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Background: Less than 50% of patients experience sufficient pain relief with current drug therapy for neuropathic pain. Minocycline shows promising results in rodent models of neuropathic pain but was not studied in humans with regard to the treatment of neuropathic pain., Methods: In this randomized, double-blind, placebo-controlled clinical trial, patients with subacute lumbar radicular pain received placebo, amitriptyline 25 mg, or minocycline 100 mg once a day (n = 20 per group) for 14 days. Primary outcome measure was the pain intensity in the leg as measured by a numeric rating scale ranging from 0 to 10 on days 7 and 14. Secondary outcome measures were the reduction of neuropathic pain symptoms in the leg as determined with a neuropathic pain questionnaire, consumption of rescue medication, and adverse events on days 7 and 14., Results: Sixty patients were randomized and included in an intention-to-treat analysis. After 14 days, patients in the minocycline and amitriptyline groups reported a reduction of 1.47 (95% confidence interval, 0.16-2.83; P = 0.035) and 1.41 (95% confidence interval, 0.05-2.78; P = 0.043), respectively, in the numeric rating scale compared to the placebo group. No differences were seen in the neuropathic pain questionnaire values at any time point during treatment between the three groups. The rate of adverse events in the amitriptyline group was 10% versus none in the minocycline and placebo groups. No differences were noted in the consumption of rescue medication., Conclusions: Although both groups differed from placebo, their effect size was small and therefore not likely to be clinically meaningful.

Orexin is a neuropeptide that has been implicated in several processes, such as induction of appetite, arousal and alertness and sleep/wake regulation. Multiple lines of evidence also suggest that orexin is involved in the stress response. When orexin is administered intracerebroventricular it activates the hypothalamic pituitary adrenal (HPA)-axis, which is the main regulator of the stress response. The HPA-axis is not the only player in the stress response evidence suggests that urocortin 1 (Ucn1), a member of the corticotropin releasing factor (CRF) neuropeptide family, also plays an important role in the stress response adaptation. Ucn1 is primarily synthetized in the centrally projecting Edinger-Westphal nucleus (EWcp), which also receives dense innervation by orexin terminals. In this study we tested the hypothesis that orexin would directly shape the response of EWcp-Ucn1 neurons to acute cold stress. To test this hypothesis, we first assessed whether orexinergic axon terminals would innervate EWcp-Ucn1/CART neurons, and next we exposed orexin deficient (orexin-KO) male mice and their male wild-type (WT) littermates to acute cold stress for 2h. We also assessed stress-associated changes in plasma corticosterone (CORT), as well as the activation of Ucn1/CART neurons in the EWcp nucleus. We found that orexin immunoreactive axon terminals were juxtaposed to EWcp-Ucn1/CART neurons, which also expressed orexin receptor 1 mRNA. Furthermore, acute stress strongly activated the EWcp-Ucn1/CART neurons and increased plasma CORT in both WT littermates and orexin-KO mice, however no genotype effect was found on these indices. Taken together our data show that orexin in general is not involved in the animal's acute stress response (plasma CORT) and it does not play a direct role in shaping the response of EWcp-Ucn1 neurons to acute stress either., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.)

Neuropathic pain is strongly associated with mood disorders like anxiety and depression. Corticotropin-releasing factor (CRF) plays a prominent role in these disorders as it is up-regulated in limbic structures such as the amygdala, upon experimentally induced neuropathy. This review discusses recent literature on the role of CRF in pain processing and highlights the amygdala as a potential hot spot in supraspinal descending pain control. Many studies have demonstrated analgesic effects of CRF following local and systemic administration, but more recently also hyperalgesic effects were shown upon endogenous amygdalar CRF increase or by blocking the CRF type 1 receptor (CRFR1). On the basis of the reviewed literature, we postulate a central mechanism for pain control in which the amygdala plays a critical role by switching on and off chronic pain. In this mechanism, upon pain stimuli, CRFR1 in the amygdala is activated by CRF to induce hyperalgesia. When the activated CRFR1 is internalized (pain initiation), it triggers the translocation of the cytoplasmic CRF type 2 receptor (CRFR2) to the plasma membrane. Here, CRFR2 can be recruited by either high (pharmacological) concentrations of CRF or by endogenous CRFR2 ligands, the urocortins, leading to analgesia (pain termination). This on-off switching of pain is completed by redistribution of the CRF receptors to their initial activity state. We furthermore propose that in neuropathic pain, this mechanism is dysregulated and causes a state of permanent hyperalgesia, and present an integrative (patho)physiological model for the way disturbed CRF receptor signalling in the amygdala could initiate neuropathic pain., (© 2011 European Federation of International Association for the Study of Pain Chapters.)

Neuropathic pain is often accompanied by stress, anxiety and depression. Although there is evidence for involvement of corticotropin-releasing factor (CRF), the detailed neuronal basis of these pain-related mood alterations is unknown. This study shows that peripheral mononeuropathy was accompanied by changes in limbic forebrain CRF, but did not lead to changes in the functioning of the hypothalamo-pituitary-adrenal axis and the midbrain Edinger-Westphal centrally projecting (EWcp) neuron population, which play main roles in the organism's response to acute pain. Twenty-four days after chronic constriction injury (CCI) of the rat sciatic nerve, the oval bed nucleus of the stria terminalis (BSTov) contained substantially more Crf mRNA as did the central amygdala (CeA), which, in addition, possessed more CRF. In contrast, Crf mRNA and CRF contents of the hypothalamic paraventricular nucleus (PVN) were unaffected by CCI. Similarly, EWcp neurons, producing the CRF family member urocortin 1 (Ucn1) and constitutively activated by various stressors including acute pain, did not show an effect of CCI on Ucn1 mRNA or Ucn1. Also, the immediate early gene products cFos and deltaFosB in the EWcp were unaffected by CCI. These results indicate that neuropathic pain does not act via the HPA-axis or the EWcp, but includes a main role of Crf in the limbic system, which is in clear contrast to stressors like acute and chronic pain, which primarily act on the PVN and the EWcp., (© 2011 European Federation of International Association for the Study of Pain Chapters.)

It has been hypothesized that corticotropin-releasing factor (CRF) and its related neuropeptide urocortin 1 (Ucn1) play different roles in the initiation and adaptive phases of the stress response, which implies different temporal dynamics of these neuropeptides in response to stressors. We have tested the hypothesis that acute pain stress (APS) differentially changes the dynamics of CRF expression in the paraventricular nucleus of the hypothalamus (PVN), oval subdivision of the bed nucleus of the stria terminalis (BSTov) and central amygdala (CeA), and the dynamics of Ucn1 expression in the midbrain non-preganglionic Edinger-Westphal nucleus (npEW). Thirty minutes after APS, induced by a formalin injection into the left hind paw, PVN, BSTov, CeA and npEW all showed a peak in cFos mRNA expression that was followed by a robust increase in cFos protein-immunoreactivity, indicating a rapid increase in (immediate early) gene expression in all four brain nuclei. CRF-dynamics, however, were affected by APS in a brain nucleus-specific way: in the PVN, CRF-immunoreactivity was minimal at 60 min after APS and concomitant with a marked increase in plasma corticosterone, whereas in the BSTov not CRF peptide but CRF mRNA peaked at 60 min, and in the CeA a surge of CRF peptide occurred as late as 240 min. The npEW differed from the other centers, as Ucn1 mRNA and Ucn1 peptide peaked at 120 min. These results support our hypothesis that each of the four brain centers responds to APS with CRF/Ucn1 dynamics that are specific as to nature and timing. In particular, we propose that CRF in the PVN plays a major role in the initiation phase, whereas Ucn1 in the npEW may act in the later, termination phase of the adaptation response to APS., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

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.)

Cocaine- and amphetamine-regulated transcript (CART) and nesfatin-1/nucleobindin 2 (NUCB2) are assumed to play a role in feeding and adaptation to stress. Both peptides are highly expressed in the midbrain non-preganglionic Edinger-Westphal nucleus (npEW), a center implicated in the regulation of stress adaptation and in the pathogenesis of stress-induced brain disorders, in a sex-specific manner. The present study was undertaken to test whether CART and nesfatin are involved in these actions of the npEW in the rat. Acute restraint and chronic variable mild stress were used. Following stress, physiological parameters (serum corticosterone levels, body, adrenal and thymus weights) were determined, CART and nesfatin-like immunoreactivity (LI) as well as mRNA expression were analyzed in the npEW nucleus. Our results depict the following changes: (1) Acute stress resulted in an increase in serum corticosterone levels that was higher in females; (2) In males, data on corticosterone and body weight gain and in females, data on body weight gain revealed an effect of chronic stress; (3) Both acute and chronic stress activated npEW neurons expressing CART and nesfatin-LI, as shown by increased cFos immunoreactivity; (4) Chronic, but not acute stress increased the amount of CART and nesfatin-LI in both males and females; (5) Neither acute nor chronic stress had an effect on CART and NUCB2 mRNA contents of npEW neurons in either sex. Taken together, our data suggest that CART and nesfatin are involved in the response of npEW neurons to chronic stress., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Even in present day pain therapy, neuropathic pain remains a challenge for clinicians to treat and a challenge for researchers to investigate. Different animal models have been developed to mimic neuropathic pain. Neurotrophins such as nerve growth factor, brain-derived neurotrophic factor and neurotrophin 3 have been studied extensively in these models, yet few review articles concerning brain-derived neurotrophic factor have been published. This article reassesses the literature concerning brain-derived neurotrophic factor expression in the sciatic nerve chronic constriction injury model, the sciatic nerve transection model, the spinal nerve ligation model and the spinal nerve transection model and discusses differences in regulation of brain-derived neurotrophic factor between these models and their causality with neuropathic pain., (Copyright 2009 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved.)

Leptin is critical for normal food intake and energy metabolism. While leptin receptor (ObR) function has been well studied in hypothalamic feeding circuitries, the functional relevance of ObR in extrahypothalamic areas is largely unknown. Central regulatory pathways involved in food intake utilize various neuropeptides, such as urocortin 1 (Ucn1), cocaine- and amphetamine-regulated transcript peptide (CART) and nesfatin-1. Ucn1 is most abundantly expressed in the non-preganglionic Edinger-Westphal nucleus (npEW). In addition to Ucn1, other satiety signals, such as CART and nesfatin-1, are highly expressed in neurons of the npEW. Using immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR), we here show the presence of short and long forms of ObR in the rat npEW. Then, we tested our hypothesis that a change in plasma leptin will modulate the activity of npEW neurons containing Ucn1, CART and nesfatin-1. First, by double-labeling immunocytochemistry, we observed that almost all npEW neurons colocalizing Ucn1, CART and nesfatin-1 also contain ObR. Fasting rats for two days caused a marked body weight loss and reduced leptin plasma level in both genders. Ucn1 mRNA and CART mRNA were upregulated after fasting in males (3.3 and 2.4 times, respectively; P<0.05) but not in females. However, their peptide levels were not significantly changed. The peptide level and mRNA of nesfatin-1 were unaffected by fasting. We conclude that npEW-neurons containing Ucn1, CART and nesfatin-1 co-express ObR, and may be involved in leptin-mediated feeding control in male rats only.

Transgenic animals are commonly employed to explore the function of individual proteins. Transgenic animal models include the mouse, the zebrafish, and the South African clawed toad Xenopus laevis. In contrast to mice and zebrafish, with Xenopus transgenesis DNA integration is mostly achieved in the one-cell stage. Moreover, Xenopus (as well as zebrafish) eggs are relatively large, the embryos are transparent, a large offspring is generated, and maintenance of the offspring is easy. In our transgenic studies in Xenopus, we focus on the well-characterized neuroendocrine melanotrope cells of the pituitary pars intermedia that are regulated during the process of adaptation of Xenopus to a changing environment. When the animal is placed on a black background, the melanotrope cells produce and process large amounts of the prohormone proopiomelanocortin (POMC). We apply stable melanotrope-specific transgenesis that is achieved by mixing a Xenopus POMC-promoter/transgene construct with sperm nuclei and injecting this mixture into unfertilized eggs. Since in the melanotrope cells the POMC promoter is much more active in black-adapted animals, the level of transgene expression can be manipulated by placing the animal on either a black or a white background. In this paper we review the possibilities of the Xenopus melanotrope-specific transgenic approach. Following a brief overview of the functioning of Xenopus melanotrope cells, stable melanotrope-specific transgenesis is discussed and our transgenic studies on brain-derived neurotrophic factor and secretory pathway components are described as examples of the transgenic approach in a physiological context and close to the in vivo situation.

Period 2 (Per2) is an important clock gene involved in the regulation of the major circadian clock in the mammalian central nervous system, the suprachiasmatic nucleus. In addition, Per2 is expressed in many other stress-sensitive brain structures. We have previously showed that the non-preganglionic Edinger-Westphal nucleus (npEW) is the main site of the corticotropin-releasing factor peptide family member urocortin 1 (Ucn1) and that this peptide undergoes conspicuous expression changes in response to various stressors. Here, we hypothesized that in the rat npEW both Per2 and Ucn1 would be produced in a diurnal, rhythmical fashion. This hypothesis was tested by following this expected rhythm on two days in rats killed at four time points each day (Zeitgeber times 0, 6, 12, and 18). We showed the co-existence of Per2 and Ucn1 in the npEW with double-label immunofluorescence and demonstrated with quantitative RT-PCR and semi-quantitative immunocytochemistry diurnal rhythms in Per2 mRNA expression and Per2 protein content, each on a single different day, with a minimum at lights-off and a maximum at lights-on. We furthermore revealed a diurnal rhythm in the number of Ucn1-immunopositive neurones and in their Ucn1 peptide content, with a minimum at night and at the beginning of the light period and a peak at lights-off, while the Ucn1 mRNA content paralleled the Per2 mRNA rhythm. The rhythms were accompanied by a diurnal rhythm in plasma corticosterone concentration. Our results are in line with the hypothesis that both Per2 and Ucn1 in the rat npEW are produced in a diurnal fashion, a phenomenon that may be relevant for the regulation of the diurnal rhythm in the stress response.

Housekeeping gene (HKG) mRNAs are used to normalize expression data of genes of interest in quantitative reverse transcriptase polymerase chain reaction studies. Such normalization assumes constant HKG gene expression under all circumstances. Although sporadic evidence suggests that HKG expression may not always fulfill this requirement and, therefore, such normalization may lead readily to erroneous results, this fact is generally not sufficiently appreciated by investigators. Here, we have systematically analyzed the expression of three common HKGs, glyceraldehyde-3-phosphate dehydrogenase, ribosomal subunit 18S and beta-actin, in two different stress paradigms, in various brain areas, in male and in female rats. HKG expressions differed considerably with respect to brain area, type of stressor and gender, in an HKG-specific manner. Therefore, we conclude that before final experimentation, pilot expression studies are necessary to select an HKG which expression is unaffected by the experimental factor(s), allowing reliable interpretation of expression data of genes of interest.

Stressful stimuli evoke neuronal and neuroendocrine responses helping an organism to adapt to changed environmental conditions. Chronic stressors may induce maladaptive responses leading to psychiatric diseases, such as anxiety and major depression. A suitable animal model to unravel mechanisms involved in the control of adaptation to chronic stress is the psychological subordination stress in the male tree shrew. Subordinate male tree shrews exhibit chronic hypothalamo-pituitary-adrenal (HPA) activation as reflected in continuously elevated cortisol secretion, and structural changes in the hippocampal formation. Corticotropin-releasing factor (CRF) is the major peptide released upon activation of the HPA axis in response to stress. Recent evidence suggests that besides CRF, urocortin 1 (Ucn1) also plays a role in stress adaptation. We have tested the significance of CRF and Ucn1 in adaptation to chronic psychosocial stress in male tree shrews exposed for 35 days to daily psychosocial conflict, by performing semi-quantitative immunocytochemistry for CRF in the parvocellular hypothalamic paraventricular nucleus (pPVN), extended amygdala, viz. central extended amygdala (CeA) and dorsolateral nucleus of the bed nucleus of the stria terminalis (BNSTdl) as well as that for Ucn1 in the non-preganglionic Edinger-Westphal nucleus (npEW). Compared to unstressed animals, psychosocial stress resulted in an immediate and sustained activation of the HPA axis and sympathetic tone as well as reduced testosterone concentration and decreased body and testis weights vs. non-stressed tree shrews. In the pPVN, the number of CRF-immunoreactive neurons and the specific signal density of CRF-immunoreactive fiber terminals in the CeA were strongly reduced (-300 and -40%, respectively; P<0.05), whereas no significant difference in CRF fiber density was found in BNSTdl. The npEW revealed 4 times less Ucn1-immunoreactive neurons (P<0.05). These clear effects on both Ucn1- and CRF-neuropeptide contents may reflect a crucial mechanism enabling the animal to adapt successfully to the stressors, and point to the significance of the pPVN, CeA and npEW in stress-induced brain diseases.

In postmortem brains of patients with major depression, the expression of corticotrophin-releasing factor (CRF) is enhanced and that of brain-derived neurotrophic factor (BDNF) decreased. In mice over-expressing neuronal CRF (an animal model for depression) the expression of urocortin 1 (Ucn1) in the non-preganglionic Edinger-Westphal nucleus (npEW) is strongly down-regulated. Therefore, we hypothesized that an altered activity of Ucn1 neurons in the npEW would contribute to the pathogenesis of major depression. To test this hypothesis we measured Ucn1 mRNA and BDNF mRNA levels in the npEW of seven male and four female, drug-free suicide victims with major depression, and compared the data with those obtained from 10 male and seven female individuals without neurological and psychiatric disorders (controls). We show that compared with controls, the Ucn1-mRNA level in npEW neurons is about 9.12 times higher in male but unchanged in female suicide victims. Furthermore, BDNF mRNA expression in microdissections of npEW was 3.36 times lower in male suicide victims, but 5.27 times higher in female victims, compared with controls. Our data also show that male suicide victims had almost 11.47 times more Ucn1 and 4.26 times less BDNF mRNA in the npEW than female suicide victims. We discuss the significance of these data for npEW Ucn1 and BDNF, and propose that altered expressions of Ucn1 and BDNF in the npEW contribute to the pathogenesis of major depression and/or suicidality in a gender-specific manner.

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.

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.

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.

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.

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.

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.

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.

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.

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., (Copyright 2004 Wiley Periodicals, Inc. J Neurobiol 58: 467-478, 2004)

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.

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.

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.

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.

In recent years a large body of evidence has emerged linking chronic stress with increased vulnerability for depression and anxiety disorders. As corticotropin-releasing factor (CRF) is hypersecreted under these psychological conditions, we used our CRF-overexpressing (CRF-OE) mouse line to study underlying brain mechanisms possibly causing these disorders. Urocortin (Ucn), a recently discovered member of the CRF peptide family may play a role in the pathophysiology of stress-induced disorders. Stressors recruit Ucn-immunoreactive neurons in the Edinger-Westphal nucleus (E-WN), which is the major site of Ucn expression. Furthermore, E-WN Ucn mRNA levels are upregulated in CRF-deficient mice. Based on these findings, we hypothesized the down-regulation of E-WN Ucn in CRF-OE mice and consequently, altered responsiveness to stressful stimuli. Our results support this hypothesis as we found weaker immunohistochemical labeling with anti-Ucn and a six times weaker Ucn mRNA signal in E-WN in CRF-OE mice. Moreover, E-WN Ucn-expressing neurons mounted a response to acute challenge in CRF-OE mice too. From these results it is concluded that the CRF and E-WN Ucn neuronal systems work in concert in response to acute challenges, but are inversely regulated in their activities during chronic hyperactivity of the hypothalamo-pituitary-adrenal axis.

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.

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.

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.

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.

TRH is a neuropeptide that activates phospholipase C and, when acting on secretory cells, usually induces a biphasic response consisting of a transitory increase in secretion (due to IP(3) mobilization of Ca(2+) from intracellular stores), followed by a sustained plateau phase of stimulated secretion (by protein kinase C-dependent influx of extracellular Ca(2+) through voltage-operated Ca(2+) channels). The melanotrope cell of the amphibian Xenopus laevis displays a unique secretory response to TRH, namely a broad transient but no sustained second phase, consistent with the observation that TRH induces a single Ca(2+) transient rather than the classic biphasic increase in [Ca(2+)](i). The purpose of the present study was to determine the signal transduction mechanism utilized by TRH in generating this Ca(2+) signaling response. Our hypothesis was that the transient reflects the operation of only one of the two signaling arms of the lipase (i.e., either IP(3)-induced mobilization of internal Ca(2+) or PKC-dependent influx of external Ca(2+)). Using video-imaging microscopy it is shown that the TRH-induced Ca(2+) transient is dramatically attenuated under Ca(2+)-free conditions and that thapsigargin has no noticeable effect on the TRH-induced transient. These observations indicate that an IP(3)-dependent mechanism plays no important role in the action of TRH. PKC also does not seem to be involved because an activator of PKC did not induce a Ca(2+) transient and an inhibitor of PKC did not affect the TRH response. Experiments with a bis-oxonol membrane potential probe showed that the TRH response also does not underlie a PKC-independent mechanism that would induce membrane depolarization. We conclude that the action of TRH on the Xenopus melanotrope does not rely on the classical phospholipase C-dependent mechanism.

Some amphibian brain-melanotrope cell systems are used to study how neuronal and (neuro)endocrine mechanisms convert environmental signals into physiological responses. Pituitary melanotropes release alpha-melanophore-stimulating hormone (alpha-MSH), which controls skin color in response to background light stimuli. Xenopus laevis suprachiasmatic neurons receive optic input and inhibit melanotrope activity by releasing neuropeptide Y (NPY), dopamine (DA) and gamma-aminobutyric acid (GABA) when animals are placed on a light background. Under this condition, they strengthen their synaptic contacts with the melanotropes and enhance their secretory machinery by upregulating exocytosis-related proteins (e.g. SNAP-25). The inhibitory transmitters converge on the adenylyl cyclase system, regulating Ca(2+) channel activity. Other messengers like thyrotropin-releasing hormone (TRH) and corticotropin-releasing hormone (CRH, from the magnocellular nucleus), noradrenalin (from the locus coeruleus), serotonin (from the raphe nucleus) and acetylcholine (from the melanotropes themselves) stimulate melanotrope activity. Ca(2+) enters the cell and the resulting Ca(2+) oscillations trigger alpha-MSH secretion. These intracellular Ca(2+) dynamics can be described by a mathematical model. The oscillations travel as a wave through the cytoplasm and enter the nucleus where they may induce the expression of genes involved in biosynthesis and processing (7B2, PC2) of pro-opiomelanocortin (POMC) and release (SNAP-25, munc18) of its end-products. We propose that various environmental factors (e.g. light and temperature) act via distinct brain centers in order to release various neuronal messengers that act on the melanotrope to control distinct subcellular events (e.g. hormone biosynthesis, processing and release) by specifically shaping the pattern of melanotrope Ca(2+) oscillations.

Light and temperature stimuli act via various brain centers and neurochemical messengers on the pituitary melanotrope cells of Xenopus laevis to control distinct subcellular activities such as the biosynthesis, processing, and release of alpha-melanophore-stimulating hormone (alphaMSH). The melanotrope signal transduction involves the action of a large repertoire of neurotransmitter and neuropeptide receptors and the second messengers cAMP and Ca(2+). Here we briefly review this signaling mechanism and then present new data on two aspects of this process, viz. the presence of a stimulatory beta-adrenergic receptor acting via cAMP and the egress of cAMP from the melanotrope upon a change of alphaMSH release activity., ((c) 2002 Elsevier Science (USA).)

The pituitary melanotrope cell of Xenopus laevis displays cytosolic Ca2+ oscillations that arise for the interplay between the burst-like openings of voltage-operated Ca2+ channels and Ca2+-extrusion mechanisms. We have previously shown that Ca2+-extrusion rates increase with increases in [Ca2+]i, suggesting that Ca2+ itself plays a role in shaping the Ca2+ oscillations. The purpose of the present study was to test this hypothesis by manipulating the intracellular Ca2+ buffering capacity of the cell and determining the consequences of such manipulations for the shape of the Ca2+ oscillations. We manipulated the cytosolic buffering capacity by loading the fast Ca2+ chelator BAPTA into cells. During loading the [Ca2+]i was dynamically imaged with confocal laser scanning microscopy. The basal [Ca2+]i was reduced with BAPTA loading and this reduction was associated with lower Ca2+-extrusion rates, a broadening of the Ca2+ oscillations and declined oscillation frequencies. Short loading periods of the buffer led to new, stable patterns of Ca2+ signaling and to reduced but stable levels of peptide secretion. We propose that the cytosolic Ca2+ buffer capacity, and thus by inference the profile of intracellular Ca2+ buffering proteins, is an important factor in setting the frequency and shape of Ca2+ oscillations.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a novel peptide of the secretin/glucagon/vasoactive intestinal polypeptide superfamily, has been initially characterized in mammals in 1989 and, only 2 years later, its counterpart has been isolated in amphibians. A number of studies conducted in the frog Rana ridibunda have demonstrated that PACAP is widely distributed in the central nervous system (particularly in the hypothalamus and the median eminence) and in peripheral organs including the adrenal gland. The cDNAs encoding the PACAP precursor and 3 types of PACAP receptors have been cloned in amphibians and their distribution has been determined by in situ hybridization histochemistry. Ontogenetic studies have revealed that PACAP is expressed early in the brain of tadpoles, soon after hatching. In the frog Rana ridibunda, PACAP exerts a large array of biological effects in the brain, pituitary, adrenal gland, and ovary, suggesting that, in amphibians as in mammals, PACAP may act as neurotrophic factor, a neurotransmitter and a neurohormone., (Copyright 2001 Wiley-Liss, Inc.)

This review deals particularly with the recent literature on the structural and functional aspects of the retino-brain-pituitary system that controls the physiological process of background adaptation in the aquatic toad Xenopus laevis. Taking together the large amount of multidisciplinary data, a consistent picture emerges of a highly plastic system that efficiently responds to changes in the environmental light condition by releasing POMC-derived peptides, such as the peptide alpha-melanophore-stimulating hormone (alpha-MSH), into the circulation. This plasticity is exhibited by both the central nervous system and the pituitary pars intermedia, at the level of molecules, subcellular structures, synapses, and cells. Signal transduction in the pars intermedia of the pituitary gland of Xenopus laevis appears to be a complex event, involving various environmental factors (e.g., light and temperature) that act via distinct brain centres and neuronal messengers converging on the melanotrope cells. In the melanotropes, these messages are translated by specific receptors and second messenger systems, in particular via Ca(2+) oscillations, controlling main secretory events such as gene transcription, POMC-precursor translation and processing, posttranslational peptide modifications, and release of a bouquet of POMC-derived peptides. In conclusion, the Xenopus hypothalamo-hypophyseal system involved in background adaptation reveals how neuronal plasticity at the molecular, cellular and organismal levels, enable an organism to respond adequately to the continuously changing environmental factors demanding physiological adaptation., (Copyright 2001 Wiley-Liss, Inc.)

An important aspect of Ca(2+) signaling is the ability of cells to generate intracellular Ca(2+) waves. In this study we have analyzed the cellular and subcellular kinetics of Ca(2+) waves in a neuroendocrine transducer cell, the melanotrope of Xenopus laevis, using the ratiometric Ca(2+) probe indo-1 and video-rate UV confocal laser-scanning microscopy. The purpose of the present study was to investigate how local Ca(2+) changes contribute to a global Ca(2+) signal; subsequently we quantified how a Ca(2+) wave is kinetically reshaped as it is propagated through the cell. The combined kinetics of all subcellular Ca(2+) signals determined the shape of the total cellular Ca(2+) signal, but each subcellular contribution to the cellular signal was not constant in time. Near the plasma membrane, [Ca(2+)](i) increased and decreased rapidly, processes that can be described by a linear and exponential function, respectively. In more central parts of the cell slower kinetics were observed that were best described by a Hill equation. This reshaping of the Ca(2+) wave was modeled with an equation derived from a low-pass RC filter. We propose that the differences in spatial kinetics of the Ca(2+) signal serves as a mechanism by which the same cellular Ca(2+) signal carries different regulatory information to different subcellular regions of the cell, thus evoking differential cellular responses.

In mammals, the brain-specific protein munc18-1 regulates synaptic vesicle exocytosis at the synaptic junction, in a step before vesicle fusion. We hypothesize that the rate of biosynthesis of munc18-1 messenger RNA (mRNA) and the amount of munc18-1 present in neurons and neuroendocrine cells are related to the physiologically controlled state of activity. To test this hypothesis, the homolog of munc18-1 in the clawed toad Xenopus laevis, xunc18, was studied in the brain and in the neuroendocrine melanotrope cells in the intermediate lobe of the pituitary gland, at both the mRNA and the protein level. In toads adapted to a black background, the melanotropes release the peptide alpha-melanophore-stimulating hormone (alpha-MSH), which induces darkening of the skin, whereas in animals adapted to a white background the cells hardly release but store alpha-MSH, making the animal's skin look pale. The intermediate pituitary lobe of black-adapted animals revealed a strong hybridization reaction with the xunc18 mRNA probe, whereas a much weaker hybridization was observed in the intermediate lobe of white-adapted animals (optical density black: 3.4 +/- 0.2 vs. white: 0.8 +/- 0.1; P < 0.02). Immunocytochemically, Xenopus munc18-like protein has been detected throughout the brain, in identified neuronal perikarya as well as in axon tracts. Western blot analysis and immunocytochemistry further demonstrated the presence of xunc18 in the neural, intermediate and distal lobe of the pituitary gland. Xunc18 protein was furthermore determined in immunoblots of homogenates of melanotropes dissociated from the pituitary gland. In melanotropes of toads adapted to a black background, the integrated optical density of the xunc18 immunosignal was 2.7 +/- 0.5 times higher than in cells of white-adapted toads (P < 0.0001). It is concluded that, in Xenopus melanotrope cells, the amounts of both xunc18 mRNA and xunc18 protein are up-regulated in conjunction with the induction of exocytosis of alpha-MSH as a result of a physiological stimulation (environmental light condition). We propose that xunc18 is involved in physiologically controlled exocytotic secretion of neuroendocrine messengers.

The process of background adaptation in the toad Xenopus laevis is controlled by neurons in the suprachiasmatic nucleus (SC) that inhibit the release of alpha-melanophore-stimulating hormone from the neuroendocrine melanotrope cells in the pituitary gland. We have identified the structural and functional organization of different neuropeptide Y (NPY)-containing cell groups in the Xenopus SC in relation to background adaptation. A ventrolateral, a dorsomedial, and a caudal group were distinguished, differing in location as well as in number, size, and shape of their cells. They also show different degrees of NPY immunoreactivity in response to different background adaptation conditions. In situ hybridization using a Xenopus mRNA probe for the exocytosis protein DOC2 revealed that melanotrope cells of black-adapted animals have a much higher expression of DOC2-mRNA than white-adapted ones. This establishes that the degree of DOC2-mRNA expression is a good parameter to measure cellular secretory activity in Xenopus. We show that in the ventrolateral SC group, more NPY-positive neurons express DOC2-mRNA in white- than in black-adapted animals. In contrast, NPY-positive neurons in the dorsomedial group have a high secretory activity under the black-adaptation condition. We propose that in black-adapted animals, NPY-positive neurons in the ventrolateral group, known to inhibit the melanotrope cells in white-adapted animals synaptically, are inhibited by NPY-containing interneurons in the dorsmedial group. NPY-positive neurons in the caudal group have similar secretory dynamics as the dorsomedial NPY neurons, indicating that they also play a role in background adaptation, distinct from that exerted by the ventrolateral and dorsomedial group., (Copyright 2001 Wiley-Liss, Inc.)

A minimal model is presented to explain changes in frequency, shape, and amplitude of Ca2+ oscillations in the neuroendocrine melanotrope cell of Xenopus Laevis. It describes the cell as a plasma membrane oscillator with influx of extracellular Ca2+ via voltage-gated Ca2+ channels in the plasma membrane. The Ca2+ oscillations in the Xenopus melanotrope show specific features that cannot be explained by previous models for electrically bursting cells using one set of parameters. The model assumes a KCa-channel with slow Ca2+-dependent gating kinetics that initiates and terminates the bursts. The slow kinetics of this channel cause an activation of the Kca-channel with a phase shift relative to the intracellular Ca2+ concentration. The phase shift, together with the presence of a Na+ channel that has a lower threshold than the Ca2+ channel, generate the characteristic features of the Ca2+ oscillations in the Xenopus melanotrope cell.

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.

We have previously shown that the stimulatory effect of TRH on alpha-MSH secretion from the frog pars intermedia is associated with Ca2+ influx through voltage-dependent Ca2+ channels, activation of a phospholipase C and mobilization of intracellular Ca2+ stores. The aim of the present study was to investigate the contribution of protein kinase C (PKC), adenylyl cyclase (AC), Ca2+/calmodulin-dependent protein kinase II (CAM KII), phospholipase A2, and protein tyrosine kinase (PTK) in TRH-induced alpha-MSH release. Incubation of frog neurointermediate lobes (NILs) with phorbol 12-myristate-13-acetate (24 h), which causes desensitization of PKC, or with the PKC inhibitor NPC-15437, reduced by approximately 50% of the effect of TRH on alpha-MSH release. In most melanotrope cells, TRH induces a sustained and biphasic increase in cytosolic Ca2+ concentration ([Ca2+]i). Preincubation with phorbol 12-myristate-13-acetate or NPC-15437 suppressed the plateau phase of the Ca2+ response. Incubation of NILs with TRH (10(-6) M; 20 min) had no effect on cAMP production. In addition, the AC inhibitor SQ 22,536 did not affect the secretory response of NILs to TRH. These data indicate that the phospholipase C/PKC pathway, but not the AC/protein kinase A pathway, is involved in TRH-induced alpha-MSH release. The calmodulin inhibitor W-7 and the CAM KII inhibitor KN-93 did not significantly reduce the response to TRH. Similarly, the phospholipase A2 inhibitors quinacrine and 7-7'-DEA did not impair the effect of TRH on alpha-MSH secretion. The PTK inhibitors ST638 and Tyr-A23 had no effect on TRH-induced [Ca2+]i increase but inhibited in a dose-dependent manner TRH-evoked alpha-MSH release (ED50 = 1.22x10(-5) M and ED50 = 1.47x10(-5) M, respectively). Taken together, these data indicate that, in frog melanotrope cells, PKC and PTK are involved in TRH-induced alpha-MSH secretion. Activation of PKC is responsible for the sustained phase of the increase in [Ca2+]i, whereas activation of PTK does not affect Ca2+ mobilization.

The neuroendocrine melanotrope cell displays Ca2+ oscillations that are build up by several discrete Ca2+ rises ('steps'). Each step is linked to Ca2+-entry across the plasma membrane via voltage-operated calcium channels and associated with a fast Ca2+-wave travelling from the plasma membrane to the central parts of the cell. Previously, linescanning with confocal laser scanning microscopy (CLSM) supported that these waves have high speeds (between 30 and 80 microm/s), which is considered indicative of the involvement of a calcium-induced calcium release (CICR) mechanism in fast-wave propagation. However, to firmly establish the presence of a CICR mechanism one must rule out the possibility that the Ca2+ signal is artifactually accelerated by the presence of a highly mobile Ca2+ probe and also eliminate imaging artifacts inherent to single wavelength imaging. In the present study both problems are addressed. Mobility and intracellular distribution of a generally used Ca2+ probe, Oregon-green 488 BAPTA-1 (O-green-1), were established using fluorescence correlation microscopy. We then used the ratio signal of co-loaded O-green-1 and Fura-Red to quantify the relative [Ca2+]i during linescanning. It was found that O-green-1 displays different diffusion times when regions near the plasma membrane and in the center of the cell are compared. However, the calculated diffusion constant of the probe was too low to account for the observed high speed of the Ca2+ wave. In conclusion, we established the authenticity of the high speed of Ca2+-waves in Xenopus melanotropes, providing evidence for the involvement of a CICR mechanism in wave propagation.

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.