Caudodorsal Cells Research Articles

The effects of mating on the fine structure of neurosecretory caudodorsal cells in Helisoma duryi (Mollusca)

The hermaphroditic snail Helisoma duryi does not lay eggs if raised as a virgin in isolation or when castrated. The fine structure of its neurosecretory caudodorsal cells (CDC), which produce the putative egg-laying hormone, was studied in reproductively inactive virgin and castrated snails and compared with that of the reproductively active mated snails, using electron microscopy, tannic acid incubation technique, and electron microscope autoradiography after injection of [3H]leucine. The CDC of virgin and castrated snails accumulate large numbers of elementary granules and appear synthetically inactive, whereas the CDC of mated snails contain fewer elementary granules and possess features characteristic of increased protein synthesis. The flattened cisternae of the rough endoplasmic reticulum of the CDC of the virgins become swollen 3 h after first mating. In tannic acid incubated tissues, more released granules were seen in CDC from snails 12 h after first mating than in CDC from virgin and castrated snails. Autoradiography showed more silver grains on the CDC from snails 12–48 h after first mating than on those from snails 3–6 h after first mating. It is suggested that, in Helisoma, mating provides a stimulus to the CDC for increased protein synthesis and release of the putative egg-laying hormone.

Simultaneous detection of different mRNA sequences coding for neuropeptide hormones by double in situ hybridization using FITC- and biotin-labeled oligonucleotides.

Oligonucleotides labeled with FITC or biotin were applied for detection of specific mRNAs in microscopic preparations by in situ hybridization. The oligonucleotides were labeled with one FITC or biotin molecule at the 5' end or with a tail of biotin molecules at the 3' end. The target sequences were mRNAs coding for an ovulation hormone (CDCH) in the caudodorsal cells (CDC) of the pond snail Lymnaea stagnalis and a molluscan insulin-like peptide (MIP) in the light green cells (LGC) of the same organism. The hybridized oligonucleotides were detected either directly after the hybridization procedure by fluorescence microscopy or indirectly after an immunocytochemical procedure to visualize the biotin or FITC moiety. The results indicate that the detectability of the mRNA sequences is at least partially dependent on the accessibility of the target sequences for the immunocytochemical detection systems. The positive hybridization results obtained with oligonucleotides containing different labels enabled us to perform double hybridization experiments for simultaneous detection of CDCH and MIP mRNAs in one tissue section. Using FITC- and biotin-labeled oligonucleotides, we also demonstrated simultaneously different sequences on the same mRNA molecule.

Discharge induction in molluscan peptidergic cells requires a specific set of autoexcitatory neuropeptides

The peptidergic caudodorsal cells of the pond snail Lymnaea stagnalis generate long lasting discharges of synchronous spiking activity to release their products. During caudodorsal cell discharges a peptide factor is released which induces similar discharges in silent caudodorsal cells [Ter Maat A. et al. (1988) Brain Res. 438, 77–82]. To identify this factor, the electrophysiological effects of putative caudodorsal cell gene products, calfluxin, caudodorsal cell hormone, four a caudodorsal cell peptides and three βcaudodorsal cell peptides, were tested individually and in various combinations. Calfluxin, a caudodorsal cell peptide 3–9 and β 1caudodorsal cell peptide each had no effect on membrane potential or excitability of the caudodorsal cells. All other caudodorsal cell peptides caused excitatory responses, but did not induce discharges. Instead, only a specific combination of four caudodorsal cell peptides, caudodorsal cell hormone and a caudodorsal cell peptide (1–11, 3–11 and 3–10), evoked caudodorsal cell discharges with similar characteristics to electrically evoked discharges. Incomplete versions of this combination failed to cause a discharge. In addition, antibodies to caudodorsal cell hormone or αcaudodorsal cell peptide reduced caudodorsal cell excitability and prevented the generation of discharges by electrical stimulation. These results suggest that excitatory autotransmission caused by four caudodorsal cell peptides provides a means to amplify excitatory inputs, thus leading to the generation of the all-or-nothing caudodorsal cell discharge.

Mating increases the synthetic activity of the neurosecretory caudodorsal cells of Helisoma duryi (Mollusca: Pulmonata)

In Helisoma duryi, virgin snails lay significantly fewer eggs than mated snails. It is generally accepted that in basommatophoran pulmonates, the neurosecretory caudodorsal cells located in the cerebral ganglia produce a hormone that regulates egg laying. The synthetic activity of the caudodorsal cells in Helisoma has been measured in vitro and in vivo using tritiated leucine. Virgin and castrated snails (reproductively inactive) showed significantly reduced levels of [3H]leucine incorporation compared with first-mated snails (24 and 48 h postmating). This increase in synthetic activity following mating was corroborated by autoradiography at the light microscope level which localized transport of caudodorsal cell neurosecretory proteins to the cerebral commissure, the neurohaemal area. Mating triggers an increase in synthetic activity of the caudodorsal cells.

Spontaneous and induced egg laying behavior of the pond snail,Lymnaea stagnalis

InLymnaea egg laying is initiated by the discharge of the neurosecretory caudodorsal cells (CDCs), which release an ovulatory hormone (caudodorsal cell hormone; CDCH) and other peptides. Egg laying is a complex behavioral pattern composed of various postures and action patterns. This paper describes the egg laying behavior of intact and freely behaving animals as it occurs spontaneously after an environmental stimulus (clean water stimulus; CWS), and following injections of highly purified CDCH. CDC discharges were monitored in vivo with chronically implanted electrodes. In these animals the egg laying behavior following spontaneous and electrically elicited CDC discharges was analyzed. 1. The egg laying behavior induced by the CWS consisted of three separate phases that occurred in a fixed temporal sequence. Shortly after transferral to clean water, animals became quiescent (resting). This was followed by an active phase (turning) during which behaviors were performed to prepare the substrate for the subsequent oviposition phase. Following oviposition, animals examined the deposited egg mass. 2. After spontaneous CDC discharges and CDC discharges elicited by selective electrical stimulation of the CDCs (with a chronically implanted fine wire electrode) the egg laying behavior was the same as that following the CWS. All phases of egg laying behavior followed the CDC discharge. 3. In contrast, the egg laying behavior following injections of highly purified CDCH was different to that following the CWS. These animals showed no resting phase. Instead, they continued locomoting and entered directly into the turning phase. The animals that did not lay eggs after injection because they were refractory did not show egg laying behavior. 4. The durations of the joint turning and oviposition phases depended strongly on clutch size, suggesting that the ovulated eggs are instrumental in causing this behavior. 5. We conclude that whenever egg laying is preceded by a CDC discharge, either occurring spontaneously or induced by CWS or electrical stimulation, the behavior differs from that following injections of CDCH directly into the blood. This demonstrates that the presence of CDCH in the blood is not sufficient to produce the full complement of egg laying behaviors and suggests that CDCH, or one of the other peptides released during a CDC discharge, has important local effects within the CNS.

Developmental and comparative aspects of nonsynaptic release by the egg-laying controlling caudodorsal cells of basommatophoran snails

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 ≥ 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.

Quantitative immunoelectron microscopy and tannic acid study of dynamics of neurohaemal and non-synaptic peptide release by the caudodorsal cells of Lymnaea stagnalis

The caudodorsal cells (CDCs) of the freshwater snail Lymnaea stagnalis control egg laying and egg-laying associated behaviours by releasing various peptides including the ovulation hormone CDCH. Previously it has been shown that release occurs (1) into the haemolymph from neurohaemal axon terminals in the outer compartment of the cerebral commissure, and (2) into the intercellular space of the central nervous system from non-synaptic release sites of axon collaterals in the inner compartment of the commissure. Outer and inner compartments are separated by a sheath of glial cells. In the present study the secretory dynamics of neurohaemal and collateral release have been studied. Immunoelectron microscopy with an antibody against a synthesized fragment of the egg-laying hormone [CDCH (21–36)] indicates that CDCH is released by exocytosis from both sites: positive immunoreaction was found for the contents of secretory granules and contents that underwent exocytosis, and furthermore in the intercellular spaces of the inner and outer compartments. Quantitative (immunogold) electron microscopy combined with either the tannic acid-glutaraldehyde-osmium tetroxide (TAGO) method or the tannic acid-Ringer incubation (TARI) method for the visualization and quantification of exocytosis of CDCH, shows different dynamics of neurohaemal and collateral CDCH release. Neurohaemal release is strongly increased during electrical activity of the CDCs (active state). This increase does not only appear from an increased number of (immunopositive) exocytoses (3×) but also from increases in (1) the percentages of all stationary and all exocytosing granule contents that are immunopositive (both increase from 70% to 85%), (2) the degree of immunopositivity per exteriorized granule content (2×) and (3) the degree of immunopositivity in the intercellular space of the neurohaemal area (5×). Collaterals show a different picture: CDCH release particularly occurs during electrical silence (resting and inhibited states). No effect was noted of the electrical state of the CDCs on the percentages of CDCH-immunoreactive stationary or exteriorized granule contents, nor on the degree of immunopositivity of the exteriorized contents. Furthermore, the degree of immunopositivity in the intercellular space of the inner compartment is drastically decreased (8×). Finally, both in the resting and the active state, the percentage of CDCH-positive exocytosing contents in the collaterals is smaller than that of CDCH-positive stationary contents, whereas in the neurohaemal area these percentages do not differ. It is concluded that the CDCs establish dual secretory dynamics in the neurohaemal and collateral system, not only with respect to the intensity of exocytosis but also with respect to the amount of CDCH released per secretory granule. Possible cellular mechanisms underlying these dynamics are discussed. The dual secretory capacity may be crucial for the CDCs in timing and coordinating the activities of reproductive organs (by peptide release from the neurohaemal area) and of central neurons that control egg-laying-associated behaviours (by peptides released non-synaptically from the collaterals).

One receptor type mediates two independent effects of FMRFa on neurosecretory cells of Lymnaea

Structure activity relations (SAR) of FMRFa on the transient hyperpolarizing response and long lasting depression of excitability of neurosecretory caudo dorsal cells (CDCs) of the pond snail Lymnaea stagnalis were examined. Although these effects to FMRFa occur independently, the SARs for the induction of both responses were identical suggesting that CDCs possess a single type of FMRFa receptors. Native GDPFLRFa and SDPFLRFa were equipotent to FMRFa receptors. It is concluded that activation of the receptor requires [Arg 3-Phe 4]-NH 2, whereas N-terminal amino acids are involved in binding.

Morphology and electrophysiological characteristics of caudodorsal cells of Lymnaea stagnalis in dissociated cell culture

1. 1. The neuroendocrine caudodorsal cells (CDCs) of Lymnaea stagnalis are a network of about 100 electrotonically coupled neurones. The CDCs release multiple peptides, including an ovulation hormone, during a period of electrical activity, the CDC-discharge. 2. 2. In isolated brains, a similar period of electrical activity (the afterdischarge) can be induced in all CDCs by a period of intracellular repetitive suprathreshold stimulation of one CDC. 3. 3. In order to study the regulation of this electrical behaviour in the absence of electrical interactions and in a controlled environment, experiments were performed on CDCs in dissociated cell culture. 4. 4. Methods for isolation and cell culture are described. Cell cultures had long-term viability and outgrowth of neurities occurred under serum-free conditions. 5. 5. CDCs in cell culture maintained their capability of producing afterdischarges upon electrical stimulation. Cells in culture appeared more excitable than cells in the intact isolated brain. 6. 6. The characteristic responses of CDCs in intact isolated brains to acetylcholine and FMRFamide were preserved in cultured CDCs. Both agents induced a transient hyperpolarization of the membrane, inexcitability and inhibition of an ongoing discharge. 7. 7. In experiments where isolated CDCs were closely apposed, but physically separate, it was found that an afterdischarge in one CDC could induce a discharge in the other CDC. 8. 8. These results confirm previous results which showed that an excitatory factor is released from the brain during the afterdischarge (Ter Maat et al., 1988, Brain Res., 43, 77–82), and demonstrate that this excitatory factor is released from the CDCs themselves.

The role of cAMP in regulation of electrical activity of the neuroendocrine caudodorsal cells of Lymnaea stagnalis

The neuroendocrine caudodorsal cells (CDCs) of the pond snail Lymnaea stagnalis release a number of peptides, including the ovulation hormone, caudodorsal cell hormone (CDCH), during a period of high electrical activity (the CDC-discharge). Earlier studies have shown that during the CDC-discharge adenylate cyclase activity is increased, and that the cyclic adenosine monophosphate (cAMP) analogue 8-chlorophenylthio (8-CPT)-cAMP induces exocytosis and release of peptides from the CDCs. Here, we have investigated the role of cAMP, adenylate cyclase and phosphodiesterase in determining the state of excitability of the CDCs. The cAMP analogue 8-CPT-cAMP induced long-lasting discharges in CDCs. Simultaneous inhibition of the phosphodiesterase by 3-isobutyl-1-methylxanthine (IBMX) and activation of the adenylate cyclase by forskolin gave similar results. These agents also induced discharges of CDCs in dissociated cell culture, indicating that the responses to an increase of cAMP were an endogenous property of the cells. The CDC-afterdischarge can be induced by a period of repetitive electrical1stimulation. Inhibition of the phosphodiesterase-activity by IBMX did not change the resting membrane potential, but increased the proportion of preparations that responded to this stimulation with an afterdischarge by more than 200%. It is suggested that cAMP-regulating enzymes are involved in stimulus-response coupling of the afterdischarge in CDCs. The induction of an afterdischarge probably requires both a low phosphodiesterase-activity and the activation of the adenylate cyclase. The low excitability of the CDCs following an afterdischarge might originate from a refractoriness in the activation of the adenylate cyclase.

Expression of the egg-laying hormone genes in peripheral neurons and exocrine cells in the reproductive tract of the mollusc Lymnaea stagnalis

The neuroendocrine caudodorsal cells play an important role in the control of reproduction in Lyrmaea stagnalis. These neurons produce at least nine neuropeptides which are encoded by caudodorsal cell hormone-I and -II genes. The role of some of these peptides in the control of reproduction has been established. The present study demonstrates that the transcription and translation of the caudodorsal cell hormone genes also proceeds abundantly in the reproductive tract of this hermaphroditic animal. In the female part of the reproductive tract neurons were found to express gene I. These neurons are most likely involved in the control of transport of the eggs and egg-masses and in the regulation of secretory activity from the female accessory sex glands. In the male part of the reproductive tract exocrine secretory cells express gene I or gene II. The gene products are secreted into the male duct and transferred to the female copulant during copulation. Furthermore, putative sensory neurons in the skin were found to express gene I. The results indicate that in L. stagnalis the complex process of reproduction is regulated—at least in part—by a set of neuropeptides which are encoded by a small multigene family, viz. the caudodorsal cell gene family.

Central and peripheral expression of genes coding for egg-laying inducing and insulin-related peptides in a snail.

Egg laying in the hermaphrodite freshwater snail Lymnaea stagnalis is a highly complex activity, including a series of internal activities (ovulation, egg and egg mass formation) which are closely correlated to a pattern of behaviours (alteration of locomotion and feeding, specific postures, oviposition). In this snail egg laying is induced by the neuroendocrine caudodorsal cells (CDCs), consisting of two homogeneous clusters at a total of 100 neurons. At egg laying these neurons release their products during a 60 min period of firing. The genes coding for these products have been cloned and characterized. There are two genes, CDCH-I and -II. Each gene codes for 12 peptides; one of these is the ovulation hormone (CDCH). The genes display over 90% homology. The most striking difference is a 17 bp deletion near the carboxy-terminal region. With immunocytochemistry and in situ hybridization both CDCH genes appeared to be expressed in the CDC and in paired groups of ectopic CDC-like neurons in the pleural ganglia, while a group of small neurons in the cerebral ganglia expresses the CDCH-I gene only. In addition, a widespread expression of the CDCH genes has been demonstrated in peripheral tissues. In the female part of the reproductive tract neurons were found to express the CDCH-I gene. In the male part of the tract exocrine secretory cells express the CDCH-I or -II gene. The gene products are secreted into the male tract and transferred to the female partner during copulation. Finally, sensory neurons in the head skin and mantle edge were found to express the CDCH-I gene. The presence of insulin-related peptides has been demonstrated in the brain as well as the digestive system of Lymnaea. The brain insulin-related peptides are produced in 4 groups of 50 giant neurons each (Light green cells, LGC). These neurons are involved in various physiological activities, related to body growth and glycogen metabolism. The major gene products expressed in the LGC have been cloned and characterized. It appeared that the predicted proteins represent three types of insulin-related molecules (MIP, molluscan insulin-related peptide). In these MIPs, those elements important in the determination of the tertiary structure, have been conserved. The MIP of the digestive system has been characterized up to now only at the peptide level. The snail gut MIP is more hydrophobic compared to bovine insulin. Cells containing MIP have been identified immunocytochemically in the gut epithelium.

Detection of mRNA molecules coding for neuropeptide hormones of the pond snail Lymnaea stagnalis by radioactive and non-radioactive in situ hybridization: a model study for mRNA detection.

To develop and optimize non-radioactive in situ hybridization techniques for mRNA detection, we used the neuropeptidergic system of the pond snail Lymnaea stagnalis as a biological model system. First, we investigated the in situ hybridization procedure using radioactive-labeled cDNA and synthetic oligonucleotide probes specific for egg-laying hormone (ELH) mRNA and molluscan insulin-like peptide (MIP) mRNA. The results show an intense grain deposit above the caudodorsal cells and light-green cells expressing, respectively, ELH mRNA and MIP mRNA. Good results with relation to signal strength and tissue morphology were obtained with freeze-dry paraformaldehyde vapor fixation. The necessity to perform tissue pre-treatment appeared to be dependent on the cell type of interest. The optimized in situ hybridization protocol proved to be applicable using probes that are either sulfonated/transaminated or labeled with acetylaminofluorene (AAF). In situ hybridization of such haptenized probes led to intense and specific staining of the cytoplasm of the caudodorsal cells. Egg-laying hormone mRNA appeared not to be homogeneously distributed in the cytoplasm but showed a "patch-like" pattern. Nuclear and axoplasmic staining for mRNA was also observed.

Isolation, characterization, and evolutionary aspects of a cDNA clone encoding multiple neuropeptides involved in the stereotyped egg-laying behavior of the freshwater snail Lymnaea stagnalis

The cerebral neurosecretory caudodorsal cells (CDCs) of the freshwater pulmonate snail Lymnaea stagnalis control egg laying, an event that involves a pattern of stereotyped behaviors. The CDCs synthesize and release multiple peptides, among which is the ovulation hormone (CDCH). It is thought that each peptide controls a specific aspect of the processes involved in egg laying. We isolated and characterized a CDC-specific cDNA clone that encodes the ovulation hormone (CDCH). RNA blot analysis and in situ hybridization experiments demonstrated that the CDCs are the major cell groups in the cerebral ganglia that transcribe the CDCH gene. In addition to CDCH, the 259-amino acid-long CDCH preprohormone contains 11 other predicted peptides. The overall homology of the CDCH preprohormone with the egg-laying hormone (ELH) preprohormones of the marine opisthobranch snails Aplysia californica and A. parvula is very low (29 and 26%, respectively). However, a more detailed comparison revealed a highly differential pattern of conservation of peptide regions. Significant homology was found between the regions containing (1) CDCH and ELH, (2) repeated pentapeptides, (3) alpha-caudodorsal cell peptide and alpha-bag cell peptide, and (4) 2 regions representing as yet unidentified peptides. Insignificant homology was found when comparing regions containing the other predicted peptides. The conserved peptides probably control similar aspects of the egg-laying fixed action patterns in these distantly related gastropod species. The pentapeptide region exhibits the highest level of homology (75%); in addition, an extra pentapeptide has been generated on the CDCH precursor. This indicates a vital function of these peptides in Aplysia, as well as in Lymnaea species.

Structural aspects, potassium stimulation and calcium dependence of nonsynaptic neuropeptide release by the egg laying controlling caudodorsal cells of Lymnaea stagnalis

The cerebral peptidergic caudodorsal cells of the freshwater snail Lymnaea stagnalis control egg laying and egg-laying behaviour by releasing peptides into (1) the haemolymph, from neurohaemal axon terminals in the periphery of the cerebral commissure and (2) the intercellular space of the central nervous system, from collaterals in the inner compartment of this commissure. Recently, it was shown that collateral release occurs from nonsynaptic release sites, which lack the morphological specializations that are characteristic of classical synapses. Probably, these sites enable the caudodorsal cells to communicate with central neurons in a nonsynaptic (“paracrine”, “diffuse”, “hormone-like”) fashion. The structural and ionic bases of nonsynaptic release were studied using the tannic acid-Ringer incubation-method for the detection of exocytotic release of secretory granule contents in vitro. Elevation of the extracellular potassium concentration strongly stimulates exocytotic activity in the collaterals. No stimulation was found in the absence of extracellular calcium ions. Similar results have been obtained for the neurohaemal axon terminals. Electron-dense material occurs apposed at the cytoplasmic side of the axolemma of collaterals (ethanolic phosphotungstic acid method). This material appears homologous with the presynaptic dense projections forming the “vesicular grid” in classical synapses. Such projections are also present in the neurohaemal axon terminals. It is concluded that secretion from nonsynaptic release sites in caudodorsal cell collaterals shares fundamental characteristics with secretion from conventional neuronal release sites (neurohaemal axon terminals and classical synapses); release occurs by exocytosis of secretory granules, is associated with a vesicular grid, is stimulated by membrane depolarization, and depends on the presence of extracellular calcium ions. The results underline the importance of nonsynaptic release sites for interneuronal communication within the central nervous system.

Dual inhibitory action of FMRFamide on neurosecretory cells controlling egg laying behavior in the pond snail

We describe here the electrophysiological characterization of a dual inhibitory action of FMRFamide (FMRFa, Phe-Met-Arg-Phe-NH 2) on the caudodorsal cells (CDCs) of the pond snail Lymnaen stagnalis: (i) a transient hyperpolarizing response (H-response) and (ii) a suppression of the excitability of the cells, which lasted as long as the peptide was present. Both effects of FMRFa occurred in silent, excitable cells as well as discharging cells. The effects were reversible and dose-dependent in the range of 10 −9 to 10 −5 M. The H-response was not blocked by any of the antagonists to classical neurotransmitters that were tested. The reversal potential of the H-response was dependent on the [K +] o, which suggests that K + is the major charge carrier in this response. 4-Aminopyridine (4-AP) blocked the H-response but did not affect the suppression of the excitability by FMRFa. This indicates that the effects of the peptide on these cells are independent. Experiments on the mechanism of the inhibition of the excitability indicated that FMRFa blocks the cAMP-dependent activation of the pacemaking mechanism of the CDCs. In experiments with isolated cells it was demonstrated that the actions of FMRFa are mediated directlly through receptors on CDCs (H-response: ED 50 = 10 −8 M). Finally, anti-FMRFa-positive varicosities and axons close to the somata, the axons and the neurohaemal endings of the CDCs were demonstrated immunocytochemically. The duality of the action of FMRFa on the neural activity of CDCs indicates its role of high priority in the regulation of egg laying behavior.

Postembryonic Development of Endocrine Dorsal Bodies and Neuroendocrine Egg Laying and Growth Hormone Producing Neurons ofLymnaea stagnalis

Summary The postembryonic development of three (neuro) endocrine centres in the snail Lymnaea stagnalis has been studied: (l) the endocrine Dorsal Bodies (DB), which are involved in the stimulation of vitellogenesis, and of growth, differentiation and secretory activity of the female accessory sex glands, (2) the Caudodorsal Cells (CDC), which control egg laying and egg-laying behaviour, and (3) the Light Green Cells (LGC), which stimulate body growth. Light microscopy, including immunocytochemistry with monoclonal antibodies, and electron microscopy show that the DB and LGC are already present in snails of 1 mm shell height, whereas CDC first appear in snails of 3 mm shell height. In such juvenile snails all cell types have a small size and occur in low numbers, but they show clear, morphological signs of formation of secretory granules. During development the cells strongly increase in size and, especially the DB cells, in number. The diameter of the secretory granules also increases, particularly in CD...

Localization of ovulation hormone-like neuropeptide in the central nervous system of the snail Lymnaea stagnalis by means of immunocytochemistry and in situ hybridization.

The caudo-dorsal cells (CDC) in the cerebral ganglia of the pond snail Lymnaea stagnalis synthesize the 36-amino acid ovulation hormone (CDCH). We have used immuno-cytochemistry and in situ hybridization to reveal the localization of neurons and axons containing CDCH-like material. A monoclonal antibody to a fragment of CDCH and a cDNA probe encoding CDCH reacted with the CDC-system, with specific cell groups in the cerebral and pleural ganglia, and with individually occurring neurons throughout the central nervous system. The cells in the pleural ganglia, which were found in about 50% of the preparations studied, are considered as "ectopic" CDC. They are morphologically similar to CDC in their somal dimensions and axonal organization. By means of immuno-electron microscopy it was shown that these neurons contain secretory vesicles that are similar to those of the CDC. The neurons of the bilateral groups occurring in the cerebral ganglia in addition to the CDC are smaller and more intensely stained than the CDC. Axons of these small neurons probably have varicosities located on the CDC axons in the neuropil of the cerebral ganglion, indicating synaptic contacts. Two major axon tracts could be followed from (or toward) the neuropil of the cerebral ganglion. One tract runs from the cerebral ganglion via the pleural and parietal ganglia to the visceral ganglion, giving off branches to most nerves emanating from these ganglia. The other tract could be traced through the cerebro-pedal connective to the pedal ganglia. Only in the right pedal ganglion was extensive axonal branching observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemically mediated positive feedback generates long-lasting afterdischarge in a molluscan neuroendocrine system

The peptidergic neuroendocrine caudodorsal cells (CDCs) of Lymnaea stagnalis control egg laying. The CDC network consists of 100 electroninically coupled neurons that form two clusters in the cerebral ganglia. Upon prolonged, repeated, intracellular stimulation of one CDC, excitation spreads over the network and leads to a 30-min period of spiking activity: the afterdischarge. During the afterdischarge a number of peptides, including the ovulation hormone, are released. When two ganglia rings from different animals were pinned down next to each other, an afterdischarge initiated in the CDCs of one CNS activated the CDCs of the other CNS, indicating that excitation spreads in the absence of physical contact between the CDCs. A single isolated intercerebral commissure (COM), the neurohaemal area of the CDCs, displayed the same discharge-inducing capability when brought in the vicinity of a second, intact, CNS. Other parts of the CNS did not possess this property. CDC afterdischarges could also induce repetitive spiking in adjacent isolated CDC somata showing that the effect can be directly on the CDCs themselves. The discharge-inducing factor was well separated from the ovulation hormone on a Bio-Gel P-6 column. The factor was pronase-degradable and inhibitors of proteolytic enzymes increased the factor's longevity. It is concluded that, contingent upon the CDC-discharge, a small (⩽ 1500 Da) excitatory peptide is released that acts directly on the CDCs. Its function is argued to be: (1) the spread of excitation from a subset of CDCs, receiving external input, over the entire CDC network; and (2) to provide a positive feedback to generate a maximum (all-or-none) response.

Immuno-electron microscopy of formation, degradation and exocytosis of the ovulation neurohormone in Lymnaea stagnalis

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.