Single-cell analysis of peptide expression and electrophysiology of right parietal neurons involved in male copulation behavior of a simultaneous hermaphrodite.

Male copulation is a complex behavior that requires coordinated communication between the nervous system and the peripheral reproductive organs involved in mating. In hermaphroditic animals, such as the freshwater snail Lymnaea stagnalis, this complexity increases since the animal can behave both as male and female. The performance of the sexual role as a male is coordinated via a neuronal communication regulated by many peptidergic neurons, clustered in the cerebral and pedal ganglia and dispersed in the pleural and parietal ganglia. By combining single-cell matrix-assisted laser mass spectrometry with retrograde staining and electrophysiology, we analyzed neuropeptide expression of single neurons of the right parietal ganglion and their axonal projections into the penial nerve. Based on the neuropeptide profile of these neurons, we were able to reconstruct a chemical map of the right parietal ganglion revealing a striking correlation with the earlier electrophysiological and neuroanatomical studies. Neurons can be divided into two main groups: (i) neurons that express heptapeptides and (ii) neurons that do not. The neuronal projection of the different neurons into the penial nerve reveals a pattern where (spontaneous) activity is related to branching pattern. This heterogeneity in both neurochemical anatomy and branching pattern of the parietal neurons reflects the complexity of the peptidergic neurotransmission involved in the regulation of male mating behavior in this simultaneous hermaphrodite.

Functional analysis of a recurrent missense mutation in Notch3 in CADASIL.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited vascular dementia characterised by recurrent ischemic strokes in the deep white matter. Mutations in the gene encoding the cell surface receptor, Notch3, have been identified in CADASIL patients, and accumulation of the extracellular domain of Notch3 has been demonstrated in affected vessels. Almost all CADASIL mutations alter the number of cysteine residues in the epidermal growth factor (EGF)-like repeats in the extracellular domain of the protein. OBJECTIVES: To understand the functional consequences of a recurrent CADASIL mutation on furin processing, cell surface expression, ligand binding, and activation of a downstream effector CBF1 by the Notch3 receptor. METHODS: We expressed wild type and mutant Notch3 receptors in cultured cells and examined cell surface expression of the proteins. We also applied a new flow cytometry based approach to semi-quantitatively measure binding to three Notch ligands. Additionally, we used a well characterised co-culture system to examine ligand dependent activation of transcription from a CBF1-luciferase reporter construct. RESULTS: These studies revealed subtle abnormalities in furin processing of the mutant receptor, although both heterodimeric and full length receptors are present on the cell surface, are capable of interacting with soluble forms of three ligands, Delta1, Delta4, and Jagged1, and retain the ability to activate CBF1 in a ligand dependent manner. CONCLUSIONS: By comparison with other mutant forms of Notch3, these data indicate that individual CADASIL mutations can have disparate effects on Notch3 expression and function.

Altered brain stem responsivity to duodenal pain after a single stressful experience.

A single session of foot shock stress produces stable and long lasting sensitization of behavioral, hormonal and intestinal motility responses to novel stressful stimuli in laboratory rats. This is reflected in increased expression of the activity marker protein Fos in brain areas involved, following an external stressor. We present data from awake, freely moving rats in which a silicone balloon was surgically implanted in the duodenum. Firstly, cardiovascular reflexes to distentions were studied using telemetry with surgically implanted transmitters, 2 weeks after a single, 15-min session of foot shocks. The distentions caused characteristic, bi-phasic responses in both mean arterial blood pressure and heart rate that were not different between preshocked and control animals. Secondly, the numbers of Fos immunopositive cells were quantified in selected brain areas, 1 h after repeated distention of the duodenum. We found an increase in distention-induced Fos in preshocked rats in the nucleus tractus solitarius and a weaker effect in the central nucleus of the amygdala. This could be a first indication that altered visceral afferent processing in previously stressed rats, found earlier for the colon, may be a general and not an organ-specific phenomenon.

Two modes of vesicle recycling in the rat calyx of Held.

Vesicle recycling was studied in the rat calyx of Held, a giant brainstem terminal involved in sound localization. Stimulation of brain slices containing the calyx-type synapse with a high extracellular potassium ion concentration in the presence of horseradish peroxidase resulted within several minutes in a reduction of the number of neurotransmitter vesicles and in the appearance of labeled endosome-like structures. After returning to normal solution, the endosome-like structures disappeared over a period of several minutes, whereas simultaneously the number of labeled vesicles increased. A comparison with afferent stimulation suggested that the endosome-like structures normally do not participate in the vesicle cycle. Afferent stimulation at 5 Hz resulted in sustained synaptic transmission, without vesicle depletion but with an estimated endocytotic activity of <0.2 synaptic vesicles per active zone per second. At 20 Hz, the presynaptic action potentials generally failed during prolonged stimulation. In identified synapses, the number of vesicles labeled by photoconversion after stimulation at 5 Hz in the presence of the styryl dye RH414 was much lower than the number of vesicles that were released, as determined by measuring EPSCs. No more than approximately 5% of the vesicles were labeled after 20 min stimulation at 5 Hz, whereas this stimulation protocol was sufficient to largely destain a terminal after previous loading. The results support a scheme for recycling in which two different modes coexist. At physiological demands, a pool of approximately 5% of all vesicles provides sufficient vesicles for release. During intense stimulation, such as occurs in the presence of high extracellular K+, the synapse resorts to bulk endocytosis, a very slow mode of recycling.

Sequential changes in synaptic vesicle pools and endosome-like organelles during depolarization near the active zone of central nerve terminals.

During periods of high-frequency stimulation the maintenance of synaptic transmission depends on a continued supply of synaptic vesicles. Local recycling in the terminals ensures synaptic vesicle replenishment, but the intermediate steps are still a matter of debate. We analyzed changes in synaptic vesicle pools and endosome-like organelles near the active zone in central nerve terminals during depolarization at the ultrastructural level by electron microscopy. A short, 100 ms, depolarization-induced recruitment of synaptic vesicles was observed from a reserve pool to a recruited pool, within 150 nm of the active zone, and the docked pool at the active zone was increased as well. Prolonged, 15 s or 3 min, depolarization decreased the total amount of synaptic vesicles, which was accompanied by a parallel increase in size and amount of endosome-like organelles. After a period of rest, the number of endosome-like organelles decreased and the amount of synaptic vesicles was restored to control level. The endocytotic nature of part of the endosome-like organelles after 15 s and 3 min depolarization was indicated by their labeling with extracellularly added horseradish peroxidase (HRP). In addition, a small number of synaptic vesicles entrapped HRP under these conditions. After repolarization, the number of HRP-loaded endosome-like structures decreased. Simultaneously, a strong increase in amount of HRP-loaded small vesicles did occur. These results indicate that during sub-second depolarization, synaptic vesicles were rapidly recruited from the reserve pool to replenish the releasable pool, whereas prolonged depolarization (s-min) induced local endocytosis in at least two ways, i.e. either directly as vesicles or via endosome-like organelles from which synaptic vesicles were reformed.

Lack of association between apolipoprotein E genoype and ischaemic stroke in a Scottish population.

BACKGROUND: Apolipoprotein E epsilon4 allele has been associated with increased risk of coronary heart disease, and is also a major genetic susceptibility locus for Alzheimer's disease. Some studies have shown an association between apoE genotype and ischaemic stroke or outcome following stroke, while other studies have failed to do so. Materials and methods Using PCR and the Taqman fluorescence system to detect polymorphisms we examined apoE genotype in 266 ischaemic stroke cases and in a control population. RESULTS: We found no association between apoeE epsilon 4 allele distribution and ischaemic stroke, or with outcome following stroke as measured using the Rankin score. Conclusion This study disagrees with a recent meta-analysis, and suggests that further studies are required to clarify the exact relationship between apoE genotype and ischaemic stroke.

Mapping and sequencing rat dishevelled-1: a candidate gene for cerebral ischaemic insult in a rat model of stroke.

A quantitative trait locus on chromosome 5 in the rat is linked to sensitivity to brain ischemia in the stroke-prone spontaneously hypertensive rat (SHRSP). The genes encoding atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) that map to this location have been excluded as candidate genes. We examined dishevelled-1 (DVL-1) as a further candidate gene. DVL-1 had not yet been identified in the rat, but Anp, Bnp, and DVL-1 map to the homologous regions of the rat chromosome 5 quantitative trait locus in both mice and man. Furthermore, DVL-1 is involved in the Notch signalling system, which plays a role in the disorder cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, the symptoms of, which include ischaemic stroke. We show with radiation hybrid mapping that rat DVL-1 indeed maps to chromosome 5, where it is positioned immediately next to microsatellite marker D5Rat49. We sequenced the complete coding sequence and a large part of the intronic genomic sequence for the SHRSP strain and its reference Wistar-Kyoto strain. The DVL-1 sequence in the two strains was identical. Our results essentially exclude the DVL-1 gene as the cause for sensitivity to cerebral ischaemic insult in this rat model of stroke.

Target-dependent differentiation and development of molluscan neurons and neuroendocrine cells: use of parasitisation as a tool.

Specimens of the freshwater snail Lymnaea stagnalis infected with the schistosome parasite Trichobilharzia ocellata show a strongly inhibited development of their reproductive tract. We hypothesised that the effects of the underdevelopment of targets are reflected at the level of the neuronal development of (i) the motor neurons innervating the male copulation organ and (ii) neuroendocrine cells regulating the gonad. We determined the state of neuronal development by measuring cell number, cell size and neuropeptide gene expression. Our results show that the neuronal development of both copulation controlling anterior lobe motor neurons of the right cerebral ganglion and neuroendocrine caudodorsal cells, which produce neuropeptides regulating ovulation, egg laying and accompanying behaviour, are affected in parasitised animals in which their respective target organs were not developed. The cell bodies were smaller and fewer cells were found to express neuropeptide genes compared to those in non-parasitised animals. These effects were not observed in the appropriate controls. Backfills and lesions of the penis nerve have shown that the inhibited development of central motor neurons in parasitised snails is target dependent; neighbouring neurons that have no connection with the male copulation organ are not affected. Our data suggest that this effect is established by target-derived neurotrophic factors that need this connection for being transported to the innervating motor neurons. We propose that the effect on the neuroendocrine caudodorsal cells is mediated by a humoral factor, since they have no known connection with their target. We have shown that the size and gene expression of motor neurons controlling copulation behaviour in the pond snail Lymnaea stagnalis are related to the size of their target, the copulation organ, and depend on the connection with this target.

Diagnosing CADASIL using MRI: evidence from families with known mutations of Notch 3 gene.

Clinical data and MRI findings are presented on 18 subjects from two families with neuropathologically confirmed CADASIL. DNA analysis revealed mutations in exon 4 of Notch 3 gene in both families. All family members with mutations in Notch 3 gene had extensive abnormalities on MRI, principally lesions in the white matter of the frontal lobes and in the external capsules. Of several family members in whom a diagnosis of CADASIL was suspected on the basis of minor symptoms, one had MRI changes consistent with CADASIL; none of these cases carried a mutation in the Notch 3 gene. MRI and clinical features that may alert the radiologist to the diagnosis of CADASIL are reviewed. However, a wide differential diagnosis exists for the MRI appearances of CADASIL, including multiple sclerosis and small-vessel disease secondary to hypertension. The definitive diagnosis cannot be made on MRI alone and requires additional evidence, where available, from a positive family history and by screening DNA for mutations of Notch 3 gene.

Detection of complement factor B in the cerebrospinal fluid of patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy disease using two-dimensional gel electrophoresis and mass spectrometry.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary condition with onset in mid-adulthood and is associated with mutations in the Notch-3 gene. (Joutel, A., Corepechot, C., Ducros, A., Vahedi, K., Chabriat, H., Mouton, P., Alamowitch, S., Domenda, V., Cecilion, M., Marechal, J., Vayssiere, C., Cruaud, C., Cabanis, E.A., Ruchoux, M.M. , Weissenvach, J., Bach, J.F., Bousser, M.G. and Tournier-Lasserve, E., Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia. Nature, 383 (1996) 707-710) Ultrastructural examination of the pathology of the cerebral infarcts reveals deposits in the vascular smooth muscle cells of the small arteries of the brain, but there is no obvious indication how the Notch-3 mutations give rise the observed pathology, nor is there any information on the exact nature of the deposits. We have investigated cerebrospinal fluid (CSF) from three CADASIL cases with known mutations in Notch-3 using two-dimensional gel electrophoresis. CSF from these patients was compared to that of six controls. We detected a single spot in the protein maps of patients which was absent from all the controls. In-gel tryptic digestion of this protein followed by mass spectrometric analysis of the tryptic fragments and a database search identified the spot as human complement factor B. These preliminary findings suggest that the alternative complement pathway may play a role in the pathogenesis of CADASIL.

Transmitter identification in neurons involved in male copulation behavior in Lymnaea stagnalis.

In this paper, we have mapped the cellular localization of various transmitters onto the central neurons which are involved in male copulation behavior in Lymnaea stagnalis, by combining retrograde tracing with immunocytochemistry and in situ hybridization. Evidence is provided that neurons which were backfilled from the penis nerve, the sole nerve to innervate the male copulatory organ, synthesize a multitude of neuropeptides (APGWamide, Lymnaea neuropeptide tyrosin [LNPY], conopressin, pedal peptide, SEEPLY, DEILSR, myomodulin, and Lymnaea inhibitory peptide [LIP]) as well as the classical neurotransmitter, serotonin. In the anterior lobe, the backfilled neurons mainly contain the tetrapeptide APGWamide and conopressin, and not LNPY or pedal peptide. The results suggest a central role in the regulation of copulation activity for the anterior lobe neurons that produce APGWamide and conopressin. Immunostainings of backfilled nervous systems revealed immunopositive axons originating from these neurons to form varicosities on the cell somata of neurons in the other clusters contributing to the innervation of the male sexual system. Neurons from the right parietal ganglion projecting into the penis nerve were electrophysiologically and morphologically identified by simultaneously recording from the cell body intracellularly and the penis nerve extracellularly and subsequently filling them with an anterograde tracer and subjecting them to immunocytochemistry. This method has provided links between morphology, physiology, and the transmitter contents of these neurons.

Localization of the neuropeptide APGWamide in gastropod molluscs by in situ hybridization and immunocytochemistry.

The amidated tetrapeptide Ala-Pro-Gly-Trp-NH2 (APGWamide) plays a key role in the control of male copulation behavior in the basommatophoran pulmonate freshwater snail Lymnaea stagnalis. The morphological basis for a conserved role of APGWamide in the control of male reproduction in gastropod molluscs is presented. The prosobranch Littorina littorea, the opisthobranch Aplysia californica, the basommatophoran pulmonate Bulinus truncatus, and the stylommatophoran pulmonates Arion ater and Limax maximus have been examined for the presence of APGWamide producing neurons using immunocytochemistry and in situ hybridization. In all species investigated a cluster of APGWamide expressing neurons is present in the anteromedial region of the cerebral ganglia. The asymmetrical distribution which exists in Lymnaea and which coincides with the innervation of the asymmetrically located penial complex is also found in the opisthobranch Aplysia, as well as in the stylommatophoran pulmonate slugs Arion and Limax, in which APGWamide immunoreactive neurons are only found in the mesocerebrum of the right cerebral ganglion. APGWamide immunoreactive varicose fibers innervate muscles of the male accessory sex organs in Bulinus and Aplysia, confirming the hypothesis that APGWamide may be a biochemically and functionally conserved factor in the regulation of gastropod mollusc reproduction.

Multi-messenger innervation of the male sexual system of Lymnaea stagnalis.

The male copulation behaviour of the hermaphrodite pond snail Lymnaea stagnalis is under the control of five groups of central neurons that produce a variety of neuropeptides and a classical transmitter, 5-hydroxy tryptamine (5HT). In this article, we describe how the male sexual organs of this snail are innervated by axons from these central neurons. We carried out immunocytochemistry with antisera against the tetra peptide Ala-Pro-Gly-TRP-NH2 (APGWamide), the Lymnaea form of neuropeptide tyrosine (LNPY), conopressin, pedal peptide, the FRMFamide copeptide SEEPLY, the GDPFLRFamide co-peptide DEILSR, myomodulin, Lymnaea inhibitory peptide, and 5HT on tissue sections of the following male sexual organs that receive input from the penis nerve: the prostate gland, vas deferens, preputium, and penis. The results demonstrate that the axons of the separate muscle systems contain particular combinations of transmitters. In addition, two networks of peripheral neurons were revealed. In the tip of the everted preputium lies what appears to be a network of conopressin-containing sensory neurons, which is possibly involved in probing; probing is the part of copulation behaviour in which the male searches for the female genital pore. The other network of peripheral neurons surrounds the most proximal part of the vas deferens and is most likely involved in the pacemaker control of vas deferens motility. On the basis of the data obtained, we hypothesize how the preputium and penis are everted during copulation and which transmitters and central neurons might be involved.

Diversity in cell specific co-expression of four neuropeptide genes involved in control of male copulation behaviour in Lymnaea stagnalis.

We report here the neuron-specific co-expression of four genes coding for neuropeptides involved in the control of male behaviour. These neurons are located in the anterior lobe of the right cerebral ganglion in the central nervous system of Lymnaea stagnalis and project via the penis nerve to the penial complex. In order to accomplish optimal assurance we applied in situ hybridization, immunocytochemistry and matrix-assisted laser desorption ionization mass spectrometry. The anterior lobe neurons express the gene encoding the amidated tetrapeptide APGWamide. Subsets of these cells are now shown to co-express the APGWamide gene exclusively with one of three other neuropeptide genes, encoding Lymnaea neuropeptide Y, conopressin or pedal peptide, respectively. All four genes are also expressed in other neurons in other centres projecting to the penial complex, but in these cells co-expression was not observed. The neuropeptides encoded by the genes could be identified in the anterior lobe cell bodies on the basis of immunocytochemistry and mass spectrometrical analysis. The neuropeptides APGWamide and Lymnaea neuropeptide Y, which are co-localized in the anterior lobe cells as well as in axons innervating the penis retractor muscle, do not induce muscle contraction but have a modulatory action by affecting the relaxation rate and amplitude of the contraction. APGWamide and conopressin had earlier been suggested to modulate peristalsis of the vas deferens. Thus, it seems that the neurons co-expressing the various combinations of neuropeptide genes in the anterior lobe represent functional units, each acting in the fine tuning of different muscles involved in specific aspects of male copulation behaviour.

Role of neuropeptides encoded on CDCH-1 gene in the organization of egg-laying behavior in the pond snail, Lymnaea stagnalis.

Egg laying in the pond snail Lymnaea stagnalis is triggered by a discharge of the neuroendocrine caudodorsal cells (CDCs). The CDCs expresses three different caudorsal cell hormone (CDCH) genes. This gene family expresses, in total, 11 different peptides among which is the ovulation hormone. Besides the CDCs, the CDCH gene family is expressed in other central and peripheral neurons. In this study, we investigated the roles the different CDCH peptides play in the organization of egg-laying behavior. Egg-laying behavior is a sequence of stereotyped movements in which three phases can be distinguished: resting, turning, and oviposition. We have used the excitation of right pedal N (RPeN) motor neurons as a simple analogue of shell-turning behavior, one of the elements of egg-laying behavior. RPeN motor neurons were inhibited during the resting phase of egg laying but were subsequently excited at the onset of and during the turning phase. The excitatory effect could be evoked by application of beta3-CDCP on RPeN motor neurons in the CNS as well as in isolation but not by the ovulation hormone, alpha-CDCP or Calfluxin, the other CDCH-1 peptides tested. The ovulation hormone itself caused inhibition of RPeN motor neurons. Anti-CDCH-1 positive fiber tracts were found close to the cell bodies and axons of the RPeN motor neurons. Electrical stimulation of a nerve that contains these fibers resulted in excitation of the RPeN motor neurons. The effects of injection of CDCH-1 peptides into intact animals correlated well with the effects of these peptides on RPeN motor neurons. Injection of beta3-CDCP or alpha-CDCP into intact animals resulted in immediate turning behavior in the absence of egg laying itself. The ovulation hormone and Calfluxin had no immediate effect on the behavior. Furthermore, our data indicate that the individual CDCH-1 peptides act on different targets.

Co-localized neuropeptides conopressin and ALA-PRO-GLY-TRP-NH2 have antagonistic effects on the vas deferens of Lymnaea.

We examined functional aspects of co-localization of neuropeptides involved in the regulation of male copulation behaviour in the simultaneous hermaphrodite snail Lymnaea stagnalis. The copulation behaviour is controlled by several types of peptidergic neurons that include a cluster of neurons in the anterior lobe of the right cerebral ganglion. All anterior lobe neurons express the gene encoding Ala-Pro-Gly-Trp-NH2 (APGWamide), and a subset of neurons also express the vasopressin-related conopressin gene. Immunocytochemical and peptide chemical experiments show that both APGWamide and conopressin are transported to the penis complex and the vas deferens via the penis nerve. Co-localization of the two peptides was also observed in some, but not all, axon bundles that run along the vas deferens. APGWamide and conopressin were structurally identified from the penis complex with vas deferens. Conopressin excites the vas deferens in vitro, whereas APGWamide inhibits the excitatory effects of conopressin, both in a dose-dependent fashion. We propose that the antagonistic effects of these peptides on the vas deferens underlie its peristalsis. Thus, these peptides play an important role in the control of ejaculation of semen during copulation.

Mutually exclusive neuronal expression of peptides encoded by the FMRFa gene underlies a differential control of copulation in Lymnaea.

An innovative method, direct peptide profiling of small samples of nervous tissue by matrix-assisted laser desorption ionization mass spectrometry, in combination with peptide characterization, immunocytochemistry in conjunction with specific neuronal labeling by backfilling of the penis nerve, and bioassay of peptides was used to study the intrinsic neuronal expression patterns of distinct sets of related FMRFa peptides and their significance for the organization of male copulation behavior in the mollusk, Lymnaea stagnalis. Previous studies indicate that the sets of FMRFa-related and GDPFLRFa-related peptides are encoded by two alternatively spliced transcripts of the single FMRFa gene. Direct mass spectrometry revealed that both FMRFa-related and GDPFLRFa-related peptides are present in the penis nerve, the sole nerve that innervates the penis complex. Accordingly, authentic FMRFa, GDPFLRFa, and related peptides were purified from the penis complex. The loci of synthesis of FMRFa and related peptides could be traced to the right cerebral ventral lobe, those of GDPFLRFa and related peptides to the B group neurons in the right parietal ganglion and to a few unidentified neurons in the right pleural ganglion. Notwithstanding their related structures, the two sets of peptides have distinctly different actions on the penis retractor muscle.

Structural and functional evolution of the vasopressin/oxytocin superfamily: vasopressin-related conopressin is the only member present in Lymnaea, and is involved in the control of sexual behavior.

It has been suggested that the gene duplication that led to the formation of the vasopressin/oxytocin two-gene family occurred early during vertebrate evolution. However, the existence of both vasopressin- and oxytocin-related peptides in invertebrates suggests that this duplication may have occurred much earlier, although there is no evidence for the co-occurrence of vasopressin- and oxytocin-related peptides in the same invertebrate species. We report here that in Lymnaea only the vasopressin-related peptide Lys-conopressin, but not an oxytocin-related peptide, is present. Moreover, it is very likely that an oxytocin-like cDNA or gene is absent. The conopressin gene is expressed in neurons that control male sexual behavior, and its gene products are present in the penis nerve and the vas deferens. Conopressin induces muscular contractions of the vas deferens and inhibits central neurons that control female reproductive behavior. Thus, although structurally related to vasopressin, conopressin has functional and behavioral characteristics typical for oxytocin. Physiological and receptor binding data suggest that conopressin and [Ile8]-conopressin, a synthetic oxytocin-like analog of conopressin, are functionally equivalent in Lymnaea, and that the chemical nature of the amino acid residue at position 8 does not result in a functional difference. Therefore, we suggest that invertebrates contain only a single member of the vasopressin/oxytocin gene family and that the amino acid change that distinguishes vasopressin from oxytocin is functionally neutral in invertebrates.

Expression and translation of the egg-laying neuropeptide hormone genes during post-embryonic development of the pond snail Lymnaea stagnalis.

The neuroendocrine caudodorsal cells of Lymnaea stagnalis express two homologous genes, each encoding a polypeptide precursor. The precursors give rise to "cocktails" of neuropeptides that regulate egg-laying. The expression and translation of both egg-laying hormone genes during post-embryonic development were investigated by in situ hybridization and by electron-microscopic immunocytochemistry. Gene-II-specific transcripts and translation products were not found in caudodorsal cells in animals with shell heights smaller than 10 mm, in contrast to gene-I products that were present even at 3-mm shell height. The onset of expression of gene II coincides with the onset of release of products from the caudodorsal cells into the blood. Large electron-dense granules were found in caudodorsal cells of snails of all developmental stages investigated. These granules form part of the Golgi sorting and packaging pathway. Their presence suggests that differential sorting and packaging is possible during post-embryonic development, like in adults. The relationship of the differential expression of the two genes to the development of the caudodorsal cell system and its targets is discussed.