Multiple control and dynamic response of the Xenopus melanotrope cell

Comparative Biochemistry and Physiology Part B 132 (2002) 257–268

Multiple control and dynamic response of the Xenopus melanotrope cell夞 S.M. Kolk, B.M.R. Kramer, L.N. Cornelisse, W.J.J.M. Scheenen, B.G. Jenks, E.W. Roubos* University of Nijmegen, Nijmegen Institute for Neurosciences and Institute of Cellular Signaling, Department of Cellular Animal Physiology, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands Received 27 January 2001; received in revised form 9 May 2001; accepted 11 May 2001

Abstract 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 a-melanophore-stimulating hormone (a-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 g-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 Ca2q 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. Ca2q enters the cell and the resulting Ca2q oscillations trigger a-MSH secretion. These intracellular Ca2q 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 Ca2q oscillations. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: a-MSH; Background adaptation; Exocytosis proteins; Ca2q oscillations; Pro-opiomelanocortin; SNAP-25; Suprachiasmatic nucleus; Synaptic plasticity

1. Introduction

夞 This paper was submitted as part of the proceedings of the 20th Conference of European Comparative Endocrinologists, organized under the auspices of the European Society of Comparative Endocrinology, held in Faro, Portugal, September 5–9, 2000. *Corresponding author. Tel. q31 24 365 2360; fax q31 24 365 2714. E-mail address: [email protected] (E.W. Roubos).

Living organisms have the conspicuous ability to adapt to the continuously changing environment. In this way they can resist unfavorable conditions (e.g. low temperatures, scarcity of food, or a predator’s attack) and survive relatively undisturbed. Obviously, in view of the diversity of such conditions, an animal needs different adaptation systems, which vary from slow, metabolic changes

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to rapid, muscle-driven actions. However, irrespective of their different nature, most animal adaptation mechanisms share the involvement of the central nervous system and often include endocrine activity. For this reason, studies on adaptation processes can provide useful insights into the functioning of the central nervous and endocrine systems. In this respect, a wealth of interesting data has been obtained in neuroendocrine research on amphibians, e.g. as to the plasticity of behavior (Denver, 1997), the identification of novel neuropeptides (Chartrel et al., 1999), their mechanisms ´ et al., 2000; Yon et al., 2001), of action (Montero the structure and development of the hypothalamo–hypophyseal system (Jansen et al., 1997; Aida et al., 1999; Kikuyama et al., 1999) and pituitary cell heterogeneity (Gracia-Navarro et al., ´ 1998; Gonzalez de Aguilar et al., 1999) (for refs, see also Vaudry and Eberle, 1993; Vaudry et al., 1998). An example of an effective animal adaptation system in amphibians is the neuroendocrine integration at the level of the pituitary pars intermedia of the South-African toad Xenopus laevis (see Jenks and van Zoest, 1990; Jenks et al., 1993b; Roubos, 1992, 1997; Roubos et al., 1999; Kramer et al., 2001a). The Xenopus pars intermedia contains endocrine melanotrope cells that secrete the peptide a-melanophore-stimulating hormone (aMSH). This hormone owes its name to its action on dermal melanophores, stimulating the dispersion of melanin-containing pigment granules. In this way, animals placed on a black background are able to camouflage themselves by turning the color of their skin black, whereas on a white background the low blood level of a-MSH will make the animal look pale. However, the secretion of a-MSH is not only triggered by placing the animal on a black background but also by other external factors such as low temperatures (Tonosaki, unpubl. res.; Kramer et al., 2001a). In this review, we will discuss how the melanotrope cell of Xenopus laevis transduces external light information into an appropriate response. Special reference will be made to the secretory mechanisms and intracellular signaling systems that enable the melanotrope cell to integrate neuronal messages from various brain centers and transduce them into different subcellular responses, such as the transcription of the gene of the a-MSH precursor, proopiomelanocortin (POMC), the translation of POMC mRNA into POMC, the posttranslational

processing of POMC into biologically active peptides (including a-MSH), and the secretion of such peptides by exocytosis. As will be discussed below in some detail, the Xenopus melanotrope cells receive a large number of different neuronal stimulatory and inhibitory inputs. This is in accordance with the fact that four brain centers are known to be involved in regulating melanotrope cell activity (e.g. Jenks et al., 1993b; Roubos, 1997; Kramer et al., 2001a). The melanotropes are pivotal in the regulation of the physiological process of background adaptation as they are the source of a-MSH, which is posttranslationally processed from POMC. 2. Various brain centers and neuronal messengers control melanotrope cell activity Superfusion studies with intact neurointermediate lobes and with single melanotrope cells dissociated from this lobe have shown that the release of a-MSH can be either stimulated or inhibited by a large number of classical neurotransmitters and neuropeptides. The same effects were found for the release of b-endorphins and for other POMCderived peptides, all of which are co-released with a-MSH (Jenks and van Zoest, 1990; Jenks et al., 1993a,b) (Fig. 1). The stimulatory factors are corticotropin-releasing hormone (CRH), sauvagine, i.e. the amphibian homologue of CRH, thyrotropin-releasing hormone (TRH), noradrenalin (NA), serotonin and acetylcholine (ACh), whereas dopamine (DA), g-aminobutyric acid (GABA), and neuropeptide Y (NPY) inhibit the release of the POMC-derived end-products. Of these regulatory factors only ACh originates from inside the pars intermedia. It was found that the melanotrope cells synthesize choline acetyltransferase and release ACh and, moreover, possess ACh receptors of the muscarinic M1 type. This indicates that the cells regulate each other in a paracrine way, and probably even stimulate themselves in an autoexcitatory fashion (Van Strien et al., 1996b). Below we will discuss the various brain centers involved in the control of the Xenopus pars intermedia (Fig. 1). 2.1. The suprachiasmatic nucleus Three neurotransmitters are known to inhibit the activity of the Xenopus melanotrope cells: DA, NPY and GABA. Retrograde tract tracing studies have shown that these transmitters coexist in neu-

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Fig. 1. Schematic representation of identified neurochemical inputs to the melanotrope cell of Xenopus laevis. CRH, corticotropin-releasing hormone; DA, dopamine; GABA, g-aminobutyric acid; IN, interneurons; LC locus coeruleus; Mg, magnocellular nucleus; NA, noradrenaline; NPY, neuropeptide Y; PI, pars intermedia; PN, pars nervosa; POMC, pro-opiomelanocortin; Ra, raphe nucleus; SC, suprachiasmatic nucleus; SMIN, suprachiasmatic melanotrope-inhibiting neuron; TRH, thyrotropin-releasing hormone; 5HT, serotonin. (Modified after Ubink et al., 1998; Kramer et al., 2001a).

rons in the ventrolateral part of the suprachiasmatic nucleus (SC) (Tuinhof et al., 1994; Ubink et al., 1998). These so-called SMINs (suprachiasmatic melanotrope-inhibiting neurons) form synaptic contacts on the melanotropes (De Rijk et al., 1990a, 1992; Van Strien et al., 1991). The synapses store DA and NPY within large dense-cored vesicles, whereas GABA is present within small electron-lucent vesicles. Quantitative immunocytochemistry and in situ hybridization studies have revealed differences in SMINs of white- vs. black-adapted animals in the degree of NPY gene expression: in black-adapted animals the level of NPY mRNA is very low and NPY-immunoreactivity is low to absent, whereas in animals on a white background, high NPY mRNA expression and strong NPY immunoreactivity are found (Tuinhof et al., 1993; Kramer et al., 2001a,c). These data indicate that the neurons in the SC are involved in the background light intensity-dependent inhibition of a-MSH release from the melanotrope cells. This conclusion was recently confirmed by showing that the SMINs express more mRNA encoding for the exocytosis-related protein DOC2, a marker for

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neuronal secretory activity (Berghs et al., 1999), in white- than in black-adapted animals (Kramer et al., 2001a,c). In contrast, NPY-positive neurons in a dorsomedial group of the SC have a high secretory activity under the black adaptation condition. It is proposed that these cells, which project ventrolaterally towards the SMINs (Ubink et al., 1998; Kramer et al., 2001c), are interneurons that inhibit the SMINs in black-adapted animals. This idea is in line with the fact that NPY-containing synapses contacting the SMINs are preferentially present in black-adapted animals (Kramer et al., 2001b, this volume). As to the optical regulation of the SC, HRP-labeling of the optic nerve has made likely that the SMINs receive input from the retina (Tuinhof et al., 1994). So, a direct retinosuprachiasmatic pathway seems to control the melanotrope cells. The ultrastructural dynamics of the SMIN axon terminals in the pars intermedia have received special attention with respect to their high degree of plasticity in response to changes in background light condition. In white-adapted animals the density of the synapse population is about twice as high as in black-adapted ones and the synaptic profiles are significantly larger as well. Also, the numerical density of the active zones of these synapses is about twice as large and the zones are approximately 50% larger in white-adapted animals as compared to black-adapted ones (Berghs and Roubos, 1996). It seems likely that under the condition of adaptation to a white background the varicosities increase their capacity to release neurotransmitters, which is reflected in a higher level of immunoreactivity for the exocytosis-related protein SNAP-25 (Kolk et al., 2000) (Fig. 2). 2.2. The magnocellular nucleus In Xenopus, the magnocellular nucleus (Mg) contains TRH-positive and CRH-positive neurons. Both these neuropeptides stimulate a-MSH release from superfused neurointermediate lobes (VerburgVan Kemenade et al., 1987a,c). TRH was shown in the Mg of Xenopus laevis tadpoles (Goos, 1978), and in this nucleus also CRH-positive neurons occur (Olivereau et al., 1987; VerburgVan Kemenade et al., 1987a). Injection of retrograde fluorescent markers into the neural lobe highlighted neurons in the Mg (Tuinhof et al., 1994). In Xenopus, a direct innervation of the intermediate lobe by fibers containing CRH and

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Fig. 2. Schematic representation of synaptic and endocrine plasticity occurring at the level of the pars intermedia in the process of background adaptation in the amphibian Xenopus laevis. Insets represent SNAP-25 immunoreactivity in black- and white-adapted animals, in the neuronal network contacting the melanotropes (upper panel; A, B; arrowheads indicate SNAP-25-immunoreactive varicosities in the pars intermedia) and the melanotropes themselves (lower panel; C, D), which is upregulated when a stronger release activity is needed to adjust to a changed environment (B, the inhibiting fiber network of the SMINs; C, the melanotropes). (Based on Berghs and Roubos, 1996; Kolk et al., 2000, 2001).

TRH has not been found (Olivereau et al., 1987) and, therefore, it is assumed that these neuropeptides are released from the neural lobe and reach the melanotropes by diffusion (Jenks et al., 1993b). Thus, it appears that the Mg of Xenopus, and probably of amphibians in general, is not only involved in the neurohormonal release of vasotocin and mesotocin, but also stimulates the secretion of a-MSH from the melanotrope cells through the release of CRH and TRH. 2.3. The locus coeruleus Neurons in the locus coeruleus (LC) become labeled when DiI is injected into the pars intermedia (Tuinhof et al., 1994; Ubink et al., 1999), indicating that these neurons project to the melanotrope cells. LC neurons as well as fibers in the pars intermedia are immunopositive for NA (Gon´ zalez and Smeets, 1993). While superfusion experiments with intact neurointermediate lobes suggest

that NA has an inhibitory action on a-MSH-release via a dopamine D2 receptor (Verburg-Van Kemenade et al., 1986c), it was recently shown that isolated melanotrope cells are stimulated by NA via a b-adrenergic receptor (Jenks, unpubl. res.). The way LC neurons release NA to the melanotrope cells is unknown because up to now, NA has not been identified at the ultrastructural level in the Xenopus pars intermedia (Berghs, unpubl. res.). It remains to be seen whether the LC plays a role in light-controlled background adaptation or that it is involved in stress-dependent regulation of the secretion of a-MSH, like in rat (Tuinhof et al., 1994). 2.4. The raphe nucleus Serotonin has a dose-dependent stimulatory effect on the release of radiolabeled peptides from melanotrope cells in vitro (Ubink et al., 1999). In the intermediate lobe serotonin-immunoreactive

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nerve fibers occur and application of FAST DiI to the pars intermedia labels neurons not only in the SC and LC but also neurons in the raphe nucleus. Only neurons in the latter nucleus reveal serotoninimmunoreactivity. Therefore, it can be concluded that the melanotrope cells in the Xenopus pars intermedia are innervated by a serotonergic network originating in neurons in the raphe nucleus, and that serotonin released from this network stimulates melanotrope cell activity (Ubink et al., 1999). 3. Melanotrope subcellular processes 3.1. Receptors and second messengers There is a wide variety of receptors involved in the transmembrane signaling by the Xenopus melanotrope cell. DA acts via a D2-like receptor (Verburg-Van Kemenade et al., 1986a,c; Martens et al., 1991), NPY via a Y1-type receptor (Blomqvist et al., 1995; Scheenen et al., 1995), GABA via a GABAA and a GABAB receptor (Verburg-Van Kemenade et al., 1986a; De Koning et al., 1993), NA via a b-adrenergic receptor (Jenks, unpubl. res.) and ACh via a muscarinic type 1 receptor (Van Strien et al., 1996b). The GABAA receptor is coupled to a chloride channel. The main second messengers that transduce receptor activations into subcellular responses are cAMP and the Ca2q ion. The D2 receptor, GABAB receptor and Y1 receptor are negatively coupled to adenylyl cyclase, whereas CRH, serotonin and NA stimulate this cyclase (Verburg-Van Kemenade et al., 1986b; Jenks et al., 1991, 1993a,b; De Koning et al., 1993). Eventually, all neuronal factors influencing Xenopus melanotrope cell activity control the intracellular Ca2q concentration (wCa2qxi) (Kongsamut et al., 1993; Scheenen et al., 1993, 1994a,b,c, 1995, 1996; Van Strien et al., 1996b). This control may be the key process by which the neuronal factors regulate subcellular melanotrope activities (e.g. Lieste et al., 1996, 1998; Koopman et al., 1996, 1999). 3.2. Role of calcium ions in control of melanotrope activity In vitro, most of the Xenopus melanotrope cells reveal spontaneous fluctuations of the wCa2qxi, the so-called calcium oscillations (Shibuya and Douglas, 1993a,b,c; Scheenen et al., 1994a,b,c, 1995,

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1996; Koopman et al., 1996, 1999; Jenks et al., 1999) (Fig. 3). Opening of N-type voltage-operated Ca2q channels in the plasma membrane permits Ca2q ions to enter the cell and stepwise build up the wCa2qxi, leading to the rise phase of a Ca2q oscillation. Dynamic video imaging of the wCa2qxi has revealed that the known secreto-inhibitors of the Xenopus melanotrope inhibit the oscillations whereas the secreto-stimulators affect the pattern of the oscillations, for instance, by changing the frequency or the amplitude of the oscillations or the number of stepwise increases per oscillation (Shibuya and Douglas, 1993b; Scheenen et al., 1994b). These findings indicate that the Ca2q oscillations are the driving force for secretion. Recently, a mathematical model for the Ca2q oscillations and the electrical bursting of the Xenopus melanotrope cell was made, which includes all main features of the oscillations, like steps in the rise phase, a plateau, a smooth exponential decline and an abrupt transition from the decline to the rise phase (Cornelisse et al., 2000) (Fig. 3). The model contains a combination of standard Hodgkin–Huxley type voltage-gated Naq, Ca2q and Kq channels to generate Ca2q action potentials spontaneously, and an atypic Kq channel with slow Ca2q-dependent gating kinetics that hyperpolarises the plasma membrane as a result of cytoplasmic Ca2q increase, thereby terminating the burst of action potentials. The K2q Ca channel activates with a delay in relation to wCa2qxi, which, together with the presence of a Naq channel, generates the specific features of the Ca2q oscillations in the Xenopus melanotrope. Confocal laser scanning microscopy shows that the influx of Ca2q ions through the N-type Ca2q channels initiates an intracellular calcium-induced calcium release process that propagates the Ca2q signal as a wave through the cytoplasm and into the nucleus. This observation suggests that the Ca2q wave is in some way involved in the control of an intranuclear process, e.g. (POMC) gene expression. In situ hybridization of POMC mRNA in single melanotrope cells demonstrated that cells displaying Ca2q oscillations have a higher level of POMC gene expression than cells that do not show such oscillations (Jenks and Dotman, unpubl. res.). This suggests that the oscillations not only control exocytosis but also gene expression. We hypothesize that the specificity of the action of a given neuronal messenger for a specific subcellular

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Fig. 3. Calcium oscillations in Xenopus melanotropes are built up of discrete increase phases, the so-called calcium steps (left panel; experimental; microfluorimetry with Ca2q ratioing probe; ratio (F360yF380) indicates wCa2q xi ). Each calcium step is associated with an action potential (cell-attached patch clamp; expressed in capacitative current, Icap-pA). Using a mathematical model in which certain parameters of the standard Hodgkin–Huxley model have been modified, the determinants of both calcium oscillations (expressed as wCa2qxi) and electrical membrane activity (in mV) are adequately described (right panel; model) (Cornelisse et al., 2000).

process lies in the unique coding of the pattern of the Ca2q oscillation. Such a mechanism would explain why these factors have differential effects on distinct subcellular processes like POMC biosynthesis, processing and release of POMC-peptides (Dotman et al., 1997). 3.3. Cleavage products of POMC The pars intermedia of the pituitary gland of the adult Xenopus laevis contains approximately 70 000 melanotrope cells (De Rijk et al., 1990b). The cell size, and, hence, the size of the total pars intermedia, strongly increases when Xenopus is moved from a white to a black background. This cell activation particularly concerns the biosynthetic apparatus which, in animals on a black background, becomes highly active, as appears from the large nuclei, extensive rough endoplasmic reticulum, high POMC mRNA contents, the welldeveloped Golgi apparatus and the high level of

POMC and its end-products (Hopkins, 1970; Weatherhead and Whur, 1972; Loh et al., 1985; Martens et al., 1987; De Rijk et al., 1990b; Ayoubi et al., 1992; Maruthainar et al., 1992; Dotman et al., 1998a,b). An early step in biosynthetic activation is the formation of a high amount of cFos(like) protein (Ubink et al., 1997), indicating that black background adaptation activates the cFos gene, preceding the increased expression of the two POMC genes in melanotropes. Changing the light intensity of the animal’s environment from white to black results into an increased expression of both POMC genes (Martens, 1986; Martens et al., 1987; Deen et al., 1991, 1992). Long-term inhibition of POMC biosynthesis is mainly controlled by DA and NPY. GABA-inhibition is only transient, apparently because of receptor desensitization or down-regulation (Dotman et al., 1996). The processing of POMC into its peptide endproducts (e.g. b-MSH, endorphins, CLIP) has been well characterized (e.g. Loh and Gainer,

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1977; Martens et al., 1981, 1982, 1987; VerburgVan Kemenade et al., 1987b; Rouille´ et al., 1989; Dores et al., 1991; Van Strien et al., 1993a,b, 1995a,b, 1996a). Of these products, a-MSH plays a key role in the background adaptation process. The melanotrope cell possesses several proteins that are associated with its biosynthetic function, such as enzymes that assist in the translocation, folding, cleavage, sorting and packaging of POMC and its end-products. The amounts of some of these proteins and their mRNAs are under control of the background light condition (e.g. Martens et al., 1989; Braks et al., 1992; Braks and Martens, 1994, 1995; Holthuis et al., 1995). For example, in animals on a black background, the precursor protein 7B2 and the POMC-cleaving enzyme, prohormone convertase 2 (PC2) are highly expressed by Xenopus melanotropes. It appears that 7B2 in its unprocessed form strongly inhibits PC2 as well as the conversion of proPC2 to mature PC2 (Braks and Martens, 1994, 1995). Together with the other POMC-derived peptides, a-MSH is packed into the melanotrope secretory granules. Immunoelectron microscopy shows that POMC is particularly present within immature electron-dense granules, whereas, as a result of POMC processing, a-MSH is abundant in the mature electron-lucent granules (Roubos and Berghs, 1993; Berghs et al., 1997, 1998). Whereas in mammalian melanotropes a-MSH is N-terminally acetylated (Eipper and Mains, 1980), in the Xenopus melanotrope this acetylation takes places just prior or during the process of secretion and, therefore, the major form of a-MSH in the pars intermedia is desacetyl-a-MSH (Martens et al., 1981; Rouille´ et al., 1989; Dores and Rothenberg, 1987; Verburg-Van Kemenade et al., 1987b). The main endorphin in the Xenopus melanotrope is a,N-acetyl-b-endorphin w1–8x (Dores et al., 1991; Maruthainar et al., 1992; Van Strien et al., 1993a). The peptide is Xenopus-specific, and is responsible for the main endorphin-immunoreactivity of the melanotrope. In contrast to a-MSH, melanotrope endorphins are intracellularly acetylated (Van Strien et al., 1995b), suggesting that there are separate acetylation mechanisms for endorphins and a-MSH. 3.4. Secretion of POMC peptides All POMC-derived peptides identified up to now are secreted from the melanotrope cell, as shown

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under in vitro conditions (Van Strien et al., 1993a, 1995a, 1996a). Except for a-MSH, which controls skin color, the physiological functions of these peptides are not known, but some have been suggested to play a role in adaptation to stressors and the control of adrenal gland activity (Jenks et al., 1993b; Roubos, 1997). Regulated secretion of POMC-peptides occurs by exocytosis of the melanotrope secretory granule contents (Roubos, 1997), and depends on activation of voltage-dependent, N-type calcium channels (Scheenen et al., 1994c). Exocytosis is the common and main mechanism by which neurons and endocrine cells release their messengers. It involves the action of a large variety of proteins (‘exocytosis-related proteins’) which regulate and mediate the various stages of exocytosis (e.g. docking, priming, fusion) eventually leading to secretion. The molecular mechanism underlying exocytosis is receiving much attention, especially in neuronal cells. Regulatory steps in this complicated process include the formation of the socalled core complex, consisting of syntaxin I, synaptobrevin and SNAP-25, implicated in medi´ ¨ ating membrane fusion (Sudhof, 1995; Fernandez´ ¨ Chacon and Sudhof, 1999). Many other proteins play a role in this complex machinery, among which are synaptotagmin, a calcium sensor, and munc18, involved in proper fusion (Verhage et al., 2000). The Xenopus brain and pituitary gland have been studied with respect to the occurrence of these proteins and their mRNAs in endocrine cells as compared to neurons (Berghs et al., 1999). Moreover, the physiological regulation of some of these proteins has been investigated (Kolk et al., 1999, 2000, 2001; Kramer et al., 2001c) (Fig. 2). Western blotting has revealed the presence of SNAP-25, syntaxin I, synaptobrevin and synaptotagmin in the brain as well as in the pituitary gland. Furthermore, on the basis of their molecular characterization, mRNA probes have been generated for the use in in situ hybridization studies. These have demonstrated the presence of Xenopus homologues of SNAP-25a and b, isoforms of SNAP-25 (Kolk et al., unpubl. res.), munc-18 (xunc-18; Kolk et al., 2001) and DOC2 (Berghs et al., 1999) throughout the brain and in the intermediate and distal pituitary lobe. These data indicate that many exocytosis-related proteins are shared by neurons and (neuro)endocrine cells. This conclusion is supported by light microscope immu-

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nocytochemical localization studies, revealing the occurrence of immunoreactive exocytosis-related proteins in neuronal cell bodies, axons and axon terminals (SNAP-25, munc18; Kolk et al., 2000, 2001) and in the melanotrope cells (SNAP-25, munc18, synaptobrevin, synaptotagmin; Kolk et al., 2000, 2001, unpubl. res.). More specifically, SNAP-25 has been localized by confocal laser scanning microscopy at the melanotrope plasma membrane and in the perinuclear and Golgi region, and a substantial amount was found in the cytosol. Ultrastructurally, SNAP-25 appeared to be in the bounding membrane of secretory granules, as shown by immunogold electron microscopy. This granular location of this initially thought plasma membrane-bound protein was also detected in the neurohemal axon terminals of the neural lobe (Kolk et al., 2000). The background light intensity condition clearly influences the expression of exocytosis-related proteins in Xenopus neurons and endocrine cells, as has been shown by a wide variety of techniques. In the SC, under a black-adaptation condition, DOC2 mRNA expression is decreased in the SMIN and increased in the dorsomedial NPY-positive neurons in the SC as well as in the melanotrope cells (Berghs et al., 1999; Kramer et al., 2001a,c). Under this adaptation condition, an upregulation of xunc18 mRNA was observed when the in situ hybridization signal was quantified. Moreover, the melanotropes also show increased expressions of SNAP-25 and xunc18 protein, as shown by quantitative Western blotting and immunofluorescence using confocal laser scanning microscopy. In contrast, the axonal network of the SMIN contacting the melanotropes reveals strongest SNAP-25 protein immunoreactivity in animals adapted to a white background, which is in line with the inhibitory action of the SMIN on the melanotropes under this condition (Kolk et al., 2000, 2001) (Fig. 2). These data concerning the presence and physiological regulation of exocytosis-related proteins in brain and pituitary of the amphibian Xenopus laevis, permit the following conclusions on exocytosis-related proteins in general. (1) They are highly conserved throughout evolution, (2) they act in both neuronal and endocrine cells and (3) their expression can be controlled at both the mRNA and protein level by physiological stimuli that increase the capacity of the molecular machinery controlling the process of exocytosis.

4. Concluding remarks The brain-pars intermedia system of the SouthAfrican toad Xenopus laevis, involved in background adaptation, has been investigated in much detail, both structurally and functionally. It reveals a high plasticity at the level of its cellular and molecular components, which is especially obvious when the system is challenged with different environmental conditions, in particular with respect to the background light intensity. We propose that environmental factors (e.g. light and temperature) act via different brain centers and specific neuronal messengers on the melanotrope cells. In this process the messengers control distinct cellular events (e.g. gene expression, and hormone biosynthesis, processing and exocytotic release) by specifically influencing the pattern of temporal changes in the intracellular Ca2q concentration of the melanotrope cell. Acknowledgments Part of the research reviewed in this article was made possible by grants from the Netherlands Organization for Scientific Research (NWO). References Aida, T., Iwamuro, S., Miura, S., Kikuyama, S., 1999. Changes of pituitary proopiomelanocortin mRNA levels during metamorphosis of the bullfrog larvae. Zool. Sci. 16, 255–260. Ayoubi, T.A.Y, Jenks, B.G., Roubos, E.W., Martens, G.J.M., 1992. Transcriptional and posttranscriptional regulation of the proopiomelanocortin gene in the pars intermedia of the pituitary gland of Xenopus laevis. Endocrinology 130, 3560–3566. Berghs, C.A.F.M., Roubos, E.W., 1996. Background adaptation and synapse plasticity in the pars intermedia of Xenopus laevis. Neuroscience 70, 833–841. Berghs, C.A.F.M, Tanaka, S., Van Strien, F.J.C., Kurabuchi, S., Roubos, E.W., 1997. The secretory granule and proopiomelanocortin processing in Xenopus melanotrope cells during background adaptation. J. Histochem. Cytochem. 45, 1673–1682. Berghs, C.A.F.M., Tanaka, S., Roubos, E.W., 1998. Neuroendocrine responses in background adaptation. In: Vaudry, H., Tonon, M.C., Roubos, E.W., De Loof, A. (eds.) Trends in Comparative Endocrinology and Neurobiology. From Molecular to Integrative Biology. Ann. N.Y. Acad. Sci. 839, 574–575. Berghs, C.A.F.M., Korteweg, N., Bouteiller, A., Tuinhof, R., Asbreuk, C., Verhage, M., et al., 1999. Co-expression in Xenopus neurons and neuroendocrine cells of messenger RNA homologues of exocytosis proteins DOC2 and munc18-1. Neuroscience 92, 763–772.

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