Search

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) m RNA expression in mouse EWcp-neurones that were Ucn1-negative. Dual- and triple-label immunocytochemistry revealed the presence of CB1 R 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 CB1 R knockout ( CB1 R- KO) mice. Acute and especially chronic stress resulted in an increase in Ucn1 content of the EWcp, which was attenuated in CB1 R- KO mice. CB1 R- 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 m RNA and peptide levels in EWcp neurones, concomitant with an augmented stress response and increased anxiety-like behaviour. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In Xenopus laevis, corticotrophin-releasing factor (CRF) and urocortin 1 are present in the brain and they both are potent stimulators of α-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 α-MSH. [ABSTRACT FROM AUTHOR]

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 Ca2+ 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 Ca2+ signalling by superfused Xenopus melanotrophs was assessed. St-Ht31 stimulated secretion but had no effect on Ca2+ signalling. We conclude Xenopus melanotrophs possess a St-Ht31-sensitive PKA II that is associated with the exocytosis machinery and, furthermore, that Ca2+ 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Melanotrope cells of Xenopus laevis generate transitory increases in intracellular Ca2+, known as Ca2+ oscillations. These oscillations arise from the influx of Ca2+ through voltage-operated Ca2+ channels (VOCCs). Such oscillations are the driving force for secretion of α-melanophore-stimulating hormone (α-MSH) from the cell. The influx of Ca2+ through VOCCs initiates the mobilization of intracellular Ca2+ to generate a Ca2+ wave. The function of the Ca2+ 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 α-MSH. To determine the possible function of Ca2+ waves in Xenopus melanotropes, we addressed whether functional VOCCs, which are an established requirement for both secretion and Ca2+ wave initiation, are also required to maintain POMC gene expression. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 pEC50 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. [ABSTRACT FROM AUTHOR]

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 α-melanotropin (α-MSH) secretion from melanotrope cells is mediated through the novel receptor subtype xTRHR3. [ABSTRACT FROM AUTHOR]

It is well-known thatα-melanophore-stimulating hormone (α-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 toadXenopus laevis, using the skin melanophore index (MI) bioassay and a radioimmunoassay to measure skin colour adaptation andα-MSH secretion, respectively. We show that temperatures below 8 °C stimulateα-MSH secretion and skin darkening, with a maximum at 5 °C, independent of the illumination state of the background. No significant stimulatory effect of low temperature on the MI andα-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 stimulatesα-MSH release from melanotrope cells located in the PI. Anin vitrosuperfusion 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-inducedα-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 °C to 5 °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 melanotropeα-MSH release. We conclude that temperatures around 5 °C inactivate the SMIN in the SC and activate TRH-neurones in the magnocellular nucleus, resulting in enhancedα-MSH secretion from the PI, darkening the skin of white-adaptedX. laevis. [ABSTRACT FROM AUTHOR]

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 α-melanophore-stimulating hormone (αMSH) 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 αMSH 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 αMSH, 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Ca2+ oscillations regulate secretion of the hormone alpha-melanphore-stimulating hormone (α-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 α-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The cerebral ganglia of the freshwater snail Lymnaea stagnalis contain two clusters of neurosecretory Caudo-Dorsal Cells (CDC). These cells produce a neurohormone which stimulates ovulation. Ganglion transplantation and quantitative electron microscopy show that neuronal isolation of the cerebral ganglia complex (CCC) results in an activation of the CDC. It was, therefore, concluded that the CDC are controlled by an inhibitory neuronal input originating outside the cerebral ganglia. Ultrastructural studies on synaptic degeneration in the CCC suggest that this input reaches the CDC via a special type of synapse-like structure, the type C-SLS. Furthermore, transplantation of CCC into acceptor snails leads to a reduced release and an increased intracellular breakdown of neurohormone in the CDC of the nervous system of the acceptors. It is supposed that these phenomena are caused by the release of an (unknown) factor from the transplanted CCC. Special attention was given to the formation and degradation of a peculiar type of neurohormone granule, the large electron dense granule. The physiological significance of the neuronal and non-neuronal control mechanisms which regulate CDC activity is discussed. [ABSTRACT FROM AUTHOR]

The process of neurosecretion in the Caudo-Dorsal Cells (CDC) of the freshwater snail Lymnaea stagnalis, which produce an ovulation hormone, shows a diurnal rhythmicity. Synthesis, transport and release of the neurosecretory material (NSM) is high during the evening and the early night and low during the rest of the day, while storage of NSM mainly occurs during the daytime. In the present study the role of the eyes in the regulation of the CDC-rhythm was investigated. During a 24-hr period, at time intervals of 6 hrs, cerebral ganglia, which contain CDC, of blinded and control snails (5 per group) were fixed and the CDC were studied with quantitative electron microscopical methods. The CDC of the controls showed a distinct diurnal rhythmicity. Blinding, on the other hand, clearly affected this rhythmicity. The results indicate that after blinding the circadian CDC-rhythms of individual snails are no longer synchronous with each other ('interanimal desynchronization'). It is suggested that the rhythm of CDC neurosecretory activity is synchronized by the natural light/dark cycle via the eyes. The information from the eyes probably reaches the CDC via a nervous pathway. True synapses and three types of synapse-like structures were found on the CDC. Their role in the regulation of CDC-activity is discussed. The effect of blinding is specific for the CDC; blinding does not influence the diurnal rhythmicity of another type of cerebral neurosecretory cells, the Light Green Cells (LGC). The CDC within a cluster act synchronously. This synchrony does not depend upon the presence of the eyes. Some structures which may be involved in establishing this synchrony, such as subsurface cisterns, desmosome-like structures and 'specific release sites', are described. [ABSTRACT FROM AUTHOR]

The presence of dopamine in the pituitary of the clawed toad Xenopus laevis was studied by light and electron microscope immunocytochemistry, using pre- and postembedding techniques. Light microscopy showed the presence of an intricate, anti-dopamine-positive fibre network throughout the pars intermedia. In preembedded stained material, dopamine appeared to occur in varicosities which make synaptic contacts with both folliculo-stellate cells and melanotrope cells. Postembedding immunogold staining of freeze-substituted material permitted the localization of anti-dopamine reactivity in electron-dense vesicles in these varicosities. This finding supports the hypothesis that dopamine is involved in the (inhibitory) control of melanotrope cell activity in X. laevis. [ABSTRACT FROM AUTHOR]

The release of neural, neuroendocrine, and endocrine secretory products by exocytosis was ultrastructurally studied by means of tissue incubation in Ringer containing tannic acid (Tannic Acid Ringer Incubation-method; TARI-method), followed by conventional fixation. Tannic acid strongly enhances the electron density of extracellular (secretory) substances. During TARI-treatment of tissues exocytosis proceeds, but the exteriorized contents of the secretory granules are immediately fixed by tannic acid and do not diffuse away into the extracellular space. In this way detection of exocytosis is markedly facilitated since the number of exocytosis phenomena visible at the ultrastructural level considerably increases with progressing incubation time. Studies of the central nervous system of the mollusc Lymnaea stagnalis show that the occurrence of exocytosis during TARI-treatment is calcium-dependent. With the TARI-method exocytosis has been clearly demonstrated in a variety of structures (endocrine cells, neurohaemal axon terminals, synapses) of L. stagnalis, the insect Locusta migratoria, and the rat, including cell types where exocytotic release had not been shown before. [ABSTRACT FROM AUTHOR]

Using in situ hybridization with a pro-opiomelanocortin (POMC)-mRNA probe and immunocytochemistry with antisera to POMC and to various POMC-derived peptides, it is shown that melanotrope cells in the pars intermedia of the hypophysis of the South African aquatic toad Xenopus laevis contain POMC, α-melanophore-stimulating hormone (α-MSH), γ-MSH, acetylated and non-acetylated endorphins and adrenocorticotropic hormone (ACTH). With the exception of γ-MSH, these peptides are also found in the corticotrope cells in the rostral pars distalis. In the Xenopus brain, neuronal cell bodies in the ventral hypothalamic nucleus express POMC, α-MSH, γ-MSH, non-acetylated endorphins and ACTH, neurones in the anterior preoptic area reveal POMC, α-MSH, γ-MSH and non-acetylated endorphin, neurones in the suprachiasmatic nucleus contain α-MSH, non-acetylated endorphin and ACTH and neurones in the posterior tubercle show α-MSH, non-acetylated endorphin and ACTH immunoreactivities. In the locus coeruleus POMC and ACTH coexist, whereas α-MSH and non-acetylated endorphin occur together in the nucleus accumbens, the striatum and the nucleus of the paraventricular organ. Finally, α-MSH alone is present in the olfactory bulb, the medial septum, the medial and lateral parts of the amygdala, the ventromedial and posterior thalamic nuclei, the optic tectum and the anteroventral tegmental nucleus, and non-acetylated endorphin alone appears in the epiphysis. It is suggested that neurones that form POMC-derived peptides may play a direct or indirect role in the control of POMC-producing hypophyseal cells and/or in the physiological processes these endocrine cells regulate. This idea is supported by the fact that the suprachiasmatic nucleus and the locus coeruleus, both involved in melanotrope cell control, show POMC and POMC-peptide expression. A possible involvement in melanotrope and/or corticotrope control of the anterior preoptic and ventral hypothalamic nuclei, which both express POMC and various POMC-derived peptides, deserves future attention. [ABSTRACT FROM AUTHOR]

Placing the clawed toad Xenopus laevis on a black background stimulates the melanotrope cells in the pars intermedia of the pituitary gland to release proopiomelanocortin (POMC)-derived peptides, including α-MSH and N-acetylβ-endorphin. In this study three types of secretory granules, electron-dense(∼130 nm Ø), moderately electron-dense (∼ 160 nm Ø) and electronlucent (∼ 180 nm Ø), have been identified in these cells. Apparently, only dark granules are formed by the Golgi apparatus and lucent granules release their contents via exocytosis. Immuno-electron microscopy (immunogold double labelling) of glutaraldehyde-fixed and freeze-substituted material shows that desacetyl-α-MSH and N-acetyl-β-endorphin coexist in all three granule types. Quantification of immunostaining revealed that immunoreactivities to these peptides are lowest in the dark granules and highest in the light ones. It is proposed that intragranular processing of POMC to immunoreactive desacetyl-α-MSH and N-acetyl-β-endorphin involves an increase in granule size and a decrease in granule electron density. Black background-induced activation of the melanotrope cell is reflected by an increase in immunoreactivity of the secretory granules to each of the antisera. This suggests that cell activation stimulates the formation of peptides by intragranular processing of POMC and/or of intermediate POMC-processing products. In addition, cell activation evoked an increase in the percentage of the granule population that reacts with anti- N-acetyl-β-endorphin, probably by stimulating intragranular acetylation of β-endorphin. Apparently, this acetylation is a regulated event that occurs in the cytoplasm, independently from the acetylation of desacetyl-α-MSH which takes place near the plasmalemma at the time of granule exocytosis. [ABSTRACT FROM AUTHOR]

Two groups of cerebral dorsal cells of the pulmonate snail Planorbarius corneus stain positively with antisera raised against synthetic fragments of the B- and C-chain of the molluscan pro-insulin-related prohormone, proMIP-I, of another pulmonate snail, Lymnaea stagnalis. At the light-microscopic level the somata of the dorsal cells and their axons and neurohemal axon terminals in the periphery of the paired median lip nerves are immunoreactive with both antisera. Furthermore, the canopy cells in the lateral lobes of the cerebral ganglia are positive. In addition, MIP-immunoreactive neurons are found in most other ganglia of the central nervous system. At the ultrastructural level, pale and dark secretory granules are found in somata and axon terminals of the dorsal cells. Dark granules are about 4 times as immunoreactive to both antisera as pale granules. Release of anti-MIP- and anti-MIP-immunopositive contents of the secretory granules by exocytosis is apparent in material treated according to the tannic acid method. It is concluded that the dorsal and canopy cells synthesize a molluscan insulin-related peptide that is packed in the cell body into secretory granules and that is subsequently transported to the neurohemal axon terminals and released into the hemolymph by exocytosis. Thus, MIP seems to act as a neurohormone on peripheral targets. On the basis of the analogy between the dorsal cells and the MIP-producing cells in L. stagnalis, it is proposed that the dorsal cells of P. corneus are involved in the control of body growth and associated processes. [ABSTRACT FROM AUTHOR]

Juvenile specimens of Lymnaea stagnalis were exposed to 0 or 4 miracidia of Trichobilharzia ocellata. Dissection followed at day 22 post exposure. The effects of infection on the activity of the female gonadotropic hormone producing Dorsal Bodies (DB) were studied by phase-contrast and electron microscopy. Morphometry shows that the relative volume of DB cells of infected snails is 1.6 times as high as in controls. This is mainly due to a 45% higher cytoplasmic volume. The number of profiles of the Golgi apparatus increases by nearly 90% and the Golgi volume by 40% as a result of infection. Numerous omega-shaped indentations of the plasma membrane of the DB cell processes indicate the release of the contents of the DB hormone containing elementary granules. It is concluded that parasitic infection causes a clear increase in the synthetic activity of the DB. Most probably, the parasites exert the inhibiting effects on reproductive activity at the level of the targets of the reproductive hormones. [ABSTRACT FROM AUTHOR]

The cerebral caudodorsal cells of the pulmonate snail Lymnaea stagnalis control egg laying and egglaying behavior by releasing various peptides derived from two precursors. The biosynthesis, storage, intracellular breakdown and release of three caudodorsal cell peptides were studied by means of immuno-electron microscopy using antisera raised to fragments of these peptides: (1) Caudodorsal Cell Hormone-I (CDCH-I; derived from precursor I), (2) Caudodorsal Cell Hormone-II (CDCH-II; from precursor II), and (3) α-Caudodorsal Cell Peptide (α CDCP; from both precursors). After affinity purification of the antisera, the specificity of the sera was confirmed with dotting immunobinding assays. From the ultrastructural immunocytochemical data it has been concluded that the precursor molecules are cleaved at the level of the Golgi apparatus after which the C-terminal parts (containing α CDCP) and N-terminal parts (containing CDCH-I or CDCH-II) are sorted and preferentially packaged into large electron-dense granules (MD 150 nm), respectively. Very probably, the content of the large electron-dense granules is degraded within the cell body. The immunoreactivity of the secretory granules increases during discharge from the Golgi apparatus, indicating further processing. At least a portion of the secretory granules contains all three peptides, as shown by double and triple immunopositive stainings whereas other granules appear to contain only one or two of these peptides. The caudodorsal cells release multiple peptides via exocytosis from neurohemal axon terminals into the hemolymph and from blindly ending axon collaterals into the intercellular space of the cerebral commissure (nonsynaptic release). [ABSTRACT FROM AUTHOR]

The cerebral caudodorsal cells (CDC) of the pulmonate snail Lymnaea stagnalis are involved in the control of egg laying and associated behaviour by releasing various peptides. One of these is the ovulation hormone (CDCH). The cellular dynamics of this peptide have been studied using an antiserum raised to a synthetic portion of CDCH comprising the 20-36 amino acid sequence. With the secondary antibody-immunogold technique, specific immunoreactivity was found in all CDC. Rough endoplasmic reticulum and Golgi apparatus showed very little reactivity as did secretory granules that were in the process of being budded off from the Golgi apparatus. However, secretory granules that were being discharged from the Golgi apparatus, were strongly reactive. Secretory granules within lysosomal structures revealed various degrees of immunoreactivity, indicating their graded breakdown. Large electrondense granules, formed by the Golgi apparatus and thought to be involved in intracellular degradation of secretory material, were only slightly reactive. In the axon terminals secretory granules released their contents into the haemolymph by the process of exocytosis. The exteriorized contents were in most cases clearly immunopositive. The possibility has been discussed that CDCH is cleaved from its polypeptide precursor within secretory granules during granule discharge from the Golgi apparatus; subsequently, the mature secretory granules would be transported towards the neurohaemal axon terminals where they release CDCH into the haemolymph. Superfluous secretory material would be degraded by the lysosomal system including the large electron-dense granules. [ABSTRACT FROM AUTHOR]

To study the cellular defense mechanism against oxygen toxicity, an oxygen-tolerant cell line from Chinese hamster ovary (CHO) was obtained by multistep adaptation to increased O levels. The hyperoxia-adapted (HA) cells were able to proliferate under an atmosphere of 99% O/1% CO, an O tension lethal to the parental (control) cells. When grown under normoxic conditions (20% O/1% CO/79% N) the cells remained tolerant for at least 8 weeks, suggesting a genetic basis for the oxygen tolerance. Compared to the parental cells, the HA cells were irregularly shaped, had larger mitochondria, contained more lipid droplets and showed a reduced growth rate. Ultrastructural morphometry revealed a 1.8-fold ( p<0.001) increase of the mitochondrial volume fraction in the HA cells, resulting from an increase in both number and average volume of the mitochondria. The volume fraction of peroxisomes was increased over two-fold in the HA cells, as appeared from a ∼1.9-fold ( p< 0.001) increase in number and a 1.2-fold ( p<0.025) increase in size. There was no evidence for ultrastructural damage in the HA cells. Specific activities of antioxygenic enzymes were considerably higher in the HA cells compared to controls: CuZn-superoxide dismutase, x 2.5; Mn-superoxide dismutase, x 2.1; catalase, x 4.0; glutathione peroxidase, x 1.9. Oxygen tolerance in CHO cells is therefore associated with increased levels of antioxygenic enzymes, confirming the proposed important role of these enzymes in the defense against oxygen toxicity. [ABSTRACT FROM AUTHOR]

The axon terminals of the neuroendocrine caudo-dorsal cells (CDC) of the freshwater snail Lymnaea stagnalis release an ovulationstimulating hormone by exocytosis in a calcium-dependent way. Ultrastructural studies of the terminals, involving the K-pyroantimonate method for the demonstration of calcium and control tests with EGTA, show that calcium occurs in mitochondria and in various types of vesicular structure. Quantitative investigations indicate that mitochondria accumulate calcium during a short period of high neurohormone-release activity ('active state'; ca. 45 min) and release it again into the axoplasm some hours later, during a period of low secretory activity ('resting state'). Probably, in this way mitochondria play an important role in the buffering of the axoplasmic calcium concentration during high hormone-release activity. HRP-incorporation studies strongly suggest that the calcium-containing vesicular structures constitute a mechanism of membrane sequestration by which the CDC axon terminals resorb, transform, and release parts of the axolemma after exocytotic hormone release. The results furthermore indicate that this mechanism also may be involved in the control of the calcium concentration of the axoplasm, by taking up calcium from the axoplasm and releasing it into the extracellular space. [ABSTRACT FROM AUTHOR]

Adenylate cyclase activity was demonstrated ultracytochemically in the neurohaemal area of the ovulation-neurohormone producing Caudo-Dorsal Cells (CDC) of the freshwater snail L. stagnalis. During electrical activity the axon terminals of the CDC release the hormone by exocytosis and show high adenylate cyclase activity on their plasma membranes. Electrically inactive terminals hardly show exocytosis and exhibit only little reaction product of adenylate cyclase. This result indicates that cAMP plays a role in the control of release of the CDC hormone. [ABSTRACT FROM AUTHOR]

The ultrastructural dynamics of exocytosis in the ovulation-stimulating neurosecretory Caudo-Dorsal Cells (CDC) of the freshwater snail L. stagnalis were studied after incubation of cerebral ganglia in Ringer's solutions with different concentrations of K and Ca. Detection of exocytosis was facilitated by the use of the tannic acid-glutaraldehyde fixation method (TAGO-method). In control Ringer (low K) the frequency of exocytosis was rather low. Exocytosis mainly occurred as 'terminal' exocytosis (TE); 'intercellular' (ICE) and, particularly, 'multiple' exocytosis (ME) took place infrequently. Incubation in high K-containing Ringer strongly increased exocytotic activity. Compared to the controls the total frequency of exocytosis was 50x as high, whereas TE, ICE and ME occurred 6 x, 47x, and more than 300x as frequently, respectively. In high K/Ca-free Ringer the total frequency of exocytosis was only 2x as high as in control Ringer. It is concluded that TE, ICE, and ME are normal, Ca-dependent exocytotic phenomena. The significance of their dynamics in response to K-stimulation is discussed. The extremely high frequency of exocytosis, as well as the presence of 'unaltered granule contents in transit', is explained by assuming that an exocytotic event in the CDC lasts rather long, viz. some minutes. The results may reflect the physiological mechanism by which the CDC release their ovulation hormone. The possible involvement of 'clear' and 'large' electron lucent vesicles in membrane reuptake after exocytosis is considered. [ABSTRACT FROM AUTHOR]

The release of neuropeptides and other proteinaceous hormones by exocytosis was studied in some detail by means of tannic acid in combination with glutaraldehyde (TAGO-method). This method strongly enhances the electron density of extracellular proteins including exocytosed secretory products, and therefore facilitates visualization of the release process in qualitative and quantitative respects. This study included a variety of neurosecretory cell types in the CNS of the freshwater snail L. stagnalis, the storage and glandular lobes of the corpora cardiaca of the locust L. migratoria, and the posterior pituitary of the rat. In addition, peptide secreting cells in the anterior pituitary were examined. The cytochemical specificity of the TAGO-method as well as the significance of 'frozen' contents of exocytosing secretory granules in mammotrophs and somatotrophs of the rat is discussed. [ABSTRACT FROM AUTHOR]

Two giant neurons (diameter 130 μm) were identified immunocytochemically by means of the unlabeled antibody enzyme technique with anti ACTH 1-39 and 1-24 in the pond snail Lymnaea stagnalis. The cells are located in the visceral and the right parietal ganglion, respectively. They contain moderately electron dense elementary granules (diameter 150-160 nm). By means of the intracellular horseradish peroxidase injection technique it was shown that the cells send fibres into the neuropiles of various ganglia and into nerves. Synapses occur on the fine fibre branches in the neuropile. Synapse-like structures were found on the cell bodies and on the major fibres. The giant neurons are electrotonically coupled. With toluidine blue staining for small peptides it was demonstrated that in the central nervous system of the pond snail numerous peptidergic neurons occur in addition to those identified with the classical staining methods for neurosecretion. [ABSTRACT FROM AUTHOR]

In the central nervous system (CNS) of the freshwater snail Lymnaea stagnalis 3 types of interneuronal contacts can be distinguished electron-microscopically, viz. true synapses, 'synapse-like structures' (SLS), and 'spinules'. Use of the electron microscope specimen tilting stage reveals numerous true synapses. Both 'terminal' and 'en passant' contacts occur on neurones and on glial cells. Furthermore 'bigeminal' synapses are present. Complex (combined) convergent and divergent synaptic arrangements are found. On the basis of the morphology of presynaptic vesicles 7 types of true synapses can be discerned. Histochemical data on the contents of the vesicles are lacking. However, vesicle morphology suggests that type IV is aminergic and type VII cholinergic. Terminal and en passant SLS may penetrate deeply into neuronal somata and large axons, and into glial cells. A cluster of synaptic vesicles is present in the presynapse-like element. Spinules (spine-coated 'evagination-invagination' specializations of the plasma membranes of 2 adjacent neuronal elements) are observed between somata, between axons, and between soma and axon. The neurosecretory Light Green Cells (LGC) and Caudo-Dorsal Cells (CDC) receive complex synaptic input. Type V true synapses, 2 types of SLS, and spinules contact the LGC. The complex morphology of the relationship between type A SLS and LGC, studied in serial sections, reveals that adjacent glial cells are also contacted by type A SLS. Type II true synapses, 3 types of SLS, and spinules are identified on the CDC. The validity of the methods of identification and classification of interneuronal contacts in the CNS of L. stagnalis, as well as the role of these contacts in the regulation of the activity of 'ordinary' neurones, neurosecretory cells, and glial cells is discussed. [ABSTRACT FROM AUTHOR]

The neurosecretory system of the freshwater snail Bulinus truncatus was investigated. With the Alcian blue-Alcian yellow (AB/AY) staining method at least 10 different types of neurosecretory cells (NSC) were distinguished in the ganglia of the central nervous system. The differences in staining properties of the NSC - with AB/AY the cells take on different shades of green and yellow - are borne out at the ultrastructural level: the NSC types contain different types of neurosecretory elementary granules. The neurosecretory system of B. truncatus is compared to that of Lymnaea stagnalis, the species which has received the most attention among the pulmonates. It appears from the comparison that the systems of both species show many similarities, although some differences are also apparent. [ABSTRACT FROM AUTHOR]

The neurosecretory Caudo-Dorsal Cells (CDC) in the cerebral ganglia of the freshwater pulmonate snail Lymnaea stagnalis produce an ovulation stimulating hormone. Previously it has been shown that neuronal and non-neuronal inputs are involved in the regulation of their activity. The degree of autonomy of these cells has been investigated by studying with morphometric methods the ultrastructure of CDC maintained in vitro. CDC of isolated cerebral ganglia which were cultured for 7 days show a considerable rate of synthesis, transport and release of neurohormone. Apparently these processes can proceed in the absence of neuronal and hormonal inputs from outside the cerebral ganglia. Completely isolated CDC, however, do not show neurosecretory activity in vitro; active Golgi zones, indicating the formation of neurosecretory elementary granules, are absent from such cells. Isolation does not seem to affect general cell functions such as protein synthesis and respiration. It is suggested that a neuronal input, originating within the cerebral ganglia, is necessary for the stimulation of CDC neurosecretory activity. Techniques are described for the isolation and culture of neurosecretory cells of L. stagnalis. [ABSTRACT FROM AUTHOR]

The neurosecretory Dark Green Cells (DGC) in the pleural and parietal ganglia of the freshwater snail Lymnaea stagnalis seem to be involved in osmoregulation. Previous experiments have indicated that changes of the osmolality of the environment induce activity changes of the DGC. Furthermore, it was shown that information on environmental osmolality reaches the DGC via the blood. In the present study right pleural and parietal ganglion complexes were cultured for 3 days in vitro under different osmotic conditions. Quantitative electron microscopy revealed that, compared with the control osmolality (130 mOsm/kg HO), osmolalities of 160 and 190 mOsm/kg HO caused a reduced synthesis and an increased storage of neurohormone in the DGC. Apparently, the activity of the DGC depended on the osmotic pressure of the medium. It is proposed that in vivo the osmotic pressure of the blood (which is related to the osmolality of the environment) regulates DGC activity. [ABSTRACT FROM AUTHOR]

The neuroendocrine Caudo-Dorsal Cells (CDC) of the snail release an egg-laying hormone (CDCH) from their axon terminals by exocytosis. The CDC show 3 states of electrical activity, resting (inactive), active (electrical discharge) and inhibited (inexcitable). Exocytosis was first studied quantitatively with the tannic acid-glutaraldebyde-OO-method (TAGO method). Exocytosis activity appeared to be low in the resting state, very high in the active state, and moderate in the inhibited state. Active CDC particularly show multigranular exocytosis. Furthermore, with the freeze- fracture technique two stages of the exocytotic fusion process were identified: 1. the pre-exocytotic stage, characterized by particle-free areas in the P- and E-faces of the secretory granule and the opposing plasma membrane, and 2. the fusion step, visible as characteristic circular membrane discontinulties. Morphometry of freeze-fractured CDC confirmed that exocytoses are scarce during electrical rest, numerous during activity, and moderate during inhibition. The differences between the numbers of exocytoses In the 3 states were not that high as In the TAGO-studies, apparently because the freeze-fracture preparations did not allow quantitation of multigranular exocytosis. It is concluded that the TAGO-metbod appears very suitable for quantitation of exocytosis activity, whereas the freeze-fracture technique is particularly useful to study membrane events during exocytosis. The significance of exocytoals in the 3 CDC states for the control of egg-laying has been discussed. [ABSTRACT FROM PUBLISHER]

The neurosecretory Dark Green Cells (DGC) in the pleural and parietal ganglia of the freshwater pulmonate Lymnaea stagnalis are involved in osmoregulation, producing a diuretic hormone. The secretory activity of these cells is dependent on the osmolarity of the environment. In order to investigate whether the regulation of this activity is under nervous control, two experiments were carried out in which the right pleural and parietal ganglion complex (PPC) was transplanted into acceptor snails. The light and electron microscopic observations in the first experiment-transplantation of the PPC into control snails, kept under normal conditions-indicate that degeneration in transplants occurs only during the first week of transplantation and is mainly restricted to neuronal processes of non-neurosecretory cells (non-NSC) of which the cell bodies are located outside the PPC. Degenerated nervous elements are eliminated by phagocytozing amoebocytes and glial cells. Degeneration of neurosecretory elements was only very seldom observed. Like most non-NSC, the DGC and the three other neurosecretory cell (NSC) types in the right parietal ganglion show a normal morphology. Moreover, up to at least 6 weeks after transplantation the NSC remain capable of synthesizing, transporting and releasing their neurosecretory material (NSM). Apparently these processes can proceed in the absence of nervous input from outside the PPC. After 3 and 6 weeks of transplantation outgrowth was noted of both non-NSC and NSC axons. In the second experiment groups of acceptor snails were exposed during one week to different environmental osmolarities. Thereafter the NSC activities of the transplanted DGC and of the DGC in the acceptor's own nervous system were determined by quantitative light and electron microscopy. The following observations apply to both transplanted and acceptor DGC. Compared with the control condition (tap water) exposure of snails to demineralized water leads to a significant increase in nuclear and cytoplasmic volumes and in the extent of the granular endoplasmic reticulum (GER) and the Golgi apparatus. Moreover, the activity of the Golgi apparatus is strongly enhanced. In the DGC axon endings located in the pleuroparietal connective the release of NSM is raised markedly above control level. Conversely, exposure to a 0.08 M NaCl solution (hypertonic to the blood) reduces the secretory activity of the DGC, as is obvious from the decrease of nuclear and cytoplasmic volumes, the smaller extent of the GER and of the Golgi apparatus and the lower Golgi activity. The number of cytosomes is increased dramatically, which accounts for the breakdown of elementary granules and of other cytoplasmic constituents. The NSM of the DGC is partly broken down in the axon endings by lysosomal action. Its release takes place at a low rate. Since these changes in neurosecretory activity occur in a similar fashion in transplanted and in acceptor DGC it is concluded that the response of the DGC to environmental osmolarity is not regulated via nervous pathways to the PPC. The only significant difference between transplanted and acceptor DGC concerns the higher number of clear vesicles in the DGC axon endings of transplants. Some explanations for this phenomenon are discussed. Possible mechanisms of regulation of DGC neurosecretory activity are considered. [ABSTRACT FROM AUTHOR]

Abstract 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 α-melanophore-stimulating hormone (α-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 α-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 α-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. [ABSTRACT FROM AUTHOR]

In the rat pineal gland the mechanism of release of secretory material was studied ultrastructurally after incubating tissues in Ringer solution containing tannic acid. The results indicate that pinealocytes release the contents of secretory vesicles into the extracellular space via exocytosis, a phenomenon that has not been visualized previously in this cell type. This finding may reflect release of polypeptides by the pineal gland. [ABSTRACT FROM AUTHOR]