized by decreased responsiveness to testosterone mediated negative feedback, but the neural mechanism for this change remains elusive. We hypothesized that decreased inhibition of the gonadotropin‐releasing hormone (GnRH) system results in increased neurosecretory activity, which includes an increase in GnRH gene expression. This study examined GnRH mRNA in male hamsters before and after puberty, and sought to determine if any increase in mRNA was specific to particular subpopulations of GnRH neurones. Brains were collected from 21‐day‐old prepubertal males (n=5) and 56‐day‐old postpubertal males (n=5). Alternate 10 μm coronal sections from fresh‐frozen brains were collected throughout the septo‐hypothalamic region, and 25% of those sections were processed for in‐situ hybridization histochemistry using an 35S‐riboprobe complementary to hamster GnRH. No differences were observed in the number of GnRH mRNA expressing cells in any region, but in the diagonal band of Broca (DBB)/organum vasculosum of the lamina terminalis (OVLT) there was a significant increase in labelling intensity (defined as area of the cell occupied by silver grains) in postpubertal males. A second analysis compared the frequency distributions of cells based on labelling intensity between prepubertal and postpubertal males. This analysis revealed significant differences between the two frequency distributions in all areas analysed (DBB/OVLT, medial septum (MS), and preoptic area (POA)). Furthermore, examining the distribution of cells in these regions revealed a shift to the right in the postpubertal population of cells, which indicated an increased number of GnRH neurones with greater labelling intensity. These data clearly demonstrate increased GnRH mRNA during puberty. Furthermore, they suggest that the previous observation of brain region specific pubertal decreases in GnRH‐immunoreactivity only within the DBB/OVLT and MS but not the POA are not due to differential levels of GnRH gene expression, but could indicate increased release from these neurones during puberty.
The Influence of Postnatal Handling on Adult Neuroendocrine and Behavioural Stress Reactivity
Authors: Meerlo  , Horvath  , Nagy  , Bohus  , Koolhaas
Published: March 2003

Environmental stimuli during early stages of life can influence the development of an organism and may result in permanent changes in adult behaviour and physiology. In the present study we investigated the influence of early postnatal handling on adult neuroendocrine and behavioural stress reactivity in Wistar rats. Pups were subjected to handling from postnatal day 1–21. The young were taken from the nest every day for 15 min and each of the pups was handled separately. Control nests were left undisturbed. When the animals had reached an adult age of 3–4 months they were individually housed and subjected to a series of tests to measure their stress reactivity. In the first experiment we established adult behavioural coping with stressors and anxiety in the following series of tests: open field test, shock prod defensive burying test, elevated plus maze and conditioned fear test. Collectively, the data clearly indicate that handled animals are characterized by a lower stress‐induced anxiety. Yet, handled and control animals do not differ in their general way of coping with stressors. Although the lower anxiety in handled animals is often reflected in a higher activity, they are not more active per se. In a second experiment, animals were provided with a permanent jugular vein canula for repeated blood sampling to determine stress hormones: noradrenaline, adrenaline, prolactin and corticosterone. Animals were subjected to a novelty test and a conditioned fear test. The neuroendocrine response profile is consistent with the conclusion that handled animals are less anxious than controls but are not different in their general strategy of coping with stressors. The handled animals showed an attenuated adrenaline, prolactin and corticosterone response. Yet, in neither of the two tests there was a difference in noradrenaline response, a typical marker for an active coping strategy. Interestingly, the differences in neuroendocrine reactivity already appeared in response to a mild novelty challenge when there were no clear behavioural differences yet. The neuroendocrine measures are in line with the behavioural data but more sensitively reflect the differences between handled and control animals.
Differential Effects of Placental Restriction on IGF‐II, ACTH Receptor and Steroidogenic Enzyme mRNA Levels in the Foetal Sheep Adrenal
Authors: Ross  , Phillips  , Simonetta  , Owens  , Robinson  , McMillen
Published: March 2003

We have investigated the effects of restriction of placental growth on foetal adrenal growth and adrenal expression of mRNAs for Insulin‐like Growth Factor II (IGF‐II), the IGF binding protein IGFBP‐2, Steroidogenic Factor 1 (SF‐1) and adrenocorticotrophic hormone (ACTH) receptor (ACTH‐R) and the steroidogenic cytochrome P‐450 enzymes: cholesterol side chain cleavage (CYP11A1), 17α‐hydroxylase (CYP17) and 21‐hydroxylase (CYP21A1); and 3β‐hydroxysteroid dehydrogenase/Δ5Δ4 isomerase (3βHSD). Endometrial caruncles were removed from non‐pregnant ewes before mating (placental restriction group; PR). The total adrenal: foetal weight ratio was higher in PR (n=6 foetuses) than in control foetuses (n=6 foetuses). There was no difference in plasma ACTH concentrations between the PR and control foetuses between 130 and 140 days gestation. Adrenal IGF‐II mRNA levels were lower (P<0.05) in the PR group, however, adrenal IGFBP‐2 mRNA levels were not different between the PR and control groups. Adrenal ACTH‐R mRNA levels were also lower whilst CYP11A1 mRNA levels were increased (P<0.005) in the PR group. We conclude that foetal adrenal growth and steroidogenesis are stimulated as a consequence of foetal growth restriction and that factors other than ACTH are important in foetal adrenal activation during chronic, sustained hypoxaemia.
Pharmacological Evidence that Prolactin Acts from Late Gestation to Promote Maternal Behaviour in Rabbits
Authors: González‐Mariscal G. , Melo A. I. , Parlow A. F. , Beyer C. , Rosenblatt J. S.
Published: March 2003

We investigated the role of prolactin and suckling stimulation in the expression of maternal behaviour of primiparous rabbits. Bromocriptine (1 mg/kg/day), given to intact mothers across postpartum days 1–5, decreased serum concentrations of prolactin to undetectable levels, reduced crouching, and increased time inside the nest. Failure of maternal nest‐building, provoked by bromocriptine injections from pregnancy day 26 to parturition or to postpartum day 5, correlated with a stronger reduction in crouching and an increased time inside the nest, measures of disturbed maternal behaviour, on postpartum days 3 and 5. Preventing suckling by thelectomy did not prevent prolactin release but reduced crouching incidence and increased the time spent inside the nest on postpartum days 3 and 5. Bromocriptine, injected in thelectomized mothers across postpartum days 1–5, further reduced the incidence of crouching and increased the time spent inside the nest on postpartum days 3 and 5. We conclude that prolactin acting prepartum facilitates maternal behaviour initiation in rabbits and, together with pup stimulation, maintains this behaviour across lactation.
The In Vitro Role of Tumour Necrosis Factor‐Alpha and Interleukin‐6 in the Hypothalamic‐Pituitary Gonadal Axis
Authors: Russell S. H. , Small C. J. , Stanley S. A. , Franks S. , Ghatei M. A. , Bloom S. R.
Published: March 2003

The adipocyte derived hormone leptin has been implicated as an important nutritional signal to the reproductive system, but the role of other adipocyte related cytokines is not clear. Tumour necrosis factor‐alpha (TNF‐α) and interleukin (IL)‐6 are present in adipose tissue and released into the circulation where plasma levels correlate positively with body mass index and body fat mass. These cytokines could play a role in signalling nutritional status to the hypothalamic‐pituitary‐gonadal axis. We investigated the effects of TNF‐α and IL‐6 on basal and luteinizing hormone releasing hormone (LHRH) stimulated luteineizing hormone (LH) release from cultured anterior pituitary cells, harvested from either proestrus female or male Wistar rats. We examined the effects of TNF‐α and IL‐6 on LHRH release from hypothalamic explants harvested from proestrus female and male rats in vitro. IL‐6 significantly suppressed LHRH stimulated LH release from male dispersed pituitaries throughout the dose range, but did not influence basal LH release. IL‐6 had no effect on basal or LHRH stimulated LH release in dispersed pituitaries from proestrus females. By contrast, TNF‐α significantly suppressed LHRH stimulated LH release in dispersed pituitaries from proestrus female rats in a dose responsive manner, but did not influence basal LH release. TNF‐α had no effect on basal or LHRH stimulated LH release in dispersed pituitaries from male rats. TNF‐α and IL‐6 had no effect on LHRH release from male hypothalamic explants in vitro. TNF‐α and IL‐6 had no effect on LHRH release from proestrus female hypothalamic explants in vitro. TNF‐α and IL‐6 have differential effects in dispersed pituitaries harvested from males and proestrus female rats. TNF‐α and IL‐6 may be important in mediating some of the nutritional effects on the reproductive axis by acting at the level of the anterior pituitary rather than the hypothalamus.
Anorexic But Not Pyrogenic Actions of Interleukin‐1 are Modulated by Central Melanocortin‐3/4 Receptors in the Rat
Authors: Lawrence C. B. , Rothwell N. J.
Published: March 2003

The cytokine interleukin‐1 (IL‐1), which mediates many responses to infection and injury, induces anorexia and fever through direct actions in the central nervous system. The melanocortin neuropeptides, such as alpha melanocyte‐stimulating hormone (α‐MSH), reportedly antagonize many actions of IL‐1, including fever and anorexia. However, it is unknown whether endogenous melanocortins modulate anorexia induced by IL‐1. The objective of the present study was to establish the effect of endogenous melanocortins on IL‐1‐induced anorexia and fever in the rat. Intracerebroventricular (i.c.v.) injection of IL‐1β caused a significant reduction in food intake and body weight gain, and a rise in core body temperature in conscious rats. Coadministration of the melanocortin‐3/4 receptor (MC3/4‐R) antagonist, SHU9119, reversed IL‐1β‐induced reductions in food intake and body weight, but did not affect the febrile response to IL‐1β. These data suggest IL‐1β may elicit its effects on food intake through the melanocortin system, predominantly via the MC3‐R or MC4‐R. In contrast, IL‐1β‐induced fever does not appear to be mediated or modulated by MC3‐R or MC4‐R activity.
Altered Regulation of Gene and Protein Expression of Hypothalamic‐Pituitary‐Adrenal Axis Components in an Immature Rat Model of Chronic Stress
Authors: Avishai‐Eliner S. , Gilles E. E. , Eghbal‐Ahmadi M. , Bar‐El Y. , Baram T. Z.
Published: March 2003

Chronic stress early in postnatal life influences hormonal and behavioural responses to stress persistently, but the mechanisms and molecular cascades that are involved in this process have not been clarified. To approach these issues, a chronic stress paradigm for the neonatal rat, using limited bedding material to alter the cage environment, was devised. In 9‐day‐old rats subjected to this chronic stress for 1 week, significant and striking changes in the expression and release patterns of key molecules that govern the neuroendocrine stress responses were observed. The presence of sustained stress was evident from enhanced activation of peripheral elements of the neuroendocrine stress response, i.e. increased basal plasma corticosterone concentrations, high adrenal weight and decreased body weight. Central regulatory elements of the neuroendocrine stress response were perturbed, including reduced expression of hypothalamic corticotropin‐releasing hormone that, surprisingly, was accompanied by reduced glucocorticoid receptor expression. Thus, the effects of chronic sustained stress in the neonatal rat on the hypothalamic‐pituitary‐adrenal axis included substantial changes in the expression and activity of major regulators of this axis. Importantly, the changes induced by this chronic stress differed substantially from those related to acute or recurrent stress, providing a novel model for studying the long‐term effects of chronic, early life stress on neuroendocrine functions throughout life.
Evidence that Orexin‐Containing Neurones Provide Direct Input to Gonadotropin‐Releasing Hormone Neurones in the Ovine Hypothalamus
Authors: Iqbal J. , Pompolo S. , Sakurai T. , Clarke I. J.
Published: March 2003

Orexins A and B (ORX) have been added recently to the growing list of neuropeptides implicated in feeding and drinking behaviour as well as neuroendocrine function. In the present study, we have used single and dual labelling immunohistochemistry and a rabbit polyclonal anti‐orexin‐A antibody, which recognizes both ORX A and B, to examine ORX pathways in the sheep hypothalamus. ORX immunoreactive cells were distributed in the dorsomedial hypothalamic nucleus, lateral hypothalamic area, zona incerta and perifornical area; a few cells were also observed in the anterior hypothalamic area. In contrast to distribution in the rat brain, most of the ORX immunoreactive cells are localized to the dorsomedial hypothalamic nucleus and perifornical area; scattered cells are found in lateral hypothalamic area. ORX immunoreactive fibres were widely distributed throughout the hypothalamus and preoptic area with dense innervation of the medial preoptic area and bed nucleus of stria terminalis. Dual labelling demonstrated widespread expression of the long form of the leptin receptor within all ORX cells that were examined. Thirty percent of the gonadotropin releasing hormone (GnRH) cells that were examined had ORX immunoreactive terminals in close contact with no regional or sex differences. FluoroGold injections into the preoptic area retrogradely labelled a subpopulation of ORX cells in the lateral hypothalamic/perifornical area, showing ORX cells of this region project to the preoptic and could potentially provide input to GnRH cells. These findings suggest an integral role for ORX in the regulation of GnRH cells in the sheep and thus provide evidence of a novel mechanism whereby leptin can influence reproductive neuroendocrine function.
Anatomical Markers of Activity in Neuroendocrine Systems: Are we all ‘Fos‐ed out’?
Authors: Hoffman G. E. , Lyo D.
Published: March 2003

It has now been nearly 15 years since the immediate early gene, c‐fos, and its protein product, Fos, were introduced as tools for determining activity changes within neurones of the nervous system. In the ensuing years, this approach was applied to neuroendocrine study with success. With it have come advances in our understanding of which neuroendocrine neurones respond to various stimuli and how other central nervous system components interact with neuroendocrine neurones. Use of combined tract‐tracing approaches, as well as double‐labelling for Fos and transmitter markers, have added to characterization of neuroendocrine circuits. The delineation of the signal transduction cascades that induce Fos expression has led to establishment of the relationship between neurone firing and Fos expression. Importantly, we can now appreciate that Fos expression is often, but not always, associated with increased neuronal firing and vice versa. There are remaining gaps in our understanding of Fos in the nervous system. To date, knowledge of what Fos does after it is expressed is still limited. The transience of Fos expression after stimulation (especially if the stimulus is persistent) complicates design of experiments to assess the function of Fos and makes Fos of little value as a marker for long‐term changes in neurone activity. In this regard, alternative approaches must be sought. Useful alternative approaches employed to date to monitor neuronal changes in activity include examination of (i) signal transduction intermediates (e.g. phosphorylated CREB); (ii) transcriptional/translational intermediates (e.g. heteronuclear RNA, messenger RNA (mRNA), prohormones); and (iii) receptor translocation. Another capitalizes on the fact that many neuroendocrine systems show striking stimulus‐transcription coupling in the regulation of their transmitter or its synthetic enzymes. Together, as we move into the 21st Century, the use of multiple approaches to study activity within neuroendocrine systems will further our understanding of these important systems.
Cerebral Endothelial Cells are a Major Source of Adrenomedullin
Authors: Kis B. , Kaiya H. , Nishi R. , Deli M. A. , Ábrahám C. S. , Yanagita T. , Isse T. , Gotoh S. , Kobayashi H. , Wada A. , Niwa M. , Kangawa K. , Greenwood J. , Yamashita H. , Ueta Y.
Published: March 2003

Adrenomedullin is a peptide hormone with multifunctional biological properties. Its most characteristic effects are the regulation of circulation and the control of fluid and electrolyte homeostasis through peripheral and central nervous system actions. Although adrenomedullin is a vasodilator of cerebral vasculature, and it may be implicated in the pathomechanism of cerebrovascular diseases, the source of adrenomedullin in the cerebral circulation has not been investigated thus far. We measured the secretion of adrenomedullin by radioimmunoassay and detected adrenomedullin mRNA expression by Northern blot analysis in primary cultures of rat cerebral endothelial cells (RCECs), pericytes and astrocytes. We also investigated the expression of specific adrenomedullin receptor components by reverse transcriptase‐polymerase chain reaction and intracellular cAMP concentrations in RCECs and pericytes. RCECs had approximately one magnitude higher adrenomedullin production (135 ± 13 fmol/105 cells per 12 h; mean ± SD, n = 10) compared to that previously reported for other cell types. RCECs secreted adrenomedullin mostly at their luminal cell membrane. Adrenomedullin production was not increased by thrombin, lipopolysaccharide or cytokines, which are known inducers of adrenomedullin release in peripheral endothelial cells, although it was stimulated by astrocyte‐derived factors. Pericytes had moderate, while astrocytes had very low basal adrenomedullin secretion. In vivo experiments showed that adrenomedullin plasma concentration in the jugular vein of rats was approximately 50% higher than that in the carotid artery or in the vena cava. Both RCECs and pericytes, which are potential targets of adrenomedullin in cerebral microcirculation, expressed adrenomedullin receptor components, and exhibited a dose‐dependent increase in intracellular cAMP concentrations after exogenous adrenomedullin administration. Antisense oligonucleotide treatment significantly reduced adrenomedullin production by RCECs and tended to decrease intraendothelial cAMP concentrations. These findings may suggest an important autocrine and paracrine role for adrenomedullin in the regulation of cerebral circulation and blood–brain barrier functions. Cerebral endothelial cells are a potential source of adrenomedullin in the central nervous system, where adrenomedullin can also be involved in the regulation of neuroendocrine functions.
Spatio‐Temporal Expression of Gonadotropin‐Releasing Hormone Receptor Subtypes in Gonadotropes, Somatotropes and Lactotropes in the Cichlid Fish
Authors: Parhar I. S. , Soga T. , Sakuma Y. , Millar R. P.
Published: March 2003

The description of two or more forms of gonadotropin‐releasing hormone (GnRH) in most vertebrates suggests multiple roles for this family of peptide hormones. In order to verify these functions, we analysed the anatomical location, time of initial expression and ontogenic changes in three distinct GnRH receptors (GnRH‐Rs) in developing and sexually mature tilapia, using antisera raised against the extracellular loop three of the receptor, which is a determinant in ligand‐selectivity and receptor coupling to signalling pathways. In all age groups, including males and females, using in situ hybridization and double‐label immunological methods, GnRH‐R type IA was colocalized in cells containing luteinizing hormone (LH) β‐subunit in the pituitary. GnRH‐R type IB was visualized in prolactin cells and LH cells. The type III GnRH‐R was expressed in growth hormone cells. On day 8 after fertilization, GnRH‐R type III was first seen in growth hormone cells and, subsequently, on day 15, GnRH‐Rs type IA and type IB were first seen in LH and prolactin cells, respectively. On day 25, the receptor occupied area per pituitary and the staining intensity of GnRH‐R type IA increased significantly, consistent with the hypothesis that differentiation of GnRH neurones and their inputs to the pituitary coincide precisely with gonadal sex differentiation and steroidogenesis in tilapia. The differential distribution of GnRH‐Rs in the pituitary provides the first clear evidence that the three native GnRH variants in tilapia have cognate receptors, each capable of regulating different pituitary endocrine cells.
Functional Subsets of Serotonergic Neurones: Implications for Control of the Hypothalamic‐Pituitary‐Adrenal Axis
Authors: Lowry C. A.
Published: March 2003

Serotonergic systems play an important role in the regulation of behavioural, autonomic and endocrine responses to stressful stimuli. This includes modulation of both the hypothalamic‐pituitary‐adrenal (HPA) axis and the hypothalamic‐spinal‐adrenal (HSA) axis, which converge at the level of the adrenal cortex to regulate glucocorticoid secretion. Paradoxically, serotonin can either facilitate or inhibit HPA axis activity and stress‐related physiological or behavioural responses. A detailed analysis of the brainstem raphe´ complex and its ascending projections reveals that facilitatory and inhibitory effects of serotonergic systems on glucocorticoid secretion may be due to influences of topographically organized and functionally diverse serotonergic systems. (i) A serotonergic system arising from the middle and caudal dorsal raphe´ nucleus and projecting to a distributed central autonomic control system and a lateral ‘emotional motor system’. Evidence suggests that serotonin can sensitize this subcortical circuit associated with autonomic arousal, anxiety and conditioned fear. (ii) A serotonergic system arising from the median raphe´ nucleus and projecting extensively and selectively to a ventral subiculum projection system. Evidence suggests that serotonin facilitates this limbic circuit associated with inhibition of ultradian, circadian and stress‐induced activity of both the HPA axis and the HSA axis. These new perspectives, based on functional anatomical considerations, provide a hypothetical framework for investigating the role of serotonergic systems in the modulation of ultradian, circadian and stress‐induced neuroendocrine function.
Nociceptin/Orphanin FQ Content is Decreased in Forebrain Neurones During Acute Stress
Authors: Devine D. P. , Hoversten M. T. , Ueda Y. , Akil H.
Published: March 2003

We examined the effects of acute and chronic stress on neurotransmission of nociceptin/orphanin FQ (N/OFQ) in a variety of brain regions. Four groups of rats were exposed to chronic variable stress, and/or a single acute stress before decapitation. Group 1 served as unstressed controls. The rats in group 2 (chronic stress/no acute stress) were exposed to a 10‐day regimen of chronic stress (two unpredictable stressors per day). These rats were decapitated 20 h after the last stressor. The rats in group 3 (no chronic stress/acute stress) were not exposed to chronic stress, but they were restrained for 30 min prior to decapitation. The rats in group 4 (chronic stress/acute stress) were chronically stressed for 10 days, and were then restrained prior to decapitation. Trunk blood was collected, and plasma adrenocorticotrophic hormone (ACTH) and corticosterone (CORT) were assayed by radioimmunoassay (RIA). The rats' brains were dissected, and N/OFQ content was measured by RIA in a variety of brain regions, and in spinal cord. Chronic stress exposure altered the hormonal responses to the acute stress exposure. In the rats that were exposed to chronic stress without acute stress (group 2), N/OFQ content did not differ from the content of the unstressed controls in any of the dissected brain regions. In the two groups that were stressed acutely just before decapitation (groups 3 and 4), N/OFQ content was decreased by 25–30% in the basal forebrain. Accordingly, the neuronal content of N/OFQ is decreased in basal forebrain neurones during acute stress exposure. In light of our previous finding that N/OFQ administration increases circulating ACTH and CORT concentrations, and augments hormonal responses to an acute stressor, the current finding raises the possibility that endogenous N/OFQ participates in neuronal regulation of hormonal responses to acute stress exposure.
Oral stereognosis in edentulous subjects rehabilitated with complete removable dentures
Authors: Mantecchini  , Bassi  , Pera  , Preti
Published: March 2003

Oral stereognosis was analysed in a group of edentulous subjects rehabilitated with complete removable dentures. Stereognosis was evaluated with and without the denture in place. The results showed that the older subjects had poorer stereognostic ability than the younger ones, whereas the duration of edentulism appeared not to influence this ability. The presence of a correct prosthetic rehabilitation appeared to improve stereognostic ability. Subjects with poorer stereognostic ability appeared more satisfied with their rehabilitation than did those with better stereognostic ability.
Ratio between vertical and horizontal mandibular range of motion
Authors: Dijkstra  , Kropmans TH. J. B. , Stegenga  , De Bont
Published: March 2003

Mandibular range of motion (ROM), vertical and horizontal, is often measured as a part of a diagnostic assessment of temporomandibular joint disorders. In the literature, a fixed ratio between the vertical and the horizontal ROM has been suggested, i.e. 4 : 1. The ratio is frequently used to predict the vertical ROM on the basis of the horizontal ROM and vice versa. However, no scientific data are available to substantiate the ratio suggested. The aim of this study was to determine whether the fixed ratio of 4 : 1 exists and, if so, whether this ratio has a predictive value. Vertical and horizontal mandibular ROM was measured in 91 healthy subjects, 59 women, and 32 men (mean age 27·2 years, s.d. 7·4 years) using vernier callipers. We found a mean ratio between vertical and horizontal ROM ranging from 6·0 : 1–6·6 : 1. On an individual basis the ratio ranged from 3·6–15·5. Correlations between vertical and horizontal ROM were weak. On the basis of the results on this study it is concluded that the ratio between vertical and horizontal ROM is approximately 6 : 1 rather than 4 : 1, and that the ratio has poor predictive value.
Electromyographic study of the temporal, masseter and suprahyoid muscles in the mandibular rest position
Authors: De Faria C. R. Sgobbi , Bérzin F.
Published: March 2003

The temporal muscles (anterior position), and the masseter and suprahyoid muscles were studied electromyographically in 15 subjects with ages ranging from 18 to 35 years, showing normal occlusion (Class I of angle), complete dentition and no dysfunction of the stomatognathic system. The volunteers sat comfortably in a chair, keeping the Frankfurt plane parallel to the floor; the muscles were analysed in the mandibular resting position through a number of stages: with minimum exteroceptive stimuli, relaxation with soft music, abolition of the intrabuccal negative pressure through a plastic tube, stress provoked by an electronic game, disocclusion of posterior teeth using the occlusal splint and finally withdrawal of this device. The electromyographic results were analysed statistically. The samples, were analysed for all of the frequencies of motor units, in a 1‐min period. It was found that there were statistically significant difference between the relaxation stages as compared with the withdrawal of negative intra‐oral pressure and mainly with the stress phase, being this only on the suprahyoid muscles, with 5% significance.
A 5‐year prospective randomized clinical trial on the influence of splinted and unsplinted oral implants retaining a mandibular overdenture: prosthetic aspects and patient satisfaction
Authors: Naert I. , Gizani S. , Vuylsteke M. , Van Steenberghe D.
Published: March 2003

Prosthetic outcome and patient satisfaction were evaluated in order to investigate whether there is a need or advantage to splint two implants in the mandible retaining a hinging overdenture. This study included 36 fully edentulous patients randomly divided into three groups according to the attachment system they received: magnets, ball attachments or straight bars (reference group). None of the implants failed during the whole observation period in any of the groups. After 5 years of observation, the Bar group presented the highest retention capacity and the least prosthetic complications but revealed more mucositis and gingival hyperplasia. Patient satisfaction rated similar for all groups although the Magnet group showed lower retention forces. All patients would repeat the same treatment even though the majority of the Magnet group would prefer a more retentive solution because of limited denture stability.
The effect of alterations in horizontal X‐ray beam angulation and bucco‐lingual cavity width on the radiographic depth of approximal cavities
Authors: Chadwick B. L. , Dummer P. M. H. , Van Der Stelt P. F.
Published: March 2003

The effects of changes in horizontal X‐ray beam angulation and different cavity shape on images of approximal cavities on bite‐wing radiographs were examined using a laboratory model. Cylindrical‐, rectangular‐ and cone‐shaped bur cut cavities were prepared in the approximal surfaces of 108 teeth and their depth determined using silicone putty impressions. Spacial relationships were controlled by fixing specimens into perspex blocks. Standardized radiographs were taken at baseline (0°) and then using pre‐angled wedges to imitate horizontal X‐ray beam movements of 5, 10 and 15° in both mesial and distal directions. Radiographic cavity depth to the nearest 0·1 mm was measured. Over the range of X‐ray beam movements, more than 72% of cavities deepened compared to baseline. As bucco‐lingual cavity width increased the magnitude of the depth change increased. For some cylindrical cavities increases equating to 200% over baseline were seen; in some rectangular cavities increases of up to 400% over baseline occurred. The present study suggests that changes in irradiation geometry from baseline are likely to result in a cavity appearing to deepen. In a clinical context over‐treatment is therefore the most likely outcome.