Jeffrey I. Goldberg

Gonadotropin-releasing hormone neuronal system of the freshwater snails Helisoma trivolvis and Lymnaea stagnalis: possible involvement in reproduction.

Peptides of the gonadotropin‐releasing hormone (GnRH) family are present in neural and nonneural tissues throughout the chordate phylum. Although GnRH peptides have been implicated in nonreproductive functions, their primary function is to control reproduction by regulating sexual behaviors and inducing gonadotropin hormone release from the pituitary. Evidence suggesting the presence of a similar peptide in the central nervous system (CNS) of the gastropod mollusc Helisoma trivolvis has recently been provided. In the present study, we examined the tissue distribution of the peptide and found that it is likely restricted to the nervous system. The neuronal system containing the endogenous GnRH‐like peptide is described further and is shown, in part, to innervate the male reproductive tract. Immunostaining in the closely related snail, Lymnaea stagnalis, showed a conservation in the locations of some immunoreactive neurons. Notably, staining occurred in and adjacent to the lateral lobes of both snails. Because these lobes contain neurons involved in the stimulation of egg laying and GnRH staining occurred in additional areas in the Helisoma CNS that are involved in reproduction, we suggest that the endogenous GnRH‐like peptide plays a role in regulating reproduction in freshwater snails. J. Comp. Neurol. 404:427–437, 1999.

Morphological identification of live gonadotropin, growth-hormone, and prolactin cells in goldfish (Carassius auratus) pituitary-cell cultures

To better understand neuroendocrine regulation and the intracellular mechanisms mediating pituitary-hormone release, it is necessary to study the physiology of identified single cells. We have developed a system to identify gonadotropin, growth-hormone, and prolactin cells in primary cultures of goldfish pituitary cells. Using Nomarski differential interference-contrast microscopy, the unique morphologies of discrete subpopulations of cells were characterized. To aid in the initial characterization of different pituitary-cell types, a discontinuous Percoll density-gradient cell-separation technique was developed. This method provided fractions enriched with functional gonadotropin, growth-hormone, and prolactin cells. The morphology of each cell type was initially characterized in enriched fractions of immunofluorescently labelled cells using differential interference-contrast microscopy. The cell type-specific morphologies were then confirmed in live pituitary-cell cultures. Gonadotropin, growth-hormone, and prolactin cells were correctly identified in live pituitary-cell cultures. Gonadotropin, growth-hormone, and prolactin cells were correctly identified in live mixed cultures in 92, 94, and 100% of the trials, respectively. The ability to directly identify cells in primary cultures allows the physiological study of identified single cells with minimal pretreatment.

Early development of an identified serotonergic neuron in Helisoma trivolvis embryos: Serotonin expression, de-expression, and uptake

In early-stage embryos of Helisoma trivolvis, a bilateral pair of identified neurons (ENC1) express serotonin and project primary descending neurites that ramify in the pedal region of the embryo prior to the formation of central ganglia. Pharmacological studies suggest that serotonin released from ENC1 acts in an autoregulatory pathway to regulate its own neurite branching and in a paracrine or synaptic pathway to regulate the activity of pedal ciliary cells. In the present study, several key features of early ENC1 development were characterized as a necessary foundation for further experimental studies on the mechanisms underlying ENC1 development and its physiological role during embryogenesis. ENC1 morphology was determined by confocal microscopy of serotonin-immunostained embryos and by differential-interference contrast (DIC) microscopy of live embryos. The soma was located at an anteriolateral superficial position and contained several distinguishing features, including a large spherical nucleus with prominent central nucleolus, large granules in the apical cytoplasm, a broad apical dendrite ending in a sensory-like structure at the embryonic surface, and a ventral neurite. ENC1 first expressed serotonin immunoreactivity around stage E13, followed immediately by the appearance of an immunoreactive neurite (stage E14). Both the intensity of immunoreactivity and primary neurite length were consistently greater in the right ENC1 at early stages. Serotonin uptake, as indicated by 5,7-dihydroxytryptamine-induced fluorescence, first occurred between stages E18 and E25. At later stages of embryogenesis (after stage E65), serotonin immunoreactivity disappeared, whereas serotonin uptake and normal cell morphology were retained.

Effect of serotonin on ciliary beating and intracellular calcium concentration in identified populations of embryonic ciliary cells

SUMMARY Embryos of the pond snail Helisoma trivolvis express three known subtypes of ciliary cells on the surface of the embryo early in development: pedal, dorsolateral and scattered single ciliary cells (SSCCs). The pedal and dorsolateral ciliary cells are innervated by a pair of serotonergic sensory-motor neurons and are responsible for generating the earliest whole-animal behavior, rotation within the egg capsule. Previous cell culture studies on unidentified ciliary cells revealed that serotonin (5-hydroxytryptamine; 5-HT) produces a significant increase in the ciliary beat frequency (CBF) in a large proportion of ciliary cells. Both Ca2+ influx and a unique isoform of protein kinase C (PKC) were implicated in the signal transduction pathway underlying the cilio-excitatory response to 5-HT. The goal of the present study was to characterize the anatomical and physiological differences between the three known populations of superficial ciliary cells. The pedal and dorsolateral ciliary cells shared common structural characteristics, including flat morphology, dense cilia and lateral accessory ciliary rootlets. By contrast, the SSCCs had a cuboidal morphology, reduced number of cilia, increased ciliary length and absence of lateral accessory rootlets. In cultures containing unidentified ciliary cells, the calcium/calmodulin-dependent enzyme inhibitor calmidazolium (2 μmol l–1) blocked the stimulatory effect of 5-HT (100 μmol l–1) on CBF. In addition, 50% of unidentified cultured cells responded to 5-HT (100 μmol l–1) with an increase in [Ca2+]i. To facilitate the functional analyses of the individual populations, we developed a method to culture identified ciliary subtypes and characterized their ciliary and calcium responses to 5-HT. In cultures containing either pedal or dorsolateral ciliary cells, 5-HT (100μ mol l–1) produced a rapid increase in CBF and a slower increase in [Ca2+]i in all cells examined. By contrast, the CBF and [Ca2+]i of SSCCs were not affected by 100μ mol l–1 5-HT. Immunohistochemistry for two putative 5-HT receptors recently cloned from Helisoma revealed that pedal and dorsolateral ciliary cells consistently express the 5-HT1Hel protein. Intense 5-HT7Hel immunoreactivity was observed in only a subset of pedal and dorsolateral ciliary cells. Cells neighboring the SSCCs, but not the ciliary cells themselves, expressed 5-HT1Hel and 5-HT7Hel immunoreactivity. These data suggest that the pedal and dorsolateral ciliary cells, but not the SSCCs are a homogeneous physiological subtype that will be useful for elucidating the signal transduction mechanisms underlying 5-HT induced cilio-excitation.

Development of Ca2+ hotspots between Lymnaea neurons during synaptogenesis

Calcium (Ca2+) channel clustering at specific presynaptic sites is a hallmark of mature synapses. However, the spatial distribution patterns of Ca2+ channels at newly formed synapses have not yet been demonstrated. Similarly, it is unclear whether Ca2+‘hotspots’ often observed at the presynaptic sites are indeed target cell contact specific and represent a specialized mechanism by which Ca2+ channels are targeted to select synaptic sites. Utilizing both soma–soma paired (synapsed) and single neurons from the mollusk Lymnaea, we have tested the hypothesis that differential gradients of voltage‐dependent Ca2+ signals develop in presynaptic neuron at its contact point with the postsynaptic neuron; and that these Ca2+ hotspots are target cell contact specific. Fura‐2 imaging, or two‐photon laser scanning microscopy of Calcium Green, was coupled with electrophysiological techniques to demonstrate that voltage‐induced Ca2+ gradients (hotspots) develop in the presynaptic cell at its contact point with the postsynaptic neuron, but not in unpaired single cells. The incidence of Ca2+ hotspots coincided with the appearance of synaptic transmission between the paired cells, and these gradients were target cell contact specific. In contrast, the voltage‐induced Ca2+ signal in unpaired neurons was uniformly distributed throughout the somata; a similar pattern of Ca2+ gradient was observed in the presynaptic neuron when it was soma–soma paired with a non‐synaptic partner cell. Moreover, voltage clamp recording techniques, in conjunction with a fast, optical differential perfusion system, were used to demonstrate that the total whole‐cell Ca2+ (or Ba2+) current density in single and paired cells was not significantly different. However, the amplitude of Ba2+ current was significantly higher in the presynaptic cell at its contact side with the postsynaptic neurons, compared with non‐contacted regions. In summary, this study demonstrates that voltage‐induced Ca2+ hotspots develop in the presynaptic cell, concomitant with the appearance of synaptic transmission between the soma–soma paired cells. The appearance of Ca2+ gradients in presynaptic neurons is target cell contact specific and is probably due to a spatial redistribution of existing channels during synaptogenesis.

Dopamine-D2 actions on voltage-dependent calcium current and gonadotropin-II secretion in cultured goldfish gonadotrophs.

Dopamine D2‐receptor activation directly inhibits GnRH‐induced gonadotropin‐II (maturational gonadotropin, GTH‐II) secretion from goldfish pituitary cells. In this study, we show that dopamine and its D2 agonist, quinpirole, reduced GTH‐II secretion induced by either high extracellular K+ concentration or the voltage‐gated Ca2+ channel agonist, Bay K 8644. These actions of dopamine were blocked by addition of the dopamine D2‐receptor antagonist, spiperone. The actions of dopamine on Ca2+ current in single identified goldfish gonadotrophs were assessed in voltage‐clamp experiments using Ba2+ as the charge carrier through voltage‐gated Ca2+ channels. Dopamine caused a concentration‐dependent reduction in Ba2+ current amplitude with an EC50 of 1.0±0.3 nM, but did not shift the current‐voltage relationship. The D2 agonist quinpirole also caused a dose‐dependent reduction in the Ba2+ current amplitude with an EC50 of 2.7±1.4 nM. Quinpirole slowed the activation and inactivation kinetics, as well as removing the steady‐state inactivation properties of the Ba2+ current. In contrast to the actions of quinpirole, the dopamine D1‐receptor agonist, SKF 38393, did not affect the Ba2+ current. The inhibitory action of dopamine on voltage‐dependent Ca2+ currents was reversed by spiperone, but not by the D1 antagonist SKF 83566. Voltage‐dependent Na+ and K+ currents were not affected by dopamine or dopamine agonists. These data indicate that dopamine D2‐receptor activation reduces Ca2+ influx through voltage‐dependent Ca2+ channels to inhibit GTH‐II secretion.

Identification, molecular structure and expression of two cloned serotonin receptors from the pond snail, Helisoma trivolvis.

SUMMARY Helisoma trivolvis has served as a model system to study the functions of serotonin (5-HT) from cellular, developmental, physiological and behavioural perspectives. To further explore the serotonin system at the molecular level, and to provide experimental knockout tools for future studies, in this study we identified serotonin receptor genes from the H. trivolvis genome, and characterized the molecular structure and expression profile of the serotonin receptor gene products. Degenerate oligonucleotide primers, based on conserved regions of the Lymnaea stagnalis 5-HT1Lym receptor, were used to amplify G protein-coupled biogenic amine receptor sequences from H. trivolvis genomic cDNA, resulting in the cloning of two putative serotonin receptors. The deduced gene products both appear to be G protein-coupled serotonin receptors, with well-conserved structure in the functional domains and high variability in the vestibule entrance of the receptor protein. Phylogenetic analysis placed these receptors in the 5-HT1 and 5-HT7 families of serotonin receptors. They are thus named the 5-HT1Hel and 5-HT7Hel receptors, respectively. In situ hybridization and immunofluorescence studies revealed that these genes and gene products are expressed most heavily in the ciliated pedal and mantle epithelia of H. trivolvis embryos. In adults, widespread expression occurred in all ganglia and connectives of the central nervous system. Expression of both receptor proteins was localized exclusively to neurites when examined in situ. In contrast, when isolated neurons were grown in culture, 5-HT1Hel and 5-HT7Hel immunoreactivity were located primarily in the cell body. This is the first study to reveal a 5-HT7 receptor in a molluscan species.

Extracellular Sodium Dependence of GnRH‐Stimulated Growth Hormone Release in Goldfish Pituitary Cells

In goldfish, gonadotropin‐releasing hormone (GnRH) stimulation of growth hormone (GH) release has been shown to involve extracellular Ca2+ entry through voltage‐sensitive Ca2+ channels and the activation of protein kinase C (PKC). In this study, the possible involvement of extracellular Na+ in mediating the GH response to GnRH was examined using dispersed pituitary cells. Perifusion with Na+‐depleted medium reversibly reduced the acute GH response to 5‐min pulses of either 10 nM salmon (s)GnRH or 10 nM chicken (c)GnRH‐II. Similarly, replacement of normal medium with Na+‐depleted medium attenuated the long‐term GH release response to sGnRH and cGnRH‐II under static incubation conditions. These results suggest that GnRH‐induced GH release requires the presence of extracellular Na+. Treatment with 5‐min pulses of the Na+‐channel agonist veratridine (10 μM) increased GH release in an extracellular Ca2+‐dependent manner, presumably due to activation of voltage‐sensitive Ca2+ channels resulting from the depolarizing effect of increased Na+ influx. On the other hand, Na+ entry through tetrodotoxin (TTX)‐sensitive, voltage‐dependent Na+ channels is not involved in GnRH‐induced GH release. Application of 250 nM TTX, which abolished the voltage‐sensitive Na+ currents in identified goldfish somatotropes, did not affect the acute GH responses to 5‐min pulses of sGnRH and cGnRH‐II. The possible participation of Na+/H+ antiport in mediating the extracellular Na+‐dependent GnRH action on GH release was then examined. In static incubation experiments, sGnRH‐ and cGnRH‐II‐induced GH secretion were reduced by inhibitors of the Na+/H+ antiport, amiloride and dimethylamiloride (DMA). Likewise, the GH response to the PKC activator, tetradecanoyl phorbol acetate, was attenuated by treatment with Na+‐depleted medium, amiloride, and DMA. The inhibitory actions of amiloride and DMA were selective as these drugs did not affect the GH response elicited by the Ca2+ ionophore ionomycin and the voltage‐sensitive Ca2+ channel agonist, Bay K 8644. Taken together, these results indicate that extracellular Na+ and the Na+/H+ exchanger are involved in the mediation of GnRH‐stimulated GH release in goldfish. Furthermore, this dependence on Na+ and Na+/H+ antiport probably occurs distal to the activation of PKC by GnRH.

INVOLVEMENT OF PROTEIN KINASE C IN 5-HT-STIMULATED CILIARY ACTIVITY IN HELISOMA TRIVOLVIS EMBRYOS

1 During development, embryos of the pulmonate gastropod, Helisoma trivolvis, undergo a rotation behaviour due to the co‐ordinated beating of three bands of ciliated epithelial cells. This behaviour is in part mediated by the neurotransmitter serotonin (5‐HT) released from a pair of identified embryonic neurons. Using time‐lapse videomicroscopy to measure ciliary beat frequency (CBF) in response to pharmacological manipulations, we determined whether protein kinase C (PKC) is involved in mediating 5‐HT‐stimulated ciliary beating. 2 Diacylglycerol (DAG) analogues sn‐1,2‐dioctanoyl glycerol (DiC8; 100 μM) and 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG; 100 μM), partially mimicked the 5‐HT‐induced increase in CBF. In contrast, application of OAG in the absence of extracellular Ca2+ did not result in an increase in CBF. 3 5‐HT‐stimulated CBF was effectively blocked by PKC inhibitors bisindolylmaleimide (10 and 100 nM) and calphostin C (10 nM). In addition, bisindolylmaleimide (100 nM) inhibited DiC8‐induced increases in CBF. At a higher concentration (200 nM), bisindolylmaleimide did not significantly reduce 5‐HT‐stimulated cilio‐excitation. 4 Two different phorbol esters, phorbol 12‐myristate 13‐acetate (TPA; 0.1, 10 or 1000 nM) and phorbol 12β, 13α‐dibenzoate (PDBn; 10 μM) did not alter basal CBF. TPA (1 μM) did not alter 5‐HT‐stimulated CBF. Likewise, the synthetic form of phosphatidylserine, N‐(6‐phenylhexyl)‐5‐chloro‐1‐naphthalenesulphonamide (SC‐9; 10 μM), did not increase CBF, whereas a strong increase in CBF was observed upon exposure to 5‐HT. 5 The results suggest that a DAG‐dependent, phorbol ester‐insensitive isoform of PKC mediates 5‐HT‐stimulated CBF in ciliated epithelial cells from embryos of Helisoma trivolvis.

Coordinated development of identified serotonergic neurons and their target ciliary cells in Helisoma trivolvis embryos

Embryonic neuron C1s (ENC1s) are bilateral serotonergic neurons that function as cilioexcitatory motor neurons in embryonic development of the pond snail, Helisoma trivolvis. Recent experiments demonstrated that these neurons stimulate cilia‐driven embryo rotation in response to hypoxia. In the present study, a comprehensive anatomic analysis of these cells and their target ciliary structures was done to address the following questions: (1) Does ENC1 have a morphology consistent with an oxygen‐sensitive sensory cell; (2) Is the development of ENC1s neurite outgrowth pathway coordinated with the development of its target effectors, the pedal and dorsolateral ciliary bands; and (3) What is the anatomic basis of ENC1–ciliary communication? By using an array of microscopic techniques on live and serotonin‐immunostained embryos, we found that each ENC1 possessed an apical dendrite that was capped with an integral dendritic knob penetrating the embryo surface. The dendritic knobs contained both microvilli and nonmotile cilia that suggested a sensory transduction role. Each ENC1 also possessed a descending projection, whose development was characterized by the rapid formation of the primary neurite pathway between stages E13 and E15, with the primary neurite of the right ENC1 developing in advance of its contralateral homologue. Secondary neurite branches formed between stages E15 and E30 in a spatiotemporal pattern that closely matched the development of the dorsolateral and pedal bands of cilia. Both dorsolateral and pedal ciliated cells formed basal processes that contacted ENC1 neurites. Finally, gap junction profiles were observed at neurite–neurite, neurite–ciliary cell, and ciliary cell–ciliary cell apposition sites, whereas putative chemical synaptic profiles were observed at neurite–neurite and neurite–ciliary cell apposition sites. J. Comp. Neurol. 457:313–325, 2003.