Biomedical subjects

E W Roubos

Publications and source records attributed to E W Roubos.

At least 19 recordsLinked to original sources

PubMed2006

Effect of starvation on Fos and neuropeptide immunoreactivities in the brain and pituitary gland of Xenopus laevis.

In mammals complex interactions between various brain structures and neuropeptides such as corticotropin-releasing factor (CRF) and urocortin 1 (Ucn1) underlay the control of feeding by the brain. Recently, in the amphibian Xenopus laevis, CRF- and Ucn1-immunoreactivities were shown in the hypothalamic magnocellular nucleus (Mg) and evidence was obtained for their involvement in food intake. To gain a better understanding of the brain structures controlling feeding in X. laevis, the effects of 16 weeks starvation on neurones immunoreactive (ir) to Fos and neuropeptides in various brain structures were quantified. In the Mg, compared to controls, starved animals showed fewer neurones immunopositive for Fos (-55.9%), Ucn1 (-44.0%), cocaine and amphetamine-regulated transcript (CART) (-94.3%) and metenkephalin (ENK) (-65.0%), whereas CRF-ir neurones were 2.1 times more numerous. These differences were mainly apparent in the ventral part of the Mg, followed by the medial and dorsal part of the nucleus. In the neural lobe of the pituitary gland a 22.5% lower optical density of CART-ir was observed. In the four other brain structures investigated, starvation had different effects. The dorsomedial part of the suprachiasmatic nucleus showed 5.9 times more NPY-ir cells and in the ventromedial thalamic area a lower number of NPY-ir cells (-33.6%) was found, whereas the Edinger-Westphal nucleus contained fewer CART-ir cells (-42.2%); no effect of starvation was seen in the ventral hypothalamic nucleus. Our results support the hypothesis that in X. laevis, the Mg plays a pivotal role in feeding-related processes and, moreover, that starvation also has neuropeptide- and brain structure-specific effects in other parts of the brain and in the pituitary gland, suggesting particular roles of these structures and their neuropeptides in physiological adaptation to starvation.

Animals↗

PubMed2006

Brain-derived neurotrophic factor in the brain of Xenopus laevis may act as a pituitary neurohormone together with mesotocin.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, occurs abundantly in the brain, where it exerts a variety of neural functions. We previously demonstrated that BDNF also exists in the endocrine melanotroph cells in the intermediate lobe of the pituitary gland of the amphibian Xenopus laevis, suggesting that BDNF, in addition to its neural actions within the brain, can act as a hormone. In the present study, we tested whether BDNF, in addition to its neural and hormonal roles, can be released as a neurohormone from the neural pituitary lobe of X. laevis. By light immunocytochemistry, we show that BDNF is present in perikarya, in ventrolaterally projecting axons of the hypothalamic magnocellular nucleus and in the neural lobe of the pituitary gland, and that it coexists in these structures with the amphibian neurohormone, mesotocin. The neural lobe was studied in detail at the ultrastructural level. Two types of neurohaemal axon terminals were observed, occurring intermingled and in similar numbers. Type A is filled with round, moderately electron-dense secretory granules with a mean diameter of approximately 145 nm. Type B terminals contain electron-dense and smaller, ellipsoid granules (long and short diameter approximately 140 and 100 nm, respectively). BDNF is exclusively present in secretory granules of type A axon terminals. Double gold-immunolabelling revealed that BDNF coexists in these granules with mesotocin. Furthermore, we demonstrate in an superfusion study performed in vitro that mesotocin stimulates peptide release from the endocrine melanotroph cells. On the basis of these data, we propose that BDNF can act on these cells as a neurohormone.

Animals↗

PubMed2006

The effects of disruption of A kinase anchoring protein-protein kinase A association on protein kinase A signalling in neuroendocrine melanotroph cells of Xenopus laevis.

The secretory activity of melanotroph cells from Xenopus laevis is regulated by multiple neurotransmitters that act through adenylyl cyclase. Cyclic adenosine monophosphate (cAMP), acting on protein kinase A (PKA), stimulates the frequency of intracellular Ca(2+) oscillations and the secretory activity of the melanotroph cell. Anchoring of PKA near target proteins is essential for many PKA-regulated processes, and the family of A kinase anchoring proteins (AKAPs) is involved in the compartmentalisation of PKA type II (PKA II) regulatory subunits. In the present study, we determined to what degree cAMP signalling in Xenopus melanotrophs depends on compartmentalised PKA II. For this purpose, a membrane-permeable stearated form of Ht31 (St-Ht31), which dislodges PKA II from AKAP (thus disrupting PKA II signalling), was used. The effect of St-Ht31 on both secretion of radiolabelled peptides and intracellular Ca(2+) signalling by superfused Xenopus melanotrophs was assessed. St-Ht31 stimulated secretion but had no effect on Ca(2+) signalling. We conclude Xenopus melanotrophs possess a St-Ht31-sensitive PKA II that is associated with the exocytosis machinery and, furthermore, that Ca(2+) signalling is regulated by an AKAP-independent signalling system. Moreover, our results support a recent proposal that AKAP participates in regulating PKA activity independently from cAMP.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Localisation and physiological regulation of corticotrophin-releasing factor receptor 1 mRNA in the Xenopus laevis brain and pituitary gland.

In Xenopus laevis, corticotrophin-releasing factor (CRF) and urocortin 1 are present in the brain and they both are potent stimulators of alpha-melanophore stimulating hormone (MSH) secretion by melanotroph cells in the pituitary gland. Because both CRF and urocortin 1 bind with high affinity to CRF receptor type 1 (CRF1) in mammals and Xenopus laevis, one of the purposes of the present study was to identify the sites of action of CRF and urocortin 1 in the Xenopus brain and pituitary gland. Moreover, we raised the hypothesis that the external light intensity is a physiological condition controlling CRF1 expression in the pituitary melanotroph cells. By in situ hybridisation, the presence of CRF1 mRNA is demonstrated in the olfactory bulb, amygdala, nucleus accumbens, preoptic area, ventral habenular nuclei, ventromedial thalamic area, suprachiasmatic nucleus, ventral hypothalamic area, posterior tuberculum, tectum mesencephali and cerebellum. In the pituitary gland, CRF1 mRNA occurs in the intermediate and distal lobe. The optical density of the CRF1 mRNA hybridisation signal in the intermediate lobe of the pituitary gland is 59.4% stronger in white-adapted animals than in black-adapted ones, supporting the hypothesis that the environmental light condition controls CRF1 mRNA expression in melanotroph cells of X. laevis, a mechanism likely to be responsible for CRF- and/or urocortin 1-stimulated secretion of alpha-MSH.

Adaptation, Physiological↗

PubMed2005

Melanotrope cells of Xenopus laevis express multiple types of high-voltage-activated Ca2+ channels.

Pituitary melanotrope cells are neuroendocrine signal transducing cells that translate physiological stimuli into adaptive hormonal responses. In this translation process, Ca2+ channels play essential roles. We have characterised which types of Ca2+ current are present in melanotropes of the amphibian Xenopus laevis, using whole-cell, voltage-clamp, patch-clamp experiments and specific blockers of the various current types. Running an activation current-voltage relationship protocol from a holding potential (HP) of -80 mV/or -110 mV, shows that Xenopus melanotropes possess only high-voltage activated (HVA) Ca2+ currents. Steady-state inactivation protocols reveal that no inactivation occurs at -80 mV, whereas 30% of the current is inactivated at -30 mV. We determined the contribution of individual channel types to the total HVA Ca2+ current, examining the effect of each channel blocker at an HP of -80 mV and -30 mV. At -80 mV, omega-conotoxin GVIA, omega-agatoxin IVA, nifedipine and SNX-482 inhibit Ca2+ currents by 21.8 +/- 4.1%, 26.1 +/- 3.1%, 24.2 +/- 2.4% and 17.9 +/- 4.7%, respectively. At -30 mV, omega-conotoxin GVIA, nifedipine and omega-agatoxin IVA inhibit Ca2+ currents by 33.8 +/- 3.0, 24.2 +/- 2.6 and 16.0 +/- 2.8%, respectively, demonstrating that these blockers substantially inhibit part of the Ca2+ current, independently from the HP. We have previously demonstrated that omega-conotoxin GVIA can block Ca2+ oscillations and steps. We now show that nifedipine and omega-agatoxin IVA do not affect the intracellular Ca2+ dynamics, whereas SNX-482 reduces the Ca2+ step amplitude. We conclude that Xenopus melanotrope cells express all four major types of HVA Ca2+ channel, as well as the resulting currents, but no low-voltage activated channels. The results provide the basis for future studies on the complex regulation of channel-mediated Ca2+ influxes into this neuroendocrine cell type as a function of its role in the animal's adaptation to external challenges.

Animals↗

PubMed2005

In situ hybridization localization of TRH precursor and TRH receptor mRNAs in the brain and pituitary of Xenopus laevis.

We examined the distribution of the mRNAs encoding proTRH and the three TRH receptor subtypes (xTRHR1, xTRHR2, and xTRHR3) in the Xenopus laevis CNS and pituitary. A positive correlation was generally observed between the expression patterns of proTRH and xTRHR mRNAs. xTRHRs were widely expressed in the telencephalon and diencephalon, where two or even three xTRHR mRNAs were often simultaneously observed within the same brain structures. In the pituitary, xTRHR2 was selectively expressed in the distal lobe, and xTRHR3 was found exclusively in the intermediate lobe of white background-adapted animals, indicating that, in amphibians, the effect of TRH on alpha-melanotropin (alpha-MSH) secretion from melanotrope cells is mediated through the novel receptor subtype xTRHR3.

Animals↗

PubMed2005

Neuroendocrine regulation of frog adrenocortical cells by neurotensin.

We previously characterized the primary structure of neurotensin (NT) from an extract of the intestine of the frog Rana esculenta. In this study, we provide evidence for the involvement of NT in the neurocrine regulation of the secretory activity of frog adrenocortical cells. Immunohistochemical studies revealed that the adrenal gland of R. esculenta is innervated by a dense network of NT-immunoreactive fibers. Graded concentrations of frog NT induced a dose-dependent stimulation of corticosterone and aldosterone secretion by frog adrenocortical explants through activation of two receptors with pEC(50) of 9.8 and 6.9. These data support the view that NT, released by nerve fibers within the frog adrenal gland, acts locally to control corticosteroid secretion.

Adrenal Cortex↗

PubMed2005

Analysis of Xenopus melanotrope cell size and POMC-gene expression.

Flow cytometry was used to separate Xenopus melanotrope cells according to their size. The cells were then submitted to real-time RT-PCR to determine the level of POMC-gene expression. The results show a positive correlation between cell size and gene expression for cells from black-background (but not white-background) adapted animals.

Animals↗

PubMed2005

Calcium influx through voltage-operated calcium channels is required for proopiomelanocortin protein expression in Xenopus melanotropes.

Melanotrope cells of Xenopus laevis generate transitory increases in intracellular Ca(2+), known as Ca(2+) oscillations. These oscillations arise from the influx of Ca(2+) through voltage-operated Ca(2+) channels (VOCCs). Such oscillations are the driving force for secretion of a-melanophore-stimulating hormone (alpha-MSH) from the cell. The influx of Ca(2+) through VOCCs initiates the mobilization of intracellular Ca(2+) to generate a Ca(2+) wave. The function of the Ca(2+) wave in the melanotrope is unknown, but its presence in the nucleus suggests a role in the regulation of gene expression, perhaps that of proopiomelanocortin (POMC), the precursor protein for alpha-MSH. To determine the possible function of Ca(2+) waves in Xenopus melanotropes, we addressed whether functional VOCCs, which are an established requirement for both secretion and Ca(2+) wave initiation, are also required to maintain POMC gene expression.

Animals↗

PubMed2004

Differential expression of high voltage-activated Ca2+ channel types in the rostral reticular thalamic nucleus of the absence epileptic WAG/Rij rat.

In the WAG/Rij rat, a model for human absence epilepsy, spike-wave discharges (SWD) and absence epileptic behavior develop after the age of 3 months. The rostral part of the reticular thalamic nucleus (rRTN) is involved in SWD. Ca(2+) channels play a central role in the initiation and maintenance of burst firing activity of thalamic cells. We hypothesize that a changed expression of alpha(1)-subunits of one or more high voltage-activated Ca(2+) channel types in the rRTN underlies the development of SWD. To test this hypothesis we compared 3- and 6-month-old WAG/Rij rats with nonepileptic, age-matched control rats. By immunocytochemistry, the expressions of alpha(1)1.3-, alpha(1)2.1-, alpha(1)2.2-, and alpha(1)2.3-subunits were shown in both strains, demonstrating the presence of Ca(v)1.3, Ca(v)2.1, Ca(v)2.2, and Ca(v)2.3 channels, respectively. Quantification of channel expression indicates that the development of SWD in WAG/Rij rats is concomitant with an increased expression of Ca(v)2.1 channels in the rRTN. These channels are mainly presynaptic, as revealed by double immunofluorescence involving the presynapse marker syntaxin. The mechanism by which this increase could be related to the occurrence of SWD has been discussed.

Animals↗

PubMed2004

Synaptology of the rostral reticular thalamic nucleus of absence epileptic WAG/Rij rats.

The adult WAG/Rij rat is a well-established animal model for human absence epilepsy characterized by the presence of spike-wave discharges (SWDs). The pacemaking activity of the rostral reticular thalamic nucleus (rRTN) has been demonstrated to be essential for SWD maintenance. We investigated if SWD maintenance can be related to the synaptic organization of the rRTN, by studying the ultrastructure of the rRTN of absence epileptic WAG/Rij rats in comparison with that of non-epileptic, age-matched ACI control rats. In WAG/Rij rats, D-, L- and F-type terminals constitute the synaptic organization of the rRTN. D-type synapses, especially axo-dendritic ones, occur frequently. L- and F-type terminals are common but less frequent than D-type terminals. Semi-quantitative observations indicate that all terminal types are present on different parts of the postsynaptic neuron, but in different numbers: they are frequent on dendrites, common on somata and axons, and occur occasionally on dendritic spines. In addition, occasionally an F-type terminal was observed on the axon hillock. The three terminal types are also involved in multiple synaptic configurations, convergent as well as divergent, with dendrites, somata, axon hillocks and axons as postsynaptic structures. Convergent synaptic configurations outnumber divergent ones. The synaptic organization of the rRTN of the non-epileptic ACI rat appears to be very similar to that of the epileptic WAG/Rij rat. This indicates that SWD maintenance in the WAG/Rij rat does not depend on a different synaptic organization of the rRTN.

Animals↗

PubMed2004

Quantification of synapse formation and maintenance in vivo in the absence of synaptic release.

Outgrowing axons in the developing nervous system secrete neurotransmitters and neuromodulatory substances, which is considered to stimulate synaptogenesis. However, some synapses develop independent of presynaptic secretion. To investigate the role of secretion in synapse formation and maintenance in vivo, we quantified synapses and their morphology in the neocortical marginal zone of munc18-1 deficient mice which lack both evoked and spontaneous secretion [Science 287 (2000) 864]. Histochemical analyses at embryonic day 18 (E18) showed that the overall organization of the neocortex and the number of cells were similar in mutants and controls. Western blot analysis revealed equal concentrations of pre- and post-synaptic marker proteins in mutants and controls and immunocytochemical analyses indicated that these markers were targeted to the neuropil of the synaptic layer in the mutant neocortex. Electron microscopy revealed that at E16 immature synapses had formed both in mutants and controls. These synapses had a similar synapse diameter, active zone length and contained similar amounts of synaptic vesicles, which were immuno-positive for two synaptic vesicle markers. However, these synapses were three times less abundant in the mutant. Two days later, E18, synapses in the controls had more total and docked vesicles, but not in the mutant. Furthermore, synapses were now five times less abundant in the mutant. In both mutant and controls, synapse-like structures were observed with irregular shaped vesicles on both sides of the synaptic cleft. These 'multivesicular structures' were immuno-positive for synaptic vesicle markers and were four times more abundant in the mutant. We conclude that in the absence of presynaptic secretion immature synapses with a normal morphology form, but fewer in number. These secretion-deficient synapses might fail to mature and instead give rise to multivesicular structures. These two observations suggest that secretion of neurotransmitters and neuromodulatory substances is required for synapse maintenance, not for synaptogenesis. Multivesicular structures may develop out of unstable synapses.

Animals↗

PubMed2004

Differential distribution and regulation of expression of synaptosomal-associated protein of 25 kDa isoforms in the Xenopus pituitary gland and brain.

Synaptosomal-associated protein of 25 kDa (SNAP-25) regulates various membrane fusion processes including exocytosis by endocrine and neural cells. To increase our understanding of the occurrence and regulation of SNAP-25 isoforms, we identified and characterized SNAP-25a and SNAP-25b mRNAs in the pituitary gland and brain of the amphibian Xenopus laevis. The proteins are strongly conserved and are resistant to botulinum neurotoxin A but not to botulinum neurotoxin E, as shown by Western blotting. The spatial distribution of the two SNAP-25 isoforms was assessed with in situ hybridization. Both SNAP-25a mRNA and SNAP-25b mRNA reside in cells in the pituitary distal lobe and, particularly, in the endocrine melanotrope cells in the pituitary intermediate lobe. The melanotrope cells are involved in the background adaptation process of the skin by releasing alpha-melanophore-stimulating hormone. Quantitation of the respective in situ hybridization signals in the Xenopus pars intermedia indicated a differential response, SNAP-25b mRNA being more highly expressed in black-adapted animals than SNAP-25a mRNA, and more than in white-adapted toads. This differential upregulation was also studied by real-time reverse transcriptase polymerase chain reaction, showing that in the intermediate pituitary lobe, both isoforms are physiologically controlled by the background light intensity stimulus, but with different intensities; in black-adapted animals SNAP-25b mRNA is upregulated by 3.33 times compared with white-adapted animals, but SNAP-25a only by 1.96 times. As to neural tissue, in situ hybridization showed that both isoforms coexist throughout the brain, sometimes with similar strengths, but in various areas either SNAP-25a mRNA or SNAP-25b mRNA expression is prevalent. It is speculated that each of the SNAP-25 isoforms in the Xenopus pituitary and brain has a distinct function in cellular fusion processes including secretion, and that their occurrence and regulation depend on the type of secreted neurotransmitter/hormone and/or the activity state of the cell.

Adaptation, Physiological↗

PubMed2004

Activity-dependent dynamics of coexisting brain-derived neurotrophic factor, pro-opiomelanocortin and alpha-melanophore-stimulating hormone in melanotrope cells of Xenopus laevis.

Brain-derived neurotrophic factor (BDNF) is involved as an autocrine factor in the regulation of the secretory activity of the neuroendocrine pituitary melanotrope cells of Xenopus laevis. We studied the subcellular distribution of BDNF in Xenopus melanotropes using a combination of high-pressure freezing, cryosubstitution and immunoelectron microscopy. Presence of BDNF, pro-opiomelanocortin (POMC) and alpha-melanophore-stimulating hormone (alphaMSH) within melanotrope secretory granules was studied by triple-labelling immunoelectron microscopy. In addition, intracellular processing of BDNF was investigated by quantifying the number of immunogold particles in different stages of secretory granule maturation, in animals adapted to black or white background light conditions. The high-pressure freezing technique provides excellent preservation of both cellular ultrastructure and antigenicity. BDNF coexists with POMC and alphaMSH within secretory granules. BDNF-immunoreactivity increases along the secretory granule maturation axis (i.e. from electron-dense, via moderately electron-dense, to electron-lucent secretory granules). Immature, low immunoreactive, electron-dense secretory granules are assumed to contain mainly or even exclusively proBDNF. Strongly immunoreactive electron-lucent secretory granules represent the mature granule stage in which proBDNF has been processed to mature BDNF. Furthermore, in moderately electron-dense secretory granules, immunoreactivity is markedly (+79%) higher in black-adapted than in white-adapted animals, indicating that stimulation of melanotrope cell activity by the black background condition speeds up processing of BDNF from its precursor in this granule stage. It is concluded that, in the Xenopus melanotrope, BDNF biosynthesis and processing occur along the secretory granule maturation axis, together with that of POMC-derived alphaMSH, and that the environmental light condition not only controls the biosynthesis and secretion of BDNF and of POMC end-products, but also regulates the rate of their intragranular processing.

Animals↗

PubMed2004

Low temperature stimulates alpha-melanophore-stimulating hormone secretion and inhibits background adaptation in Xenopus laevis.

It is well-known that alpha-melanophore-stimulating hormone (alpha-MSH) release from the amphibian pars intermedia (PI) depends on the light condition of the animal's background, permitting the animal to adapt the colour of its skin to background light intensity. In the present study, we carried out nine experiments on the effect of low temperature on this skin adaptation process in the toad Xenopus laevis, using the skin melanophore index (MI) bioassay and a radioimmunoassay to measure skin colour adaptation and alpha-MSH secretion, respectively. We show that temperatures below 8 degrees C stimulate alpha-MSH secretion and skin darkening, with a maximum at 5 degrees C, independent of the illumination state of the background. No significant stimulatory effect of low temperature on the MI and alpha-MSH plasma contents was noted when the experiment was repeated with toads from which the neurointermediate lobe (NIL) had been surgically extirpated. This indicates that low temperature stimulates alpha-MSH release from melanotrope cells located in the PI. An in vitro superfusion study with the NIL demonstrated that low temperature does not act directly on the PI. A possible role of the central nervous system in cold-induced alpha-MSH release from the PI was tested by studying the hypothalamic expression of c-Fos (as an indicator for neuronal activity) and the coexistence of c-Fos with the regulators of melanotrope cell activity, neuropeptide Y (NPY) and thyrotrophin-releasing hormone (TRH), using double fluorescence immunocytochemistry. Upon lowering temperature from 22 degrees C to 5 degrees C, in white-adapted animals c-Fos expression decreased in NPY-producing suprachiasmatic-melanotrope-inhibiting neurones (SMIN) in the ventrolateral area of the suprachiasmatic nucleus (SC) but increased in TRH-containing neurones of the magnocellular nucleus. TRH is known to stimulate melanotrope alpha-MSH release. We conclude that temperatures around 5 degrees C inactivate the SMIN in the SC and activate TRH-neurones in the magnocellular nucleus, resulting in enhanced alpha-MSH secretion from the PI, darkening the skin of white-adapted X. laevis.

Adaptation, Physiological↗

PubMed2003

NPY-mRNA expressions in the nucleus accumbens, caudate putamen and cerebral cortex of apomorphine-susceptible and apomorphine-unsusceptible rats.

Using the apomorphine-induced stereotyped gnawing response as a selection criterion, two distinct groups of rats can be distinguished, apomorphine-susceptible (APO-SUS) and apomorphine-unsusceptible (APO-UNSUS) rats. These two lines differ in several components of both striatal and extrastriatal areas. This study deals with the expression of neuropeptide Y (NPY)mRNA-expressing neurons in the nucleus accumbens, caudate putamen and cerebral cortex of both rat lines, using non-radioactive in situ hybridisation. The morphology of the neurons in the three regions is similar, viz. oblong, rectangular or triangular, with two or three processes. The neurons are homogeneously distributed in all regions, and in the nucleus accumbens they are particularly numerous ventrally to the anterior commissure. Using automated image analysis, the mean numerical density of NPYmRNA-positive neurons per brain region and the mean NPYmRNA expression level per neuron per brain region were determined. No differences appear in the numerical densities of NPYmRNA-containing neurons in the nucleus accumbens, caudate putamen and cortex between APO-SUS and APO-UNSUS rats. However, distinct differences between the rat lines are present in the level of NPYmRNA expression per neuron in the nucleus accumbens and in the caudate putamen, showing that NPY contributes to the differential neurochemical make-up of these rat lines that is responsible for their obvious differences in behaviour, physiology and immune competence.

Animals↗

PubMed2003

Development of the mouse neuromuscular junction in the absence of regulated secretion.

To investigate the role of neurotransmitter secretion in the development and stabilization of synapses, the innervation of the diaphragm and intercostal muscles was studied in munc18-1 null mutant mice, which lack regulated secretion. We found that this mutant is completely devoid of both spontaneous and evoked neuromuscular transmission throughout embryonic development. At embryonic day (E) 14, axonal targeting and main branching of the phrenic nerve were normal in this mutant, but tertiary branches were elongated and no terminal branches were observed at this stage, in contrast to control littermates. Acetylcholinesterase staining was observed in the endplate region of mutant muscle from E14 onwards, but not as dense and confined to spots as in controls. Acetylcholine receptor staining was also present in the endplate region of the mutant muscle. In this case, the staining density and the concentration in spots (clusters) were similar to controls, but the distribution of these clusters was less organized. Starting at E15, some receptor clusters co-localized with nerve terminal staining, suggesting synapses, but most clusters remained a-neural. Electron microscopical analysis confirmed the presence of synaptic structures in the mutant. Between E14 and birth, the characteristic staining pattern of nerve branches gradually disappeared in the mutant until, at E18, an elaborate meshwork of nerve fibers with no apparent organization remained. In the same period, most of the motor neuronal cell bodies in the spinal cord degenerated. In contrast, sensory ganglia in the dorsal root showed no obvious degeneration. These data suggest that regulated secretion is not essential for initial axon path finding, clustering of acetylcholine receptors, acetylcholinesterase or the formation of synapses. However, in the absence of regulated secretion, the maintenance of the motor neuronal system, organization of nerve terminal branches and stabilization of synapses is impaired and a-neural postsynaptic elements persist.

Acetylcholinesterase↗

PubMed2003

Expression and characterization of the extracellular Ca(2+)-sensing receptor in melanotrope cells of Xenopus laevis.

The extracellular Ca(2+)-sensing receptor (CaR) is expressed in many different organs in various species, ranging from mammals to fish. In some of these organs, this G protein-coupled receptor is involved in the control of systemic Ca(2+) homeostasis, whereas in other organs its role is unclear (e.g. in the pituitary gland). We have characterized the CaR in the neuroendocrine melanotrope cell of the intermediate pituitary lobe of the South African clawed toad Xenopus laevis. First, the presence of CaR mRNA was demonstrated by RT-PCR and in situ hybridization. Then it was shown that activation of the CaR by an elevated extracellular Ca(2+) concentration and different CaR-activators, including L-phenylalanine and spermine, stimulates both Ca(2+) oscillations and secretion from the melanotrope. Furthermore, it was revealed that activation of the receptor stimulates Ca(2+) oscillations through opening of voltage-operated Ca(2+) channels in the plasma membrane of the melanotropes. Finally, it was shown that the CaR activator L-phenylalanine could induce the biosynthesis of proopiomelanocortin in the intermediate lobe. Thus, in this study it is demonstrated that the CaR is present and functional in a defined cell type of the pituitary gland, the amphibian melanotrope cell.

Animals↗