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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.

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.

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 cerebral caudodorsal cells (CDC) of the pulmonate snail Lymnaea stagnalis control egg-laying and associated behaviors. They produce various peptides derived from two precursor molecules, proCDCH-I and II, one of which is calfuxin (CaFl). CaFL is involved in the control of the activity of a female accessory sex gland, the albumen gland. At the light microscope level, using an antibody raised against synthetic CaFl, immunoreactivity was demonstrated in all CDC somata as well as in the neurohemal CDC terminals in the periphery of the cerebral commissure and in the CDC axon collaterals in the inner region of the commissure. A group of small neurons in each cerebral ganglion was also immunopositive. At the ultrastructural level, secretory granules (SG) and large electron-dense granules (LG), formed by the Golgi apparatus and thought to be involved in intracellular degradation of secretory material, were clearly immunolabeled. The density of immunolabeling of LG was 3.3 times greater than that of SG, indicating that CaFl is preferentially packed into LG. In the LG, the density of immunolabeling with anti-alpha CDCP (alpha CDCP is also a peptide derived from proCDCH-I and II) was 10 times greater than in SG, suggesting that CaFl and alpha CDCP are processed and sorted in (quantitatively) different ways. In the neurohemal terminals SG release their CaFl-immunopositive contents into the hemolymph by the process of exocytosis, whereas collaterals release such contents into the intracellular space of the intercerebral commissure. It is proposed that neurohemally released CaFl acts upon the albumen gland, whereas CaFl released from the collaterals may influence the activity of central neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

The secretion of most pituitary hormones is under the control of feedback mechanisms. The feedback control of alpha-melanophore-stimulating hormone (alpha-MSH) from melanotrope cells is controversial. The possible existence of an autofeedback exerted by alpha-MSH or other POMC-derived peptides on melanotrope cells of the amphibian Xenopus laevis has been investigated. alpha-MSH or its potent agonist 4-norleucine,7-D-phenylalanine-alpha-MSH has no effect on the release of radiolabeled POMC-derived peptides or immunoreactive beta-endorphin from superfused neurointermediate pituitary lobes. Melanin concentrating hormone, previously reported to have an alpha-MSH-like effect on melanophores, did not affect alpha-MSH secretion. Neurointermediate lobe superfusate, which contains a mixture of POMC-derived peptides, failed to affect the secretory activity of melanotropes. It is concluded that in X. laevis the secretory activity of melanotropes is not under the control of short-term autofeedback mechanisms involving alpha-MSH or other POMC-derived peptides.

A study of the relation between nutritive state and female reproductive activity as affected by the ovulation hormone (CDCH) has been made in the freshwater pulmonate snail Lymnaea stagnalis. CDCH is produced by the neuroendocrine caudodorsal cells (CDC) in the cerebral ganglia. Spontaneous oviposition ceased within 6 days of the beginning of a starvation period. This is most probably partially due to a reduction in the CDC activities because (1) quantitative electron microscopy showed a nearly 80% decrease in the number of release phenomena in the CDC-axon terminals in the neurohemal area in the intercerebral commissure, and (2) a bioassay showed a considerable reduction in the amount of CDCH in this area. During starvation the ovotestis and the female accessory sex organs became progressively less sensitive and, after 25 days, were completely insensitive to injected CDCH. This was indicated by a decrease in the responses to CDCH injection and, correspondingly, by a gradual increase in the CDCH thresholds for ovulation and egg formation. It is argued that the insensitivity may be caused by a reduction in the activities of the endocrine dorsal bodies. During refeeding, CDCH injections again become effective in inducing egg mass production, followed by resumption of spontaneous oviposition. This suggests a rapid restoration of DB and CDC activities following refeeding.

In an immunoelectron microscope study the postembryonic development of the cerebral caudodorsal cells (CDC) in the freshwater snail Lymnaea stagnalis was studied as well as the development of similar neurons in other basommatophoran families. The CDC of adult L. stagnalis control egg-laying and associated behaviors by releasing various peptides, including the ovulation hormone CDCH. The CDC release peptides from neurohemal axon terminals and from nonsynaptic release sites of axon collaterals. During postembryonic development the collateral system develops synchronously with the neurohemal area. The first collaterals appear in the cerebral commissure of juvenile snails (10 mm shell height; S = 10). Up to S = 30 they gradually increase in size and length and eventually run through the entire inner compartment. Secretory granules in both collaterals and neurohemal axon terminals increase in size as well. Immunoelectron microscopy combined with the TARI-method for the demonstration of exocytosis indicates that CDCH-release from collaterals and neurohemal terminals occurs already in S = 10; exocytosis of immunoreactive granule contents takes place from nonsynaptic release sites, unspecialized areas of the axolemma of the collaterals. Release activity in the collaterals gradually increases up to S greater than or equal to 20. Neurohemal release activity shows a similar picture except for a steep increase in adult snails. A distinct glial sheath, separating the neurohemal area from the collateral system, appears around S = 15. Representatives of three families of Basommatophora, viz. the lymnaeid L. ovata, the planorbid Planorbis planorbis, and the bulinid Bulinus truncatus possess a well-developed collateral system showing many signs of exocytosis. A glial sheath separates the collaterals from the neurohemal area. Secretory granules of the CDC in L. ovata stain weakly positive with the anti-CDCH antiserum. Since the other Basommatophora did not show immunoreactivity, the chemical structure of egg laying peptides in Basommatophora seems to be genus specific. Apparently the secretory activity of both the neurohemal area and the collateral system is not only important in the sexually mature animal, being involved in the control of egg laying and egg-laying behavior, but also in the juvenile snail. The finding of a collateral system in representatives of three basommatophoran families strongly indicates the importance of the system for the control of reproduction in basommatophoran snails in general.

In a light and electron microscope study, neurosecretory cells morphologically homologous with the egg-laying hormone-producing caudodorsal cells (CDC) and growth hormone-producing dorsal cells (DC, light green cells) of the freshwater basommatophoran snail Lymnaea stagnalis have been found in five genera (seven species) of Basommatophora, viz. in Lymnaea palustris and Lymnaea ovata, in Planorbis planorbis and Planorbis vortex, in Planorbarius corneus, in Bulinus truncatus, and in Biomphalaria glabrata. It is concluded that the functions of these cells are homologous as well. The homologies of the respective neuron types regard their locations in the cerebral ganglia, their clustering in groups, location of their neurohemal area (CDC: cerebral commissure; DC: median lip nerves), and ultrastructural characteristics (e.g., abundance of rough endoplasmic reticulum, well-developed Golgi apparatus, presence of two types of neuron-specific secretory granules, and release of granule contents by exocytosis into the hemolymph). In addition, CDC show large electron-dense granules and DC reveal infoldings of the plasma membrane at the abaxonal side of the soma as well as synaptic input. On the other hand, each neuron type shows species-specific characteristics, particularly with regard to the number of cells and the structure of the neurohemal area. Furthermore, the CDC show marked differences between genera in the morphology (especially the mean diameter) of type 2 and, particularly, type 1 secretory granules. The morphology of the two types of secretory granules in the DC differs strongly between species. The possible relation between the morphology and the chemical contents of secretory granules has been discussed.

The cerebral neuroendocrine peptidergic light green cells (LGC) of the freshwater snail Lymnaea stagnalis regulate body growth. The LGC are controlled by a tentacle sensory system that consists of two types (S1 and S2) of primary sensory neuron located at the base of each tentacle. Sensory (S2) axons make synaptic contacts (type A synapse-like structures) with the somata and axons of the LGC, where they release the contents of secretory granules, by exocytosis (demonstrated with the ultrastructural tannic acid-Ringer incubation method). Ultracytochemistry indicates that the granule contents are glycoproteinaceous. Furthermore, the S2 axons release secretory material in a nonsynaptic fashion into the interneuronal space of the central nervous system (CNS), at the level of the neuropiles of the cerebral ganglia and of the cerebral commissure. This release occurs by exocytosis from nonsynaptic release sites. It is proposed that the tentacle sensory system not only (synaptically) controls LGC activity but also influences other, remote neuronal targets in the CNS in a nonsynaptic ("at long distance," "paracrine," "hormone-like") fashion. Already in newly hatched snails (with a shell height of 1 mm) S2 axons show a fair rate of exocytotic activity, in both synaptic and nonsynaptic respects. During postembryonic development the secretory capacity of the S2 sensory neurons increases markedly, by increases in (1) the number of axons, (2) the size of the secretory granules, and (3) exocytosis activity. This increased capacity may meet a growing demand of the developing CNS, including the LGC, for neurochemical input from the tentacle sensory system.

Spontaneous oviposition of the freshwater pulmonate snail Lymnaea stagnalis stopped after transfer from 20 degrees to low temperatures (8 and 4 degrees). This was due to a reduction in the activities of the neurosecretory caudodorsal cells (CDC), which produce the ovulation hormone (CDCH). Oviposition latencies (time interval between CDCH-injection and start of oviposition) increased with decreasing temperature. The ovotestis and the female accessory sex organs of snails placed at 8 degrees became less sensitive, and those at 4 degrees became completely insensitive to injected CDCH. This was probably caused by a reduction in activities of the endocrine dorsal bodies (DB), which control vitellogenesis and the activities of the female accessory sex organs. After a change from 4 to 20 degrees, CDCH injections rapidly became effective in inducing egg mass production, and spontaneous oviposition also restarted quickly, suggesting a rapid increase in DB and CDC activities.