bluuebunny/crossref_metadata_2025 huggingface dataset:
In Situ Hybridization Detection of TSHβ Subunit Gene Expression in the Serum‐Free Primary Culture of the Adult Rat Pituitary
Authors: Koibuchi Noriyuki , Ohtake Hideki , Sakai Mikako , Kato Yukio , Yamaoka Sadao
Published: September 2006

This study was designed to construct the primary culture system to detect the change in TSHβ subunit (TSHβ) gene expression in individual cells. Adult, male Wistar rats were sacrificed by transcardial perfusion of 0.25% trypsin solution under pentobarbital anesthesia (50 mg/kg body weight). Their anterior pituitaries were removed, dispersed and cultured for 1, 2, 3 or 6 days with or without 1 nM triiodothyronine (T3) under the serum‐free condition. In some cultures, TRH was added to a final concentration of 1 μM on 6, 12 or 24 h before fixation. Then the culture media were removed to measure TSH concentration. Cells were fixed with paraformaldehyde and hybridized with 35S‐labeled RNA probe complementary to TSHβ mRNA. Emulsion autoradiography was subsequently performed. T3 treatment markedly suppressed relative cellular levels of TSH4bT mRNA on 2, 3 and 6 days after the onset of culture (day 2, 3 and 6) and suppressed TSH secretion on day 3 and 6. TRH treatment increased TSHβ mRNA on 12 and 24 h after the treatment on day 2 and 3 but did not increase TSHβ mRNA on day 6. TSH concentration in the culture medium was increased by TRH treatment on 6, 12 and 24 h after the treatment on day 2, on 12 h and 24 h on day 3, and 24 h on day 6. On day 2 and 3, although T3 treatment suppressed basal level of TSHβ mRNA, TRH‐induced increase in TSHβ mRNA was not suppressed by T3 treatment. These results show that the thyroid hormone and TRH regulate TSHβ gene expression independently. Our culture system may provide a useful model to examine the action of individual substances on a specific subpopulation of the anterior pituitary cells.
Distribution of Estrogen Receptor‐lmmunoreactive Cells in the Brain of the Rainbow Trout (Oncorhynchus mykiss)
Authors: Anglade Isabelle , Pakdel Farzad , Bailhache Thierry , Petit Fabrice , Salbert Gilles , Jego Patrick , Valotaire Yves , Kah Olivier
Published: September 2006

Using antibodies against the hormone binding domain of the trout estrogen receptor (ER), the distribution of ER‐immunoreactive (ER‐IR) cells was studied in the brain of maturing diploid and triploid female rainbow trout using a streptavidin‐biotin‐peroxidase method followed by a nickel‐intensified diaminobenzidine reaction. This technique resulted in an excellent signal/background ratio allowing unambiguous identification of positive cells. In all animals, ER‐IR cells were consistently located in three brain regions, the ventral telencephalon, the anterior ventral preoptic region, and the mediobasal hypothalamus. About 250 ER‐IR cells were observed in the ventral and dorsal parts of the ventral telencephalon. In the anterior nucleus preopticus periventricularis, about 2400 ER‐IR cells were observed surrounding the preoptic recess. In the posterior hypothalamus, approximately 2700 ER‐IR cells were located in the anterior, posterior and inferior divisions of the nucleus lateralis tuberis and in the nucleus saccus vasculosus. In these regions, cell nuclei exhibiting different densities of staining were observed and absolutely no labeling of cytoplasmic processes was detected. These results are in partial agreement with those obtained either after injection of tritiated‐estradiol in other teleots species or in situ hybridization of ER mRNAs in trout. In particular, no immunoreactivity was observed in the thalamic region nor in the nucleus posterioris periventricularis. These data indicate that target cells for estradiol are essentially located in brain regions involved in the neuroendocrine control of pituitary functions and having direct connections with the hypophysis.
Diurnal Rhythm in Proopiomelanocortin mRNA in the Arcuate Nucleus of the Male Rat
Authors: Steiner Robert A. , Kabigting Emilia , Lent Karin , Clifton Donald K.
Published: September 2006

We tested the hypotheses that in the male rat, expression of proopiomelanocortin (POMC) mRNA in cells of the arcuate nucleus displays a diurnal fluctuation and that expression of this rhythm is dependent upon the secretory products of the testis. To accomplish this, we sacrificed groups of testes‐intact and castrated adult male rats throughout the day and compared levels of POMC mRNA in individual cells of the arcuate nucleus across time and between groups. Adult male rats were housed on a 12–12 L D cycle with lights on a 0600 h and were divided into groups that were either castrated or left intact. Four days later, pairs from these groups were sacrificed at 0600 h, 1200 h, 1800 h, 2400 h, and again at 0600 h (n = 4 per group at each time point). We used in situ hybridization and a computerized image analysis system to measure cellular levels of POMC mRNA, as reflected by the number of autoradiographic grains over individual cells in the rostral quarter of the arcuate nucleus (counting ∼ 30 cells per animal). Using cosinor analysis, we observed that in intact male rats, POMC mRNA levels varied significantly over the 24 h day with a nadir value at 1800 h. In contrast, there was no significant diurnal variation in POMC mRNA levels in castrated animals. POMC mRNA levels were significantly greater in the intact compared with castrated animals at every time point (P<0.01), except at 1800 h, when the groups did not differ significantly from one another. We conclude that adult male rats display at diurnal rhythm in cellular POMC mRNA levels in the arcuate nucleus, and we infer that testosterone or some other secretory product of the testis is a prerequisite for expression of this rhythm.
Differential Dependence of ACTH Secretion Induced by Various Cytokines on the Integrity of the Paraventricular Nucleus
Authors: Kovács Krisztina J. , Elenkovt Ilia J.
Published: September 2006

Effect of different cytokines, human recombinant interleukin‐la and fl (IL‐1 a, 1L‐1 j), interleukin‐6 and tumor necrosis factor‐a (TNF) on adrenocorticotropin (ACTH) secretion was compared in sham‐operated rats and those with lesions of the hypothalamic paraventricular nucleus. IL‐1 a was less active than IL‐1 b in stimulating ACTH in sham‐operated rats. Intravenous injection of IL‐1 fl in sham‐operated animals resulted in a rapid elevation of ACTH secretion. Five days after surgical lesion of the paraventricular nucleus, the main hypothalamic source of hypophysiotropic corticotropin‐releasing factor‐41, the response to IL‐1 /I was attenuated but not abolished. This suggests involvement of extra‐paraventricular releasing factors in mediation of ACTH‐releasing activity of IL‐1 P, altered responsiveness of pituitary to CRFs, and/or direct action of IL‐1 j on the corticotrope cells. TNF resulted in a biphasic stimulation of ACTH concentration, with peaks at 15 min and 90 min. In paraventricular‐lesioned, TNF injected rats both of these ACTH peaks disappeared, suggesting that CRFs from the paraventricular origin mediates ACTH‐inducing activity of TNF. IL‐6 elevated ACTH secretion much later than the other intravenously injected cytokines, the peak was at 1 h in sham‐lesioned rats. Paraventricular lesion completely prevented the increase of ACTH plasma levels after IL‐6 injection. These data suggest that: (1) Effect of TNF and IL‐6 on hypothalamo‐pituitary‐adrenal axis is mediated through the hypothalamic paraventricular nucleus and (2) IL‐lB is able to release ACTH even in the absence of hypothalamic drive.
Negative Feedback Regulation following Administration of Chronic Exogenous Corticosterone
Authors: Young Elizabeth A. , Kwak Seung P. , Kottak Juliet
Published: September 2006

Administration of exogenous glucocorticoids is known to suppress the HPA axis and has been reported to occupy brain glucocorticoid receptors, eventually leading to down‐regulation. To determine the effects of chronic corticosterone administration on HPA axis function, corticosterone was administered as both 25% and 50% corticosteronekholesterol pellets. Rats were sacrificed 6 days after corticosterone pellet implantation. The 25% corticosterone pellets produced a small increase in morning corticosterone concentrations but no change in evening ACTH or corticosterone secretion. The 50% corticosterone pellets produced constant corticosterone concentrations of 5–6 pg/dl, with no circadian variation in corticosterone, indicating inhibition of evening ACTH and corticosterone secretion. The 25% corticosterone pellets produced no significant decrease in thymus weight or in adrenal weight; 50% corticosterone pellets produced significant decreases in thymus weight and adrenal weight. Neither 25% nor 50% corticosterone pellets produced significant decreases in GR in hippocampus and cortex. The 50% corticosterone pellets treatment resulted in a decrease in anterior pituitary POMC mRNA levels, a decrease in baseline and oCRH stimulated ACTH release from the anterior pituitary, and a near complete inhibition of the AM and PM response to restraint stress. These results suggest that: 1) the HPA axis was able to adjust to the small increase in glucocorticoids produced by the 25% cort pellets with minimal disturbances in function and 2) 50% corticosterone pellets exert a significant inhibitory effect on stress and diurnal ACTH secretion which appears to be exerted at the pituitary as well as possible inhibitory effects on brain.
Nucleotides as Extracellular Signalling Molecules
Authors: Chen Zhen‐Ping , Levy Andrew , Lightman Stafford L.
Published: September 2006

There is now wide acceptance that ATP and other nucleotides are ubiquitous extracellular chemical messengers. ATP and diadenosine polyphosphates can be released from synaptosomes. They act on a large and diverse family of P2 purinoceptors, four of which have been cloned. This receptor family can be divided into two distinct classes: ligand‐gated ion channels for P2X receptors and G protein‐coupled receptors for P2Y, P2U, P2T and P2D receptors. The P2Y, P2U and P2D receptors have a fairly wide tissue distribution, while the P2X receptor is mainly found in neurons and muscles and the P2T and P2Z receptors confined to platelets and immune cells, respectively. lnositol phosphate and calcium signalling appear to be the predominant mechanisms for transducing the G‐protein linked P2 receptor signals. Multiple P2 receptors are expressed by neurons and glia in the CNS and also in neuroendocrine cells. ATP and other nucleotides may therefore have important roles not only as a neurotransmitter but also as a neuroendocrine regulatory messenger.
Corticosteroid Feedback Resistance in Rats Genetically Selected for Increased Dopamine Responsiveness
Authors: Rots Nynke Y. , Cools Alexander R. , de Jong Jeannette , De Kloet E. Ronald
Published: September 2006

Pharmacogerietically selected Wistar rat lines were used to investigate the implication of either high or low responsiveness of the dopamine system for the activity of the hypothalamus‐pituitary‐adrenal (HPA) axis. As selection criterion the gnawing response induced by the dopamine agonist apomorphine was used. This criterion allows to distinguish apomorphine susceptible (apo‐sus) rats which show a vigorous gnawing response from apomorphine unsusceptible (apo‐unsus) rats. The present study, using male animals of the 9‐1 2th generation of the two rat lines, revealed the following characteristics of the stress response system: (i) in apo‐sus rats under basal conditions corticotrophin‐releasing hormone (CRH) mRNA level in the paraventricular nucleus (PVN) and plasma adrenocorticotropin (ACTH) concentration were significantly higher; total corticosterone (B) plasma level was similar but free B level was lower; (ii) exposure to a novel environment resulted in a higher and prolonged plasma ACTH and total B response in the apo‐sus rats. Moreover, the elevated free B level was also prolonged; (iii) apo‐sus rats had increased CRH‐induced pituitary ACTH release and B secretion was also increased, but not as prolonged as during novelty. (iv) In dexamethasone‐pretreated rats an intravenous ACTH1–24 injection resulted in a similar plasma B response in rats of both lines; (v) In vitro, ACTH1–24 produced a significantly higher B secretion by adrenocortical cells of apo‐sus rats reflecting the higher in vivo ACTH priming of the adrenal glands in these animals. (vi) apo‐sus rats had higher body and thymic weight.

In conclusion, rats genetically selected for increased susceptibility of the dopamine system to apomorphine display subtle adrenocortical hyporesponsiveness and show resistance to corticosteroid hormone feedback action.
Nicotine Acts Directly on Pituitary GH3 Cells to Inhibit Prolactin Promoter Activity
Authors: Coleman Daniel T. , Bancroft Carter
Published: September 2006

We have employed the GH3 rat pituitary cell line to investigate whether nicotine can regulate prolactin (PRL) gene expression. Nicotine strongly inhibited (45%) transient expression of a construct containing the first 187 base‐pairs of the rat PRL promoter cloned upstream of the chloramphenicol acetyl transferase (CAT) gene. This implies that nicotine acts directly on the GH3 cells to inhibit transcription directed by the PRL promoter. Expression of a control reporter construct containing the CAT gene under the control of the RSV promoter was not affected by exposure of the cells to nicotine, demonstrating that the effect of nicotine is promoter‐specific. The inhibition by nicotine of PRL promoter activity was not blocked by hexamethonium, suggesting that this effect of nicotine may be mediated by a novel type of nicotine receptor previously described in frog pituitary cells. Nicotine was also observed to yield a concentration‐dependent inhibition of the stimulation by thyrotrophin‐releasing hormone (TRH) of PRL promoter activity, implying that nicotine can also interfere with hormonal regulation of the PRL gene. These results suggest that the reduced serum PRL levels that result from smoking may originate in part from decreased transcription of the PRL gene resulting from a direct effect of nicotine on pituitary PRL‐secreting cells.
Biosynthesis and Processing of the N‐Terminal Part of Proopiomelanocortin in Xenopus laevis: Characterization of γ‐MSH Peptides
Authors: van Strien Frank J. C. , Devreese Bart , Van Beeurnen Jozef , Roubos Eric W. , Jenks Bruce G.
Published: September 2006

The aim of this study was to determine the terminal products of processing of the N‐terminal part of proopiomelanocortin (POMC) in pituitary melanotrope cells of Xenopus laevis. Biosynthetic in vitro labelling studies showed that POMC is rapidly processed to form N‐terminal peptides with an estimated molecular mass of 18 kDa, 9 kDa and 4 kDa. All peptides were released into the medium, indicating that they are processing end products. An antiserum was raised against the synthetic N‐terminal eight amino acids of the putative Xenopusγ‐MSH which is present in the N‐terminal part of POMC. With immunocytochemistry we demonstrated that γ‐MSH‐immunoreactive material in the pituitary gland is restricted to the pars intermedia. A radioimmunoassay in combination with reversed‐phase HPLC revealed the presence of at least two γ‐MSH‐like peptides. Complete purification followed by electrospray ionization mass spectrometry and amino acid sequence determination showed that these peptides are γ1‐MSH and glycosylated γ3‐MSH. The amounts of these γ‐MSH peptides were low compared to the other POMC‐derived peptides, α‐MSH and β‐endorphin. Only 10% of POMC is processed into γ‐MSH peptides and the 4 kDa peptide, leaving the 18 kDa and 9 kDa peptides as the major end products.
Intraventricular Prolactin Inhibits Hypothalamic Vasoactive‐Intestinal Polypeptide‐Expression in Doves
Authors: Saldanha Colin J. , Silver Rae
Published: September 2006

While the role of prolactin in promoting the development of the crop‐sac in members of the pigeon family (Columbiformes) is well established, its action in the central nervous system is less well understood. In the present study, prolactin was administered intracerebroventricularily (i.c.v.) in ring doves, and central expression of vasoactive‐intestinal polypeptide (VIP) and gonadotropin‐releasing hormone (GnRH), and the display of sexual behavior was investigated. Ovine‐prolactin (1 μg in 2 μl o‐prl) was injected daily for six days through chronically implanted cannula either prior to a 2‐h period of courtship, or late in incubation. Control subjects were given vehicle injections and were otherwise identical to experimental animals. Prolactin administered prior to courtship resulted in a reduction of sexual behavior, and in a decrease in testicular weight but had no detectable effect on the number of neurons expressing VIP or GnRH. In contrast, i.c.v. prolactin during incubation resulted in a reduced number of infundibular VIP‐positive neurons and decreased crop weight. We conclude that during incubation prolactin regulates its own synthesis and/or release by modulating VIP expression in infundibular neurons.
The Effect of Pentobarbital on the Electrical Activity of LHRH Pulse Generator in the Ovariectomized Rat with or without Estrogen Priming
Authors: Kimura Fukuko , Sano Akane
Published: September 2006

Effects of pentobarbital sodium (PB) on the electrical activity of LHRH pulse generator were investigated in the ovariectomized (OVX) rat fitted with chronically implanted electrode arrays in the arcuate‐median eminence region of the hypothalamus. PB was injected intraperitoneally (i.p.) at a dose of 32 mg/kg bw, which has blocked the surge of LH secretion when injected at 13.45 on the day of proestrus, presumably by blocking the activity of the LHRH surge generator. After i.p. injection of PB, the first characteristic increase (volley) in the hypothalamic multiunit activity (MUA), each of which was associated with the initiation of an LH pulse, appeared at the mean interval of 42 min, which was significantly greater than the value, 30 min, in the rat given i.p. injection of saline. The second and third MUA volleys appeared, however, at similar intervals to those in rats given saline. There were no significant changes in the amplitude of LH pulses which followed. In the OVX rat implanted with a silastic tube packed with estradiol‐benzoate (E2) for 1 day, MUA volleys were unclear, mingled with high background activities, but clear volleys appeared after PB injection. The mean interval of LH pulses in the rat given i.p. injection of saline was 51 min, by 24 min longer than the value in the E2‐unimplanted OVX rat. But, after PB injection, the 1st LH pulse appeared at the interval of 37 min, which was by 25 min smaller than that after saline and the overall mean of 3 intervals was 34 min, showing a significant shortage. No PB effect was seen in the LH pulse amplitude in the E2‐implanted OVX rat. The presults show that the LHRH pulse generator is much more resistant to PB than the surge generator in the OVX rat either with or without estrogen priming, and in the rat with estrogen priming, the pulse generator is rather activated by PB.
Evidence for a Regulatory Role of Melatonin on Serotonin Release and Uptake in the Pineal Gland
Authors: Míguez Jesús M. , Simonneaux Valérie , Pévet Paul
Published: September 2006

Melatonin has been proposed to exert some regulatory actions within the pineal gland itself. The present study examined the effect of melatonin on the release of serotonin (5‐HT) and 5‐hydroxyindoleacetic acid (5‐HIAA) from rat pineal glands by using an in vitro perifusion system. Melatonin induced a concentration‐dependent stimulatory effect on 5‐HT secretion from 10−6 M to 10−3 M. Maximal effects were obtained with melatonin 10−3M and concentrations lower than 10−6 M were without effect. The secretion of 5‐HIAA was inhibited by melatonin 10−3 and 10−4M, but it was increased when pineals were incubated with 10−5 and 10−6 M of melatonin. The indoleamine secretion was also studied on peripherally denervated rat pineal glands. Basal output of 5‐HT from these glands was increased when compared with those from control rats. In contrast, the secretion of 5‐HIAA was strongly reduced after removal of the sympathetic input to the pineal gland. Melatonin 10−3 M failed to stimulate 5‐HT release from denervated pineal glands, although it inhibited 5‐HIAA secretion. In contrast, melatonin 10−5 M enhanced 5‐HT release without altering 5‐HIAA output. Fluoxetine, a 5‐HT uptake inhibitor, produced similar effects than mM concentrations of melatonin on the indoleamine secretion from control pineal glands, but it had no effect on glands taken from peripherally denervated rats. These data suggest that mM concentrations of the pineal hormone are able to stimulate 5‐HT release from the pinealocyte, while mM concentrations of melatonin increase extracellular 5‐HT by inhibiting its reuptake in the adrenergic nerve endings. These findings are discussed in relation to the possible role of melatonin regulating the intra‐ and extracellular availability of 5‐HT in the pineal gland and its significance as an autocrine factor.
Anxiolytic Effect of Progesterone is Mediated by the Neurosteroid Allopregnanolone at Brain GABAA Receptors
Authors: Bitran Daniel , Shiekh Michael , McLeod Michael
Published: September 2006

Previous studies from this laboratory have shown that progesterone (PROG) treatment in ovariectomized rats produces an anti‐anxiety response similar to that observed after the administration of prototypical anxiolytic benzodiazepine (BDZ) compounds. The PROG‐induced anxiolytic response was highly correlated with an increased level of 3α‐hydroxy‐5α‐pregnan‐20‐one (allopregnanolone) in the blood and brain, and was also associated with a facilitation of GABA‐stimulated chloride ion (Cl−) influx in cortical synaptoneurosomes. This correlative evidence suggested that the anxiolytic effect of PROG was a result of its in vivo reduction to the neuroactive steroid, allopregnanolone. In this report, a series of studies was conducted to determine the mechanism(s) by which PROG alters behavior in animal models of anxiety. In the first experiment, ovariectomized rats were injected with PROG (1 mg/0.2 ml, SC) 4 h prior to a test in the elevated plus‐maze. Some animals also received an injection of picrotoxin (0.75 mg/kg, IP), a GABAA receptor‐gated Cl− channel antagonist, whereas other animals were pretreated with RU 38486 (5 mg/0.2 ml, SC), a progestin receptor antagonist. PROG elicited anxiolytic behavior in the plus‐maze, an effect that was blocked by picrotoxin administration. Pretreatment with RU 38486 was not effective in altering PROG‐induced anxiolytic behavior in the plus‐maze. In a second experiment, the effect of PROG on behavior in the plus‐maze was determined in the presence of N,N‐diethyl‐4‐methyl‐3‐oxo‐4‐aza‐5α‐androstane‐17β‐carboxamide (4‐MA; 10 mg/0.2 ml, SC), a 5α‐reductase inhibitor. The enzyme inhibitor was potent in preventing the anxiolytic effect observed in the plus‐maze after PROG administration. In the defensive burying paradigm, PROG treatment also produced anxiolysis by reducing the duration of burying behavior, and this effect was prevented by 4‐MA pretreatment, but not by RU 38486 administration. After the completion of the behavioral assays, analysis of blood allopregnanolone levels revealed a marked increase in PROG‐treated females. The PROG‐induced elevation in circulating allopregnanolone was blocked by pretreatment with 4‐MA. In cortical synaptoneurosomes, the sensitivity (inverse of the EC50) and the maximal response (Emax) in GABA‐stimulated 36Cl− uptake were increased in PROG‐treated females. The potentiation of PROG on both of these neurochemical measures was not observed in animals pretreated with 4‐MA. Together, these studies provide evidence that the anxiolytic effect of PROG is not associated with an intracellular steroid receptor that initiates genomic‐mediated responses. The evidence is consistent with a nongenomic mechanism whereby PROG is metabolized to allopregnanolone, a neuroactive steroid that potentiates GABAA receptor‐mediated responses.
5β‐Reductase and other Androgen‐Metabolizing Enzymes in Primary Cultures of Developing Zebra Finch Telencephalon
Authors: Schlinger Barney A. , Amur‐Umarjee Shashi , Campagnoni Anthony T. , Arnold Arthur P.
Published: September 2006

Enzymes in the avian brain irreversibly catalyze the conversion of androgens into either active metabolites (aromatase and 5α‐reductase) or inactive metabolites (5β‐reductase). 5β‐reductase is thought to influence the formation of active metabolites by reducing the concentration of androgenic substrate available for these reactions. However, because these enzymes have different regional, cellular and subcellular distributions in brain, the traditional method to measure enzyme activity in brain homogenates may be inaccurate because of artificial mixing of enzymes. Recently, we have prepared primary cell cultures from the telencephalons of developing zebra finches. Cell cultures offer the advantage that enzyme activity can be measured in live cells in which the relative distribution of enzymes may more closely reflect that found in vivo. We have previously reported that aromatase is expressed at high levels in these cultures, and that it is active in both neurons and in glia. Here we report on the activities of 5α‐ and 5β‐reductase in these cell cultures. Along with aromatase, 5β‐reductase was expressed at high levels in these mixed cell cultures, including cultures highly enriched in glia. This suggests that 5β‐reductase is present in non‐neuronal cells in brain, possibly co‐localized with aromatase. However, despite the presence of 5β‐reductase, aromatase was detected reliably in vitro even when the concentration of substrate was low. Thus, 5β‐reductase does not prevent the synthesis of estrogen in the telencephalon of developing zebra finches. By contrast, 5α‐reductase activity was very low or absent in these cultures. Thus, cells expressing 5α‐reductase may be poorly represented in these cultures. Alternatively, 5α‐reductase and aromatase together may interfere with the synthesis of 5α‐reduced androgens in the zebra finch telencephalon.
Estradiol Induces Vasoactive Intestinal Peptide and Prolactin Gene Expression in the Rat Anterior Pituitary Independently of Plasma Prolactin Levels
Authors: Montagne Marie‐Noëlle , Dussaillant Monique , Chew Li‐Jin , Berod Anne , Lamberts Steven J. , Cartert David A. , Rostene William
Published: September 2006

It is well established that estrogens are potent stimulators of prolactin (PRL) secretion. It has also been demonstrated that estradiol (E2) can increase the expression and the anterior pituitary levels of the vasoactive intestinal peptide (VIP), a peptide which also acts as a potent PRL‐releasing factor. It thus remained unknown whether the effects on pituitary VIP were due to E2 itself or to E2–induced hyperprolactinemia (HPRL). In order to test this hypothesis, various plasma PRL levels were induced in rats either with ectopic pituitary grafts, PRL secreting tumours or E2 implants, and VIP mRNA expression in the anterior pituitary was measured by in situ hybridization and Northern blot analyses. Whereas decreases in VIP mRNA can be observed in pituitaries of rats with pure HPRL, a 6‐fold increase in VIP mRNA can be seen in E2‐treated rats. E2 increased both 1.0 and 1.7 Kb VIP mRNA species. The presence of the graft in E2‐treated rats significantly reduced the increase in VIP mRNA observed following E2. The direct stimulation by E2 of VIP mRNA expression was further demonstrated by the fact that statistical analysis of the data indicated that both E2 and graft were acting independently of each other, and that a new selective antiestrogen, RU 58668, almost totally blocked the effect of E2. Moreover, under similar experimental conditions, pituitary PRL mRNA levels were reduced in the graft group and a marked up‐regulation was observed similarly in both E2 and in E2 rats bearing ectopic grafts. We observed furthermore that not only levels of the mature 1.0 Kb PRL mRNA but also the 1.7 and 3.8 KbPRL precursor RNAs were increased. Such changes were associated with modifications in PRLmRNA size due to an increase in 3′polyA tail lenghts. The present data demonstrate that E2 directly affects VIP mRNA and PRL mRNA expression in the pituitary and support the hypothesis that VIP may play a role in the hypersecretion of PRL associated with the formation of E2‐induced prolactinomas.
Estrogen‐Sensitive Oxytocin Binding Sites are Differently Regulated by Progesterone in the Telencephalon and the Hypothalamus of the Rat
Authors: Krémarik P. , Freund‐Mercier M. J. , Stoeckel M. E.
Published: September 2006

The localization at the cellular level and the regulation by progesterone of the estrogen‐sensitive oxytocin binding sites was studied in the rat telencephalon and the hypothalamus by using quantitative film‐autoradiography and histoautoradiography. Male rats (castrated or not) and ovariectomized females (estradiol supplemented or not) were used to characterize these sites and to precise their localization. They were detected in the striatal cell bridges, the olfactory tubercle, the principal nucleus of the bed nucleus of the stria terminalis and the medial nucleus of the amygdala of the telencephalon and in the medial preoptic, the ventromedial and the ventral premammillary nuclei of the hypothalamus. Estrogen administration in addition induced expression of oxytocin binding sites in the major island of Calleja, the anterior hypothalamic area and the terete nucleus. The density of the estrogen‐sensitive oxytocin binding sites varied during the estrous cycle, but differently in the telencephalon and the hypothalamus. in the telencephalon it peaked at proestrus 9 h and was already decreased at proestrus 21 h, whereas in the hypothalamus it was similarly high at proestrus 9h and proestrus 21 h, suggesting the intervention of progesterone in the regulation of the hypothalamic estrogen‐sensitive oxytocin binding sites.
Localization in the Nervous System of Drosophila Melanogaster of a C‐Terminus Anti‐Peptide Antibody to a Cloned Drosophila Muscarinic Acetylcholine Receptor
Authors: Harrison J. Barrie , Chen Hong Hong , Blake Allan D. , Huskisson Neville S. , Barker Patrick , Sattelle David B.
Published: September 2006

Localization in the nervous system of Drosophila melanogaster of a cloned Drosophila muscarinic acetylcholine receptor (mAChR) was investigated using a polyclonal antiserum raised against a peptide corresponding to the predicted receptor carboxyl terminal domain. Immunocytochemical studies on fly sections indicated that the product of the Dm1 mAChR gene was localized in the antennal lobes and in other regions of the brain and thoracic nervous system. Intense staining in the glomeruli of the antennal lobes, the region of the nervous system containing terminals of antennal olfactory sensory neurones and mechanosensory neurones, indicates possible roles for this mAChR gene product in the processing of olfactory and mechanosensory signals in the fly. The staining of a discrete group of neurosecretory cells in the pars intercerebralis of the brain indicates a possible new role for this mAChR in the regulation of neurosecretion. Very little staining is detected in the thoracic nervous system.
Three Dimensional Culture of Pineal Cell Aggregates: A Model of Cell‐Cell Co‐operation
Authors: Khan Naim A. , Shacoori Valiollah , Havouis Réné , Querné Dominique , Moulinoux Jacques‐Ph. , Rault Bernard
Published: September 2006

Three dimensional (3‐D) cultures of pineal cell aggregates were obtained by constant gyratory shaking the heterogenous cell populations, obtained from the rat pineals, in the DMEM (Dulbecco's modified Eagle's medium). Within 4 days, the pineal cells became organized into a tissue like configuration appearing as a compact ball, evidenced by the scanning electron microscopy. The 3‐D aggregates seemed to be mainly composed of pinealocytes (round‐oval cells), glial (elongated cells) and other unknown cells. The heterogenous cells were separated by intercellular spaces. The ultrastructural characteristics revealed by transmission electron microscopy exhibited the presence of granular lysosomes, typical of pinealocytes actively involved in the secretion. These pineal cell aggregates secreted melatonin and other indole amines i.e. 5‐methoxytryptamine (5‐MT), indole acetic acid (IAA), 5‐methoxy‐3‐indole acetic acid (5‐MIAA), tryptophol (TOL) and 5‐methoxytryptophol (5‐MTL) in the culture medium, indicating the functional aspect of pinealocytes. The 3‐D aggregates cultures had advantages over the pineal monolayer cultures as, after 4 days of culture, the amounts of indole amines secreted by 3‐D aggregates were higher than those secreted by monolayer cultures. Besides, the 3‐D aggregates remained functional till 24 days in the gyratory culture conditions. In the continuous perifusion system, the 3‐D aggregates secreted melatonin while challanged with isoproterenol. This 3‐D model of pineal cell aggregates might be useful, in future, to perform other kinetic studies of the release of indole amines in perifusion experiments as this system allows the maintenance of pineal cells for a long period of time.
The Emerging Concept of Relaxn as a Centrally Actng Peptide Hormone with Hemodynamic Actions
Authors: Geddes Brad J. , Summerlee Alastair J. S.
Published: September 2006

The novel finding that relaxin has an action on the brain was first published in 1984. Since then, it has been shown that exogenous relaxin affects the release of a number of hypothalamo‐pituitary hormones and has a robust pressor action. In this paper, we review the accumulating evidence that relaxin affects the release of oxytocin and vasopressin by an action at the level of the brain. The potential mechanisms of this central action are discussed and the evidence presented for the interaction between relaxin and the forebrain angiotensin‐ll system. Furthermore, we articulate the possible physiological influences of relaxin on the changes in cardiovascular function that occur during pregnancy.
Central Oxytocin Mediates Stress‐Induced Tachycardia
Authors: Morris Mariana , Callahan Michael F. , Li Ping , Lucion Aldo B.
Published: September 2006

To address the role of oxytocin in the control of cardiovascular reactivity, we examined the effect of central injection of oxytocin, vasopressin and mixed base antisense oligodeoxynucleotides on stress‐induced cardiovascular and endocrine changes. Antisense oligomers were injected into the paraventricular nucleus (PVN), 4 h prior to the stress test. The oxytocin antisense abolished the tachycardia produced by 5 min of shaker stress. The blood pressure and plasma oxytocin responses were not different between the groups. PVN levels of OT were reduced in the oxytocin antisense‐treated group while brain stem levels were increased. These results demonstrate the importance of a specific peptide system, the PVN/oxytocin axis, in stress‐induced tachycardia. Further, the data illustrate the effectiveness of short‐term treatment with antisense oligomers on physiological responses.
Caloric Intake Stimulates Growth Hormone Secretion in Food‐Deprived Rats with Anterolateral Deafferentation of the Medial Basal Hypothalamus or Administered Antiserum to Somatostatin
Authors: Suzuki Nobuchika , Okada Kenmei , Sugihara Hitoshi , Minami Shiro , Wakabayashi Ichiji
Published: September 2006

In rats, food deprivation inhibits episodic growth hormone (GH) secretion. On the basis of previous studies, we hypothesized that during a recovery from prolonged fasting, caloric intake stimulates the release of GH‐releasing factor (GRF) and this process does not depend on the specific macronutrients in the meal, while protein in the meal acts to restore characteristic ultradian rhythmicity of GH secretion. To test this hypothesis, the effect of caloric intake on GH secretion was examined in fasted adult male Wistar rats devoid of somatostatin (SS) influence on GH secretion either by anterolateral deafferentation (ALC) of the medial basal hypothalamus (MBH) or administration of anti–SS goat serum (ASS). Rats were provided with an indwelling right atrial cannula and were deprived of food for 72 h. ALC was performed 2 weeks prior to the study. ASS was given i.v. 8 h and 7 h prior to refeeding, respectively. Serial blood specimens were collected every 10 min. fn rats with ALC (ALC rats) or rats given ASS (ASS rats), the blood GH level revealed irregularly occurring small fluctuations, instead of the usual high bursts and low trough level. The baseline GH level and the mean GH level of fasted ALC rats or fasted ASS rats were significantly lower than those of fed ALC rats or fed ASS rats. Feeding the isocaloric mixed meal, the protein meal or the protein‐deficient meal increased the GH pulse frequency, the pulse amplitude, the baseline GH level and the mean GH level in 72–h fasted ALC rats. These changes in GH secretory pattern persisted during the period of observation and were independent of the type of meal ingested. Following feeding the mixed meal, similar changes in the GH secretory pattern demonstrated in 72‐h fasted ALC rats were also observed in 72–h fasted ASS rats, suggesting that the stimulation of GH secretion following caloric intake is not limited to ALC rats. Since the influence of SS on GH secretion has been largely eliminated in ALC or ASS rats, it is highly unlikely that the augmentation of GH secretion following feeding after prolonged food deprivation was the consequence of inhibition of SS secretion. Although GRF measurement was not performed, it is conceivable that the signal of caloric intake is conveyed to the MBH and acts to stimulate GRF release.
Effects of Dehydroepiandrosterone (DHEA) on Pituitary Prolactin and Arcuate Nucleus Neuron Tyrosine Hydroxylase mRNA Levels in the Rat
Authors: De Yebenes Ester Garcia , Hong Min , Pelletier Georges
Published: September 2006

It is well documented that dehydroepiandrosterone (DHEA), an adrenal androgen, is converted into potent androgens and/or estrogens in peripheral tissues. Since sex steroids are involved in the regulation of prolactin (PRL) secretion, we have studied the effect of DHEA administration on PRL mRNA levels in both adult male and female rats. Since tuberoinfundibular dopaminergic (TIDA) neurons are involved in the negative regulation of PRL, we have also evaluated the effects of DHEA on the genetic expression of tyrosine hydroxylase (TH), the limiting enzyme in catecholamine biosynthesis in TIDA neurons. Sham‐operated and castrated animals of both sexes received during 2 days DHEA at the dose of 6 mg/kg/day, starting on the first day after castration. PRL and TH mRNA levels were measured by quantitative in situ hybridization. In the male rat, orchiectomy performed 3 days earlier did not modify PRL mRNA levels. DHEA administration increased the hybridization signal in both sham‐operated and orchiectomized animals. In the female, ovariectomy decreased PRL mRNA levels and, as observed in the male, DHEA treatment induced an increase in the hybridization signal in both control and ovariectomized rats. In TIDA neurons, castration increased TH mRNA levels as evaluated by number of grains over labelled neurons and the number of TH‐labelled cells per section in both male and female animals. In both sham‐operated male rats and orchiectomized animals, DHEA decreased the hybridization signal. In the female, DHEA administration completely prevented the increase in TH mRNA levels due to ovariectomy. In sham‐operated female rats, the treatment had no effect. These data clearly indicate that in both male and female rats DHEA exerts an estrogenic influence on both PRL and TH gene expression. Although these in vivo experiments do not allow to establish whether the stimulation of PRL gene expression is due to an action of the steroid on the pituitary or at the hypothalamic level or alternatively at both sites, it is likely that one of the mechanisms of action of DHEA might be related to a decrease in dopamine release following a depression of TIDA neuron activity.
A Role for the Mineralocorticoid Receptor in a Rapid and Transient Suppression of Hippocampal 5‐HT1A Receptor mRNA by Corticosterone
Authors: Meijer Onno C. , de Kloet E. Ronald
Published: September 2006

The interaction between corticosterone and hippocampal 5‐HT1A receptor mRNA expression was investigated in male rats. Two days after adrenalectomy, 5‐HT1A receptor mRNA was increased in the granule cell layer of the dentate gyrus. Injection of corticosterone led to a significant dose‐dependent and transient suppression of 5‐HT1A receptor mRNA. The effect of a low dose of corticosterone (50 μg/kg) could be blocked by RU 28318, a specific mineralocorticoid receptor antagonist, but not by RU 38486, a glucocorticoid antagonist. The effect of a higher dose of corticosterone (300 μg/kg) could be partially blocked by RU 28318, whereas there was no additional effect of RU 38486. These results point to a stringent regulation of hippocampal 5‐HT1A receptors by corticosterone that is predominantly mediated by the mineralocorticoid receptor.
Distribution of Androgen Receptor‐Like Immunoreactivity in the Brains of Cynomolgus Monkeys
Authors: Michael Richard P. , Clancy Andrew N. , Zumpe Doris
Published: September 2006

A polyclonal antibody, PA1, raised in a rabbit against fusion proteins containing fragments of the human prostatic androgen receptor (AR) was used to map the distribution of AR‐like immunoreactivity in the brains of adult male and female cynomolgus monkeys. PA1 AR‐immunoreactive (ARir) labeling occurred in the cell nuclei and, more weakly, in the cytoplasm of brain cells. The PA1 ARir labeling occurred primarily in brain regions previously shown on the basis of gonadal steroid autoradiography to contain androgen receptors. However, the distribution of PA1 ARir staining was substantially more restricted than that of autoradiographic labeling using 3H‐androgens. The pattern of PA1 ARir labeling was closely similar between animals and occurred in the lateral septum, medial preoptic area, bed nucleus of stria terminalis, anterior, cortical, accessory basal and medial amygdala, several hypothalamic nuclei including the supraoptic, anterior, paraventricular, ventromedial and arcuate nuclei, and the premammillary nucleus. No significant sex differences were observed. With the exception of the supraoptic nucleus, reported not to be labeled by autoradiography, earlier autoradiographic findings and the current immunocytochemical results, although not congruent, have noteworthy similarities.
Evidence for Short‐Loop Feedback Effects of ACTH on CRF and Vasopressin Expression in Parvocellular Neurosecretory Neurons
Authors: Sawchenko P. E. , Arias C.
Published: September 2006

Immuno‐ and hybridization histochemical methods were used to examine a possible role for adrenocorticotropic hormone (ACTH) in regulating the expression of corticotropin‐releasing peptides in rat hypothalamus. Densitometric assessments of relative levels of mRNAs encoding corticotropin releasing factor (CRF), arginine vasopressin (AVP) and oxytocin (OT) in the parvocellular division of the paraventricular nucleus (PVH) were carried out in intact, adrenalectomized (ADX) and hypophysectomized (HYPOX) animals. Both surgeries resulted in comparable increases in relative levels of CRF and AVP transcripts in the parvocellular PVH; no effects on OT mRNA in this compartment were evident. In a second experiment, ACTH or saline vehicle were administered systemically via osmotic minipump for seven days to rats submitted to both HYPOX and ADX surgeries. Lower replacement doses of ACTH reduced the number of detectable AVP‐immunoreactive (AVP‐ir) cells in the parvocellular PVH to 53% of that seen in vehicle‐treated HYPOX/ADX controls; the number of CRF‐IR cells was not significantly affected. Higher doses of ACTH resulted in counts of AVP‐ and CRF‐IR neurons that were reduced to 32% and 70%, respectively, of control values. Staining patterns for the two peptides in the external lamina of the median eminence generally followed the cell count data. Neither densitometric nor combined immunohistochemical (for CRF‐ir) and hybridization histochemical (for AVP mRNA) assays revealed any marked effect of ACTH on AVP mRNA expression in the parvocellular PVH of HYPOX/ADX rats. The results indicate that ACTH is capable of inhibiting corticotropin‐releasing peptide, but not mRNA, expression in hypophysiotropic neurons. The mechanisms underlying these effects remain to be fully clarified.
Neuropeptide Y and Luteinizing Hormone Releasing Hormone Synergize to Stimulate the Development of Cellular Follicle‐Stimulating Hormone in the Hamster Adenohypophysis
Authors: Woller Michael J. , Campbell Gary T. , Blake Charles A.
Published: September 2006

Luteinizing hormone releasing hormone (LHRH) stimulates the development of cellular FSH immunoreactivity in the perinatal hamster adenohypophysis. Because neuropeptide Y (NPY) can act directly on rat adenohypophysial cells to stimulate FSH and LH release and potentiate the stimulatory effect of LHRH on FSH and LH release, we investigated the effects of NPY alone and in combination with a low, ineffective dose of LHRH on inducing cellular FSH immunoreactivity in the neonatal hamster adenohypophysis. Neonatal female pituitary glands were grafted beneath the right renal capsules of hypophysectomized‐ovariectomized adult hamster hosts with a catheter implanted in the external jugular vein. After treatment, hosts were decapitated and graft tissue was stained for FSH and LH immunoreactivity. The mean percentage of adenohypophysial cells that stained for FSH was low (2.8%) in grafts in hosts infused continuously with heparinized saline vehicle for 7 days. In other hosts, peptides were pulsed through the catheter every 12 h for 7 days. The mean percentage of FSH cells also was low after pulsing 6 ng LHRH or 2 μg NPY but increased substantially when the two peptides were pulsed simultaneously. No differences in the mean percentage of LH cells existed between any of the groups. The results demonstrate that NPY and LHRH can synergize to induce cellular FSH immunoreactivity in the neonatal female hamster.
Bipolar localization of the group II intron Ll.LtrB is maintained in Escherichia coli deficient in nucleoid condensation, chromosome partitioning and DNA replication
Authors: Beauregard Arthur , Chalamcharla Venkata R. , Piazza Carol Lyn , Belfort Marlene , Coros Colin J.
Published: September 2006

Group II introns are mobile genetic elements that invade their cognate intron‐minus alleles via an RNA intermediate, in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. In Escherichia coli, retrotransposition of the lactococcal group II intron, Ll.LtrB, occurs preferentially within the Ori and Ter macrodomains of the E. coli chromosome. These macrodomains migrate towards the poles of the cell, where the intron‐encoded protein, LtrA, localizes. Here we investigate whether alteration of nucleoid condensation, chromosome partitioning and replication affect retrotransposition frequencies, as well as bipolar localization of the Ll.LtrB intron integration and LtrA distribution in E. coli. We thus examined these properties in the absence of the nucleoid‐associated proteins H‐NS, StpA and MukB, in variants of partitioning functions including the centromere‐like sequence migS and the actin homologue MreB, as well as in the replication mutants ΔoriC, seqA, tus and topoIV ts. Although there were some dramatic fluctuations in retrotransposition levels in these hosts, bipolar localization of integration events was maintained. LtrA was consistently found in nucleoid‐free regions, with its localization to the cellular poles being largely preserved in these hosts. Together, these results suggest that bipolar localization of group II intron retrotransposition results from the residence of the intron‐encoded protein at the poles of the cell.