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Neurones in the centrally projecting Edinger-Westphal nucleus (EWcp) are the main site of urocortin 1 (Ucn1) synthesis in the mammalian brain, and are assumed to play a role in the stress response of the animal. Because endocannabinoid signalling has also been strongly implicated in stress, we hypothesised that endocannabinoids may modulate the functioning of the urocortinergic EWcp. First, using in situ hybridisation, we demonstrated cannabinoid receptor 1 (CB1R) mRNA expression in mouse EWcp-neurones that were Ucn1-negative. Dual- and triple-label immunocytochemistry revealed the presence of CB1R in several GABA-immunopositive fibres juxtaposed to EWcp-Ucn1 neurones. To test functional aspects of such an anatomical constellation, we compared acute (1 h of restraint) and chronic (14 days of chronic mild stress) stress-induced changes in wild-type (WT) and CB1R knockout (CB1R-KO) mice. Acute and especially chronic stress resulted in an increase in Ucn1 content of the EWcp, which was attenuated in CB1R-KO mice. CB1R-KO mice had higher basal and chronic stress-induced adrenocorticotrophin and corticosterone levels and were more anxious on the elevated plus-maze versus WT. Collectively, our results show for the first time EWcp-Ucn1 neurones are putatively innervated by endocannabinoid sensitive, inhibitory, GABAergic afferents. In addition, we provide novel evidence that the absence of the CB1 receptor alters the Ucn1 mRNA and peptide levels in EWcp neurones, concomitant with an augmented stress response and increased anxiety-like behaviour., (© 2012 The Authors. Journal of Neuroendocrinology © 2012 British Society for Neuroendocrinology.)

The extracellular signal-regulated kinase (ERK) pathway is important in the regulation of neuronal plasticity, although a role for the kinase in regulating plasticity of neuroendocrine systems has not been examined. The melanotroph cells in the pars intermedia of pituitary gland of the amphibian Xenopus laevis are highly plastic, undergoing very strong growth to support the high biosynthetic and secretory activity involving α-melanophore-stimulating hormone (α-MSH), a peptide that causes pigment dispersion in dermal melanophores during the adaptation of the animal to a dark background. In the present study, we tested our hypothesis that ERK-signalling is involved in the regulation of melanotroph cell function during black-background adaptation, namely in the production of pro-opiomelanocortin (POMC), the precursor of α-MSH. Using western blot analyses, we found elevated levels of the activated (phosphorylated) form of ERK in melanotrophs of black- versus white-adapted animals. Treatment of melanotrophs in vitro with the mitogen-activated protein kinase kinase inhibitor U0126 markedly reduced ERK phosphorylation and lowered the transcription as well as the translation of POMC. This same treatment also reduced the expression of BDNF transcript IV and of the immediate early genes c-Fos and Nur77. We conclude that ERK-mediated signalling is important for the maintenance of the melanotroph cells in an active state., (© 2011 The Authors. Journal of Neuroendocrinology © 2011 Blackwell Publishing Ltd.)

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.

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.

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.

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.

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.

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.

Pituitary pars intermedia melanotrope cells are often used as a model to study mechanisms of neuroendocrine integration. In the amphibian Xenopus laevis, the synthesis and release of alpha-melanophore-stimulating hormone (alpha-MSH) from these cells is a dynamic process dependent upon the colour of background. In animals on a black background, there is a higher level of synthesis and secretion of alpha-MSH than in animals on a white background, and, consequently, there is skin darkening in animals on a black background. The melanotropes are innervated by hypothalamic neurones that produce neuropeptide Y (NPY), a peptide that inhibits alpha-MSH secretion via the NPY Y1 receptor. The inhibitory neurones have a higher expression of NPY in animals adapted to a white background and both the size and the number of inhibitory synapses on the melanotrope cells are enhanced. The purpose of the present study was to determine if this presynaptic plasticity displayed by the inhibitory neurones is reciprocated by postsynaptic plasticity (i.e. if there is an enhanced expression of the Y1 receptor in melanotropes of animals adapted to a white background). For this purpose quantitative real-time reverse transcriptase-polymerase chain reaction was used to determine the level of Y1 receptor mRNA in melanotropes of animals undergoing the process of background adaptation. The results showed that there is a higher Y1 receptor mRNA expression in melanotropes of white-adapted animals. We conclude that the inhibitory neuroendocrine interface in the Xenopus pars intermedia displays postsynaptic plasticity in response to changes of background colour. To our knowledge, this is the first demonstration of a physiological environmental change leading to changes in postsynaptic receptor expression in a fully identified vertebrate neuroendocrine reflex.

Ca2+ oscillations regulate secretion of the hormone alpha-melanphore-stimulating hormone (alpha-MSH) by the neuroendocrine pituitary melanotrope cells of the amphibian Xenopus laevis. These Ca2+ oscillations are built up by discrete increments in the intracellular Ca2+ concentration, the Ca2+ steps, which are generated by electrical membrane bursting firing activity. It has been demonstrated that the patterns of Ca2+ oscillations and kinetics of the Ca2+ steps can be modulated by changing the degree of intracellular Ca2+ buffering. We hypothesized that neurotransmitters known to regulate alpha-MSH secretion also modulate the pattern of Ca2+ oscillations and related electrical membrane activity. In this study, we tested this hypothesis for the secretagogue sauvagine. Using high temporal-resolution Ca2+ imaging, we show that sauvagine modulated the pattern of Ca2+ signalling by increasing the frequency of Ca2+ oscillations and inducing a broadening of the oscillations through its effect on various Ca2+ step parameters. Second, we demonstrate that sauvagine caused a small but significant decrease in K+ currents measured in the whole-cell voltage-clamp, whereas Ca2+ currents remained unchanged. Third, in the cell-attached patch-clamp mode, a stimulatory effect of sauvagine on action current firing was observed. Moreover, sauvagine changed the shape of individual action currents. These results support the hypothesis that the secretagogue sauvagine stimulates the frequency of Ca2+ oscillations in Xenopus melanotropes by altering Ca2+ step parameters, an action that likely is evoked by an inhibition of K+ currents.

In mammals, the synaptosomal-associated protein of 25 kDa, SNAP-25, is generally thought to play a role in synaptic exocytosis of neuronal messengers. Using a polyclonal antiserum against rat SNAP-25, we have shown the presence of a SNAP-25-like protein in the brain of the South-African clawed toad Xenopus laevis by Western blotting and immunocytochemistry. Xenopus SNAP-25 is ubiquitously present throughout the brain, where its distribution in various identified neuronal perikarya and axon tracts is described. Western blot analysis and immunocytochemistry also demonstrated the presence of SNAP-25 in the neural, intermediate and distal lobes of the pituitary gland. Intensity line plots of confocal laser scanning microscope images of isolated melanotropes indicated that SNAP-25 is produced and processed in the rough endoplasmatic reticulum and Golgi apparatus, and is associated with the plasma membrane. Immunoelectron microscopy substantiated the idea that SNAP-25 is present in the plasma membrane but also showed a close association of SNAP-25 with the bounding membrane of peptide-containing secretory granules in both the neurohemal axon terminals in the neural lobe and the endocrine melanotropes in the intermediate lobe. Quantitative Western blotting revealed that adapting Xenopus to a dark background has a clear stimulatory effect on the expression of SNAP-25 in the neural lobe and in the melanotrope cells. This background light intensity-dependent stimulation of SNAP-25 expression was confirmed by the demonstration of increased immunofluorescence recorded by confocal laser scanning microscopy of individual melanotropes of black background-adapted toads. On the basis of this study on Xenopus laevis, we conclude that SNAP-25 (i) plays a substantial role in the secretion of a wide variety of neuronal messengers; (ii) functions in the central nervous system but also in neurohormonal and endocrine systems; (iii) acts at the plasma membrane but possibly also at the membrane of synaptic vesicles and peptide-containing secretory granules; (iv) acts not only locally (as in synapses), but at various sites of the plasma membrane (as in the endocrine melanotrope cell); and (v) can be upregulated in its expression by physiological stimuli that increase the extent of the molecular machinery involved in exocytosis.

At this point three brain centres are thought to be involved in the regulation of the melanotrope cells of the pituitary pars intermedia of Xenopus laevis: the magnocellular nucleus, the suprachiasmatic nucleus and the locus coeruleus. This study aims to investigate the existence of a fourth, serotonergic, centre controlling the melanotrope cells. In-vitro superfusion studies show that serotonin has a dose-dependent stimulatory effect on peptide release (1.6 x basal level at 10(-6) M serotonin) from single melanotrope cells. Retrograde neuronal tract tracing experiments, with the membrane probe FAST Dil applied to the pars intermedia, reveals retrogradely labelled neurones in the magnocellular nucleus, the suprachiasmatic nucleus, the locus coeruleus and the raphe nucleus. Of these brain centres, after immunocytochemistry only the raphe nucleus revealed serotonin-immunoreactive cell bodies. In addition, serotonin-immunoreactive cell bodies were found in the nucleus of the paraventricular organ, the posteroventral tegmental nucleus and the reticular istmic nucleus. In the pituitary, the pars nervosa, pars intermedia and pars distalis all reveal serotonin-immunoreactive nerve fibres. With immunocytochemical double-labelling for tyrosine hydroxylase and serotonin no colocalization of serotonin and tyrosine hydroxylase was observed in cell bodies in the brain, and in the pituitary hardly any colocalization was found in the nerve fibres. However, after in-vitro loading of neurointermediate lobes with serotonin, tyrosine hydroxylase and serotonin appear to coexist in a fibre network in the pars intermedia. On the basis of these data we propose that the melanotrope cells in the Xenopus pars intermedia are innervated by a 5-HT network originating in the raphe nucleus; this network represents the first identified stimulatory input to the pars intermedia of this species.

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 Xenopus gamma-MSH which is present in the N-terminal part of POMC. With immunocytochemistry we demonstrated that gamma-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 gamma-MSH-like peptides. Complete purification followed by electrospray ionization mass spectrometry and amino acid sequence determination showed that these peptides are gamma 1-MSH and glycosylated gamma 3-MSH. The amounts of these gamma-MSH peptides were low compared to the other POMC-derived peptides, alpha-MSH and beta-endorphin. Only 10% of POMC is processed into gamma-MSH peptides and the 4 kDa peptide, leaving the 18 kDa and 9 kDa peptides as the major end products.

The secretory activity of endocrine cells largely depends on the concentration of free cytosolic calcium. We have studied the mechanisms that are involved in supplying the calcium necessary for the secretion of alpha-melanophore-stimulating hormone (alpha-MSH) from melanotrope cells in the pituitary intermediate lobe of the amphibian Xenopus laevis. Using whole-cell voltage clamp, high-voltage activated calcium currents were observed, with a peak current between 0 and +20 mV. Two types of Ca(2+)-currents appeared, depending on the experimental setup. An inactivating current, which was observed after a 10 msec depolarizing prepulse, resembled currents through N-type channels as it was clearly inhibited by 1 microM omega-conotoxin. The second type was a non-inactivating current, which was blocked up to 50% by 1 microM nifedipine, indicating its L-type nature. Only a small component of this inactivating current could be blocked by omega-conotoxin. No evidence was found for the presence of transient, low-voltage activated currents. The spontaneous secretion of alpha-MSH from superfused neurointermediate lobes was dependent on extracellular calcium, as low calcium conditions (10(-4)-10(-8) M) rapidly inhibited this process. Under these conditions, secretion was not affected by depolarizing concentrations of potassium chloride. The calcium ionophore A23187 increased secretion under low calcium conditions, but had no effect on spontaneous alpha-MSH release. These results suggest that spontaneous alpha-MSH release depends on influx of calcium through voltage-operated calcium channels.(ABSTRACT TRUNCATED AT 250 WORDS)

The existence of multiple secretory pathways within endocrine cells has been receiving increasing attention (1-5). In some cases there is evidence for independently regulated pathways, for example in prolactin-producing cells (6) and parathyroid hormone-producing cells (7, 8). These pathways concern newly synthesized hormone versus hormone sequestered in mature secretory compartments. In the present study, we have analysed secretion of newly synthesized and mature peptides from proopiomelanocortin (POMC)-producing cells of the mouse pars intermedia. Evidence has been found for independent regulation and we show that the peptide composition of the two secretory pathways differs with respect to some of the POMC-derived peptides.