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Identifying hazards and assessing associated risks can be a considerable challenge for researchers in universities and colleges. Neglecting risk management is generally one of the factors leading t...

The principle aim of the 2016 Workshop: Safety By Design was to improve laboratory safety by promoting discussions, collaborative interactions, and problem solving between researchers and environmental health and safety (EH&S) professionals. The workshop provided a platform for participants to learn about effective safety programs and innovative approaches from colleges, universities, national laboratories and chemical industry. The program included talks by leaders in safety management and safety research, panel discussions by researchers and health and safety experts, and breakout sessions in which all participants joined together in small workgroups to explore laboratory safety challenges and provide recommendations for improving safety in specific areas. The workshop participants emphasized that Principal Investigators (PI) are responsible for the health and safety of their students and staff in the laboratory, and are instrumental in shaping a strong culture of safety. The institution's administrative leadership plays an important role in promoting a positive culture of safety through recognition and support of safety activities, and through financial commitment. Recommendations for empowering researchers to engage in laboratory safety were formulated, and the importance of supporting student leadership and involvement in student-led safety programs was underlined. Analysis of the post-workshop survey showed perceptions about laboratory safety changed as a result of participating in the workshop for 85% of researchers and 53% of EH&S professionals. This outcome supports the importance of creating a structured environment where researchers and EH&S experts can exchange ideas and work toward the common goal of safety.

GnRH neurons integrate internal and external cues to control sexual maturation and fertility. Homeostasis of energy balance and food intake correlates strongly with the status of reproduction. Neuropeptides secreted by the hypothalamus involved in modulating energy balance and feeding may play additional roles in the regulation of reproduction. Hypocretin (Hcrt) (also known as orexin) is one such peptide, primarily controlling sleep/wakefulness, food intake, and reward processing. There is a growing body of evidence indicating that Hcrt/orexin (Hcrt) modulates reproduction through interacting with the hypothalamo-pituitary-gonadal axis in mammals. To explore potential morphological and functional interactions between the GnRH and Hcrt neuronal systems, we employed a variety of experimental approaches including confocal imaging, immunohistochemistry, and electrophysiology in transgenic zebrafish, in which fluorescent proteins are genetically expressed in GnRH3 and Hcrt neurons. Our imaging data revealed close apposition and direct connection between GnRH3 and Hcrt neuronal systems in the hypothalamus during larval development through adulthood. Furthermore, the Hcrt receptor (HcrtR) is expressed in GnRH3 neurons. Electrophysiological data revealed a reversible inhibitory effect of Hcrt on GnRH3 neuron electrical activity, which was blocked by the HcrtR antagonist almorexant. In addition, Hcrt had no effect on the electrical activity of GnRH3 neurons in the HcrtR null mutant zebrafish (HcrtR-/-). Our findings demonstrate a close anatomical and functional relationship between Hcrt and GnRH neuronal systems in zebrafish. It is the first demonstration of a link between neuronal circuits controlling sleeping/arousal/feeding and reproduction in zebrafish, an important animal model for investigating the molecular genetics of development.

Animal research frequently involves the use of hazardous chemicals such as carcinogens, reproductive toxins or other highly toxic substances. Animal facility workers and researchers may be exposed to these chemicals when treating animals, or handling cages or bedding of animals excreting these chemicals or their toxic intermediates. Handling of hazardous chemicals in animal facilities follows the chemical hygiene plan conventionalized for research laboratories but not animal facilities. Exposure to these chemicals in animal excretions (urine and feces) has not been fully evaluated. The goal of this case study was to understand how much and for how long hazardous select chemicals are excreted by male and female laboratory mice. We examined the metabolic fate of two hazardous chemicals commonly used in animal research: tamoxifen and bromodeoxyuridine (BrdU). In this study, excretion rates of tamoxifen and its two bioactive metabolites (hydroxytamoxifen and endoxifen) showed a dose-sex interaction; but, excreted concentrations reached very low to undetectable levels by 72 hour post treatment no matter the dose or sex of the animal. BrdU excretion rates showed little to no sex difference, and excreted concentrations reached low levels by 48 hour post treatment in both males and females. The outcomes of this study can be used by health & safety experts to develop evidence-based policies for risk management of handling cages and bedding of animals treated with tamoxifen and BrdU.

The goal of the University of California Center for Laboratory Safety (UCCLS) 2014 Workshop was to bring together researchers and Environmental Health & Safety (EHS (1) Impact of Regulations and Policies on Lab Safety; (2) Carrots versus Sticks: Impact on Compliance and Safety; and (3) Impact of Beliefs and Attitudes on Safety. Based on an exit survey, 95% of the participants rated the workshop as overall productive or very productive, and 86% generated potential research ideas on laboratory safety. An added benefit of the workshop was that 76% of the participants changed their perception on laboratory safety and 86% collected ideas for overcoming barriers to improving research safety in their home institution or lab. Following the workshop, a Request for Proposals was sent to all meeting participants to support research projects in laboratory safety.

Bisphenol A (BPA) is a well-known environmental, endocrine-disrupting chemical, and bisphenol S (BPS) has been considered a safer alternative for BPA-free products. The present study aims to evaluate the impact of BPA and BPS on the reproductive neuroendocrine system during zebrafish embryonic and larval development and to explore potential mechanisms of action associated with estrogen receptor (ER), thyroid hormone receptor (THR), and enzyme aromatase (AROM) pathways. Environmentally relevant, low levels of BPA exposure during development led to advanced hatching time, increased numbers of GnRH3 neurons in both terminal nerve and hypothalamus, increased expression of reproduction-related genes (kiss1, kiss1r, gnrh3, lhβ, fshβ, and erα), and a marker for synaptic transmission (sv2). Low levels of BPS exposure led to similar effects: increased numbers of hypothalamic GnRH3 neurons and increased expression of kiss1, gnrh3, and erα. Antagonists of ER, THRs, and AROM blocked many of the effects of BPA and BPS on reproduction-related gene expression, providing evidence that those three pathways mediate the actions of BPA and BPS on the reproductive neuroendocrine system. This study demonstrates that alternatives to BPA used in the manufacture of BPA-free products are not necessarily safer. Furthermore, this is the first study to describe the impact of low-level BPA and BPS exposure on the Kiss/Kiss receptor system during development. It is also the first report of multiple cellular pathways (ERα, THRs, and AROM) mediating the effects of BPA and BPS during embryonic development in any species.

The University of California Center for Laboratory Safety (Center) organized a laboratory safety workshop held on March 15–16, 2012 at the National Academy of Sciences’ Beckman Center in Irvine, California. The participants were a diverse group of 69 people who shared an interest in improving lab safety in a way that will be embraced by those who supervise and perform lab research ( Appendix A ). Participants were from academia, private industry, national laboratories as well as local and federal government. They were laboratory researchers from the physical sciences, engineering, and the biomedical and life sciences, and health and safety professionals. Through a series of talks, panel discussions, and breakout sessions, the workshop allowed for dialogue between all participants on the many topics under the umbrella of lab safety. One of the primary aims of the Center is to develop and optimize lab safety practices based on evidence from research, and to make the outcomes from that research broadly and readily accessible to the research and health and safety communities through peer-reviewed publications. In order to achieve this, the Center is looking to build productive collaborations and begin the process of developing research programs in lab safety. The workshop was a first step in this process.

To investigate the extent of gender bias in the volunteerism of small-group leaders amongst first-year medical students, and whether bias could be eliminated with special instructions to the students.The gender of leaders in small-group sessions in a real academic setting was monitored under two conditions: control conditions, in which basic instructions were provided to participants, and intervention conditions, in which the same basic instructions were provided plus a brief "pep talk" on the importance of experiencing a leadership role in a safe environment. During the small-group sessions, an observer noted the gender and names of group leaders for later analysis. After a class debriefing, a subset of leaders and nonleaders from both the control and intervention groups were invited to be interviewed about their perceptions of the small-group experience. Interviews were tape recorded and transcribed for analysis.In 2007-2008 and 2008-2009, disproportionately fewer women than men volunteered to become small-group leaders under control conditions. This gender bias was eliminated under intervention conditions. The interviews illustrated how a subtle change in instructions helped some female students take on a leadership role.Gender bias in leadership in the small-group setting amongst medical students-even when women make up half of the class-may persist without targeted intervention. The authors suggest that frequent and consistent intervention during medical school could be an important factor in encouraging women to identify themselves as leaders, promoting confidence to consider leadership roles in medicine.

Bisphenol-A (BPA) is a synthetic estrogen and monomer component of polycarbonate plastics and epoxy resins that are widely used in the production of food and beverage containers. It leaches into our food and drink at concentrations shown to have biological consequences. Here we show that exposure to low levels of BPA accelerated early embryonic development within 24 h of exposure, attenuated body growth, and advanced the times of hatching and reproductive maturation in medaka fish (Oryzias latipes). The acceleration in embryonic development and time to hatch were blocked by the thyroid-hormone receptor (TH-R) antagonist amiodarone, suggesting that BPA alters global developmental timing through a thyroid-hormone pathway. Our results are likely to have broad implications regarding the effects of plastic-derived contaminants on embryonic and reproductive development.

A survey on laboratory safety, conducted in 2012, provided the basis for comparing safety culture attributes of respondents from academic (n = 991), government (n = 133) and industry (n = 120) laboratories. There were institutional differences in risk assessment methods conducted before carrying out an experiment, for which fewer researchers from academia (18%) used forms issued by their organization as compared to government and industry researchers (51% and 43%, respectively). Safety training in all three institutions was reported to be similar; however, about 25% of all researchers were not trained on the specific hazard with which they worked. Risk perceptions were similar between respondents from all three institutions with respondents generally believing their personal risk to be significantly lower than what they assumed was predicated by their institution. The most striking difference between institutions was observed with self-reported PPE compliance behavior; respondents from industry labs were significantly more compliant with wearing a lab coat (87%) and eye protection (83%) than respondents from academic (66% and 61%, respectively) and government labs (73% and 76%, respectively). PPE compliance was highly associated with researchers’ perception of the level of risk in their lab; PPE compliance generally declined with lower perceived risk. In contrast to industry and government labs, PPE compliance of researchers in academia was significantly positively influenced when their safety behavior was monitored. Active safety involvement of the principal investigator (PI) or laboratory supervisor also correlated significantly with lower numbers of accidents reported by students and postdoctoral fellows in academic labs. The data support the view that laboratory safety is positively impacted by active involvement of the PI or a dedicated laboratory supervisor in academic institutions.

Gonadotropin-releasing hormone (GnRH) controls reproduction in vertebrates. Most studies have focused on the population of GnRH neurons in the hypothalamus that ultimately controls gonadal function. However, all vertebrates studied to date have two to three anatomically distinct populations of GnRH neurons that express different forms of this hormone. The purpose of the present study was to develop a new model for studying the population of GnRH neurons in the terminal nerve (TN) associated with the olfactory bulb and then to characterize their pattern of action potential firing to provide a foundation for understanding the role of these neurons in regulating reproduction. A stable line of transgenic medaka (Oryzias latipes) was generated in which a DNA construct containing the salmon GnRH (Gnrh3) promoter linked to green fluorescent protein (GFP) was expressed in TN-GnRH3 neurons. This population of GnRH neurons is located at or near the ventral surface of the brain, making them ideally situated for electrophysiological analysis. Whole-cell and loose-patch recordings in current-clamp mode were performed on these neurons from excised, intact brains of adult males in which afferent and efferent neural connections remained intact. All TN-GnRH3GFP neurons that we recorded showed a beating pattern of spontaneous action potential firing. Action potentials were blocked by tetrodotoxin, indicating they are generated by a voltage-sensitive Na þ current; however, an oscillation in subthreshold membrane potential persisted. The present results indicate that this transgenic fish will provide an excellent model for studying the cell physiology of an extrahypothalamic population of GnRH neurons.

The bag cell neurons (BCNs) of the mollusk Aplysia californica provide a simple model system for investigating cellular and molecular events regulating synthesis and secretion of a reproductive neuropeptide and their impact on physiology and behavior. The BCNs secrete a large amount of egg-laying hormone (ELH) in response to an electrical afterdischarge. The afterdischarge also triggers cellular and molecular events leading to upregulation of ELH biosynthesis to replenish the supply of releasable hormone that was lost because of secretion. In the present review, we discuss signal-transduction events that link membrane excitability to ELH biosynthesis. We present evidence that the afterdischarge stimulates ELH synthesis by upregulating translation of ELH mRNA rather than by activating ELH gene transcription. This increase in ELH synthesis is accompanied by a decrease in total protein synthesis, suggesting that the synthetic machinery is being funneled selectively toward ELH. We also discuss work showing that afterdischarge-induced ELH synthesis uses a novel mechanism of translation initiation, one involving a switch from cap-dependent to cap-independent translation initiation that activates an internal ribosome entry site (IRES) located in the 5'-untranslated region of ELH mRNA. The IRES-regulated translation provides a unique cellular mechanism to selectively upregulate synthesis of a critical reproductive hormone at the expense of nonessential proteins.

Earlier work showed that cell bodies and neurites of the peptidergic bag cell neurons of Aplysia californica contain mRNA for egg-laying hormone. The purpose of the present study was to determine if egg-laying hormone synthesis and prohormone processing is similar in the pleurovisceral connective nerves (containing neurites of bag cell neurons) and the bag cell neuron clusters (containing both cell bodies and neurites of bag cell neurons). Initial experiments confirmed by RT-PCR and sequencing that egg-laying hormone mRNA was present in the pleurovisceral connective nerves. To investigate possible regional differences in translation of mRNA and prohormone processing, clusters were separated from connective nerves and newly synthesized egg-laying hormone-immunoreactive proteins were analyzed. Results showed that synthesis and processing of prohormone occurred in both the clusters and isolated connective nerves; however, the relative abundance of prohormone, processing intermediates, and egg-laying hormone was different. Pulse-chase experiments showed that prohormone was processed more slowly in the connective nerves than in the clusters. These results show that mRNA in isolated neural processes of neuroendocrine cells can be translated, and that the cellular machinery for protein synthesis is present, but processing of the ELH prohormone is significantly compromised.

The University of California at Los Angeles (UCLA) quickly and comprehensively transformed its laboratory safety program following a chemistry accident in December 2008 that caused the death of a researcher. UCLA's Chancellor immediately announced sweeping changes in the laboratory safety program and challenged UCLA to become "Best in Class" in academic laboratory safety. Given the size of UCLA's lab researcher population and the decentralized nature of the campus, it became a daunting task to reach out and improve compliance with newly implemented safety policies. The Office of Environment, Health and Safety (EH&S) improved their operations, enhanced overall inspection procedures, and instituted mandatory laboratory safety training of PIs and researchers. A Laboratory Hazard Assessment Tool (LHAT) was implemented to aid in the identification of hazards, track laboratory space and personnel, and guide lab groups in compliance with personal protective equipment (PPE) policies. Laboratory inspections were increased by more than 4-fold in 2012 as compared to 2007 and now followed a comprehensive checklist to improve reproducibility and thoroughness of the inspection process. To provide better customer service to the research community and expedite corrective actions, all laboratory inspection reports are required to be issued the next business day. Unannounced PPE inspections began in 2010, and inspection findings showed a robust decrease in non-compliance with the PPE policy within one year of its implementation. As of the first half of 2013, all PIs of active laboratory research programs had completed the initial, in-class PI-specific laboratory safety training. In 2012, more than 20,000 EH&S safety classes were completed either in-class or online by PIs, lab supervisors, research staff, students, and visitors to UCLA's research laboratories. Overall, analysis of outcomes from changes in UCLA's lab safety program indicates rapid improvements in compliance with lab safety regulations such as increased PPE use. Lab safety changes required commitment and cooperation at all levels - from executive leadership, to health and safety experts, to deans and department chairs, and to PIs and researchers. © 2014 The Authors.

Understanding the physiological basis of environmental regulation of reproduction at the cellular level has been difficult or unfeasible in vertebrate species because of the highly complex and diffuse nature of vertebrate neuroendocrine systems. This is not the case with the simple nervous system of mollusks in which reproductive neuroendocrine cells are often readily identifiable in living tissue. Given that there are mollusks that are seasonal breeders, that the neuroendocrine cells controlling reproduction have been identified in several molluskan species, that these neurons are conducive to cell physiological analysis, and that basic features of cell biology have been highly conserved between mammals and mollusks, it seems that the mollusk would provide an excellent model system to investigate cell-physiological events that mediate effects of environmental signals on reproduction. The purpose of this review is to explore this idea in three species in which the topic of the neural basis of seasonal reproduction has been studied: the giant garden slug Limax maximus, the freshwater pond snail Lymnaea stagnalis, and the marine snail Aplysia californica.

Gonadotropin-releasing hormone (GnRH) is a neurohormone central to the regulation of reproductive functions in vertebrates. Recently, several studies have reported the presence of GnRH immunoreactivity (IR) in a number of mollusks, suggesting that the distribution of GnRH may not be restricted to Phylum Chordata. In the present study, we extend our investigations to an opisthobranch mollusk, Aplysia californica, to characterize the source, chemical nature, and biological activity of molluscan GnRH-related molecules. Specific radioimmunoassays (RIAs) of various tissue extracts of Aplysia revealed that only ovotestis, hemocytes, and hemolymph contained significant amounts of GnRH that crossreacts with antisera raised against tunicate-I (tI) and mammalian (m) GnRH. Further RIAs and extractions revealed that the GnRH-IR in the hemolymph is biochemically and immunologically distinct from the GnRH-IR in the hemocytes and ovotestis. Using reverse-phase high-performance liquid chromatography coupled with RIAs, the GnRH-IR in the hemolymph was resolved into two major peaks. The first peak eluted earlier than most known forms of vertebrate GnRH, and the later peak coeluted with m, lamprey I, chicken II, and tI-GnRH. However, both peaks were broad and may contain a heterogeneous mixture of GnRH-IR. Immunocytochemical study showed that tI-GnRH-IR was present in the connective sheath surrounding the central nervous system, with a strong presence in what appeared to be vascular space, again suggesting the close association between Aplysia GnRH-IR and circulation. Finally, treatment of the neuroendocrine bag cells with chicken II GnRH significantly decreased the duration of the afterdischarge (AD, a characteristic pattern of electrical firing in bag cell neurons) and the number of action potentials fired during an AD, indicating the presence of a corresponding GnRH receptor in the Aplysia central nervous system. Overall, the results demonstrated the presence of multiple forms of GnRH-IR that crossreact with tI- and mGnRH antisera in A. californica and the ability of a vertebrate GnRH to alter Aplysia neural activity. Together, these data suggest that GnRH may be a factor released by the ovotestis and hemocytes into the circulation to alter neural functions. GnRH-IR produced by the latter may serve as a novel mediator of the neural and immune functions in Aplysia.

The molecular links between membrane excitability and neurohormone synthesis were investigated using a simple model system: the bag cell neurons of Aplysia. We tested the hypothesis that the electrical afterdischarge, which leads to depletion of egg-laying hormone (ELH) by triggering secretion, rapidly stimulates ELH synthesis to replenish bag cell stores of hormone. Newly synthesized peptides were radiolabeled, and ELH peptide was immunoprecipitated. Within 4 h of afterdischarge, there was a 100% increase in ELH synthesis compared with that in unstimulated controls. Northern blot analysis showed that ELH messenger RNA (mRNA) levels were not altered for at least 8 h after the onset of afterdischarge. ELH mRNA levels were also not affected by 4-h treatments that inhibited either transcription (with actinomycin D) or translation (with anisomycin). Further work revealed that ELH mRNA is stable, with a half-life exceeding 32 h. Notably, the stimulatory effect of afterdischarge on ELH synthesis was blocked in response to treatment with the transcription inhibitor actinomycin D. These results suggest that the afterdischarge-induced increase in ELH synthesis is mediated by an increase in the rate of translation of already existing ELH mRNA, and that transcription of a non-ELH gene(s) is required for this effect to occur.

The neuroendocrine bag cells of Aplysia provide an excellent model system for exploring the roles of second-messenger pathways regulating peptide hormone secretion. Both the cAMP and diacylglycerol second-messenger systems and their associated protein kinases (PKA and PKC) are involved in regulating membrane excitability in bag-cell neurons of Aplysia. The purpose of the present set of experiments was to determine if PKA and PKC also play roles in regulating egg laying hormone (ELH) secretion from bag-cell neurons. Abdominal ganglia with attached bag-cell clusters and connective nerves were dissected from reproductively mature Aplysia, and ELH secretion in response to electrically stimulated afterdischarges was measured by RIA. ELH secretion from bag cells treated with protein-kinase inhibitors (Rp-cAMPS to inhibit PKA; H-7 to inhibit PKC) was compared to that from untreated controls. Our experiments showed that 100 microM Rp-cAMPS significantly attenuated ELH secretion during the nonbreeding seasons (winter and spring) of 2 consecutive years. This suggested a role for PKA in regulating ELH secretion. However, Rp-cAMPS had no effect on ELH secretion during the breeding seasons (summer and fall) of 2 consecutive years, even when the dose of Rp-cAMPS was increased to 200 microM. These findings indicate that there is a seasonal fluctuation in responsiveness to PKA inhibition. We also investigated if there was a seasonal fluctuation in the ability of the PKC inhibitor H-7 to suppress ELH secretion. During the nonbreeding season, 10-100 microM H-7 significantly inhibited ELH secretion, but during the breeding season, only the highest dose (100 microM) of H-7 inhibited ELH release. These results confirm that PKC plays a role in regulating ELH secretion and indicate that there is a seasonal fluctuation in responsiveness to PKC inhibition. Overall, our findings suggest that both the cAMP and diacylglycerol second-messenger pathways are regulated on a seasonal basis.

The neuroendocrine bag cells from Aplysia control reproductive functions through the regulated release of egg-laying hormone (ELH). The purposes of this study were to investigate (1) if synthesis of ELH in bag cells compensates for the release and degradation of the hormone, (2) the ratio of released to stored ELH, (3) the fate of newly synthesized ELH and its relative contribution to the overall pattern of ELH secretion, and (4) the consequences of blocking protein synthesis in bag cells on the pattern of ELH secretion and subsequent reproductive function. Excised bag cells were incubated with [ 3 H]leucine to radiolabel newly synthesized ELH and were then electrically stimulated to trigger secretion of the hormone on 4 successive days, mimicking bag cell activities during the breeding season. Samples of bag cell secretion were collected; total (new and old) and radiolabeled (new) ELH levels were determined. The results showed that ELH synthesis could not keep up with the daily loss of ELH through release and degradation, but the ratio of released to stored ELH remained constant over the 4-day study. In addition, releasable ELH was secreted preferentially within 24 hr after it was synthesized. Blocking protein synthesis resulted in a 50% decrease in total ELH released, suggesting that newly synthesized ELH contributes to half of the total ELH secreted. Furthermore, this lowered level of total ELH, i.e., older ELH alone, was not sufficient to stimulate egg laying in Aplysia, indicating that the contribution of newly synthesized ELH was physiologically relevant.

Understanding development of gonadotropin-releasing hormone (GnRH) neuronal circuits is fundamental to our understanding of reproduction, but not yet well understood. Most studies have been focused on GnRH neurons located in the hypothalamus and preoptic area (POA), which directly regulate the pituitary-gonadal axis. In zebrafish (Danio rerio), two forms of GnRH have been identified: GnRH2 and GnRH3. GnRH3 neurons in this species plays two roles: hypophysiotropic and neuromodulatory, depending on their location. GnRH3 neurons in in the ventral telencephalon, POA and hypothalamus control pituitary-gonadal function; in other areas (e.g., terminal nerve), they are neuromodulatory and without direct action on reproduction. To investigate the biology of GnRH neurons, a stable line of transgenic zebrafish was generated in which the GnRH3 promoter drives expression of a bright variant of green fluorescent protein (Emerald GFP, or EMD). This provides unprecedented sensitivity in detecting and imaging GnRH3 neurons during early embryogenesis in the transparent embryo. Using timelapse confocal imaging to monitor the time course of GnRH3:EMD expression in the live embryo, we describe the emergence and development of GnRH3 neurons in the olfactory region, hypothalamus, POA, and trigeminal ganglion. By 50 hours post fertilization, these diverse groups of GnRH3 neurons project broadly in the central and peripheral nervous systems and make anatomical connections with each other. Immunohistochemistry of synaptic vesicle protein 2 (a marker of synaptic transmission) in this transgenic model suggests synaptic formation is occurring during early development of the GnRH3 neural network. Electrophysiology reveals early emergence of responsiveness to the stimulatory effects of kisspeptin in terminal nerve GnRH3 neurons. Overall, our findings reveal that the GnRH3 neuronal system is comprised of multiple populations of neurons as a complicated network.

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express(1) . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons(2, 3) .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property(4-6), the frequency of which is modulated by visual cues from conspecifics(2) and the neuropeptide kisspeptin 1(5). In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein(7) to show you how to identify neurons and monitor their electrical activity in the whole brain preparation(6).

1. Despite the considerable progress made in understanding the role of electrical activity in triggering secretion, the developmental relationships between excitability and secretion are not well understood. The well-characterized bag cell neurons of Aplysia provide an advantageous system in which to investigate developmental interactions of these two key properties of neurons. 2. A prolonged afterdischarge triggers egg laying hormone (ELH) secretion in mature bag cell neurons. To investigate secretion in the developmental framework of excitability, we first examined whether immature neurons, which are incapable of the mature form of excitability (afterdischarge), contain ELH and whether this hormone is packaged in vesicles. We used immunoelectron microscopy to compare vesicular localization of ELH and to compare the size and density of ELH-containing vesicles in neurons from adult and juvenile Aplysia. This comparison revealed that immature neurons contain ELH in vesicles in the size range of secretory vesicles. However, they lack a class of large vesicles (> 250 nm in diameter) that is characteristic of mature neurons. 3. To investigate whether the ELH contained in immature bag cell neurons could be secreted in response to electrical activity, we used the potassium channel blocker tetraethylammonium (TEA) combined with nerve stimulation to depolarize neurons from both juvenile animals (ovotestes do not contain eggs) and from adult Aplysia (ovotestes contain eggs). Using radioimmunoassay, we have found that the duration and amount of ELH secreted from bag cell neurons from juvenile Aplysia in response to TEA does not depend on whether or not the cells can be induced to afterdischarge, and the amount and duration of ELH secreted from bag cell neurons of juvenile Aplysia (whether or not they afterdischarged) differed from those secreted by adult neurons. However, by normalizing for body size, we found that the final estimated hemolymph concentration of ELH would be similar in juvenile and adult animals. 4. We investigated the potential functional significance of secretion of bag cell hormones in juvenile Aplysia by attempting to bypass the bag cell neurons and directly activate downstream elements with extract from adult bag cell neurons (BCE), known to contain ELH and other peptides. We found that juvenile Aplysia exhibit at least one component of egg-laying behavior, cessation of locomotion, in response to BCE during a developmental period (as measured by weight) in which they normally would possess neurons incapable of afterdischarge. Thus developmental regulation of excitability in the bag cell neurons may prevent inappropriate hormone release and subsequent premature expression of reproductive behaviors.

Egg laying in the gastropod mollusk Aplysia californica is a temperature-dependent behavior, facilitated by warm temperature and attenuated by cold. The purpose of this work was to determine which components of the reproductive axis controlling egg laying are affected by temperature. There were three neural/reproductive tissues investigated: (1) the head ganglia that transmit neural signals to the neuroendocrine bag cells, (2) the bag cells that synthesize and secrete egg-laying hormone (ELH), and (3) the ovotestis that extrudes eggs into the hermaphroditic duct in response to ELH. There was no significant effect of temperature on the ability of ELH to stimulate egg laying, suggesting that temperature does not alter responsiveness of the ovotestis to hormonal stimulation. There was evidence that the bag cells play a secondary role in mediating the effects of temperature on egg-laying behavior, that is, using both bioassays and radioimmunoassay for detection or measurement of ELH, hormone secretion was shown to be inhibited by cooler temperatures in some preparations. However, the inconsistency of this response suggested that the primary effect of temperature is upstream from the bag cells. Results from an experiment in which the head ganglia were chemically stimulated to activate bag cell electrical activity suggested that the primary site of temperature regulation lies within the head ganglia.

Seasonal reproduction in the ewe is generated by an endogenous circannual rhythm of reproductive neuroendocrine activity. Exposure to as few as 70 days of photoperiodic information a year is sufficient to synchronize the rhythm. The present study was conducted to identify which portions of the photoperiodic cycle are utilized for synchronization. For this purpose, we used pinealectomized ewes that could not respond reproductively to changes in day length. Selected photoperiodic information was provided via infusion of melatonin, a hormone that provides the neuroendocrine code for day length in this species. Melatonin was delivered according to circadian patterns. The infusion patterns were tailored to mimic those of melatonin secretion in pineal-intact ewes during one of the four seasons: winter, spring, summer, or autumn. The infusions were provided for 90 days a year during each of the three years following pinealectomy. The ewes were ovariectomized and treated with constant-release Silastic capsules containing estradiol; reproductive neuroendocrine activity was monitored by measurement of serum concentrations of LH. In the absence of exogenous melatonin, most (19 of 24) pinealectomized controls exhibited circannual LH cycles that were not in synchrony, indicating that the rhythm was free-running. Melatonin synchronized the rhythm (such that the period was 365 days and the stages of the rhythm were both concurrent among animals and in appropriate phase with the geophysical year), but not all melatonin patterns were equally effective in this regard. The most effective melatonin patterns mimicked those of secretion during summer. Spring and autumn melatonin patterns were less effective, and winter melatonin patterns were ineffective. These results support the concept that there is a seasonal specificity with regard to the photoperiodic cues that synchronize the circannual rhythm of reproductive neuroendocrine activity in the ewe. The rhythm is synchronized most effectively by long-day photoperiodic cues perceived on or around the summer solstice.

The bag cell neurons of the marine mollusk Aplysia have been used extensively for investigating the electrophysiology and molecular biology of neurosecretory cells. However, there has been little attempt to carefully describe the pattern of secretion of the 36-amino acid peptide hormone that controls egg-laying behavior. This egg-laying hormone (ELH) is secreted in response to a highly characteristic pattern of action potential firing called the afterdischarge. The purpose of this study was to document the pattern of ELH secretion in response to the bag cell afterdischarge. To accomplish this goal, we developed the first RIA for measurement of ELH. The study was conducted in vitro in excised neural preparations that included the bilateral bag cell clusters; preparations were maintained in static culture of filtered artificial sea water (ASW) at 21-22 C. Afterdischarges were electrically stimulated, and action potentials were monitored via extracellular electrodes. The entire volume of ASW was collected every 5 min and immediately replaced with fresh ASW. In all 20 preparations, ELH levels were low to undetectable before stimulation of the afterdischarge; ELH rose above baseline within 5-10 min of the start of the afterdischarge, reached peak levels near the end or after the end of the afterdischarge, and then remained elevated long after action potentials had ceased firing. These results indicate a temporal dissociation between action potential firing and secretion. We suggest that the afterdischarge is important for triggering intracellular events that culminate in peptide release. Importantly, once these secretion-inducing events have been initiated, they can continue in the absence of further action potential stimulation.

Kisspeptin (product of the kiss1 gene) is the most potent known activator of the hypothalamo-pituitary-gonadal axis. Both kiss1 and the kisspeptin receptor are highly expressed in the hypothalamus of vertebrates, and low doses of kisspeptin have a robust and long-lasting stimulatory effect on the rate of action potential firing of hypophysiotropic gonadotropin releasing hormone-1 (GnRH1) neurons in mice. Fish have multiple populations of GnRH neurons distinguished by their location in the brain and the GnRH gene that they express. GnRH3 neurons located in the terminal nerve (TN) associated with the olfactory bulb are neuromodulatory and do not play a direct role in regulating pituitary-gonadal function. In medaka fish, the electrical activity of TN-GnRH3 neurons is modulated by visual cues from conspecifics, and is thought to act as a transmitter of information from the external environment to the central nervous system. TN-GnRH3 neurons also play a role in sexual motivation and arousal states, making them an important population of neurons to study for understanding coordination of complex behaviors. We investigated the role of kisspeptin in regulating electrical activity of TN-GnRH3 neurons in adult medaka. Using electrophysiology in an intact brain preparation, we show that a relatively brief treatment with 100 nM of kisspeptin had a long-lasting stimulatory effect on the electrical activity of an extrahypothalamic population of GnRH neurons. Dose-response analysis suggests a relatively narrow activational range of this neuropeptide. Further, blocking action potential firing with tetrodotoxin and blocking synaptic transmission with a low Ca2+/high Mg2+solution inhibited the stimulatory action of kisspeptin on electrical activity, indicating that kisspeptin is acting indirectly through synaptic regulation to excite TN-GnRH3 neurons. Our findings provide a new perspective on kisspeptin's broader functions within the central nervous system, through its regulation of an extrahypothalamic population of GnRH neurons involved in multiple neuromodulatory functions. © 2012 Zhao, Wayne.

The aim of the present work was to generate information that allows further exploration of mechanisms regulating release of egg-laying hormone (ELH) from neuroendocrine bag cells of Aplysia. To start, experiments that document the pattern of ELH secretion in response to an electrical afterdischarge from excised neural preparations maintained in vitro, as well as from freely-behaving animals arc described. Preliminary studies invcstigating the role of the cAMP second-messenger system in regulating ELH secretion are also discussed. Concentrations of ELH, from either medium (in vitro preparations) or hemolymph (in vivo preparations), were measured by radioimmunoassay. In both types of preparations, the electrical afterdischarge triggered ELH secretion, but peak levels were reached near the end of or following termination of the afterdischarge. All preparations showed prolonged durations of secretion that extended at least 43 min past the end of the afterdischarge, indicating that action potentials arc not required for maintaining secretion of ELH. What cellular processes regulate this prolonged secretion? The cAMP second-messenger system is a likely candidate because of its suggested role in excitation of bag-cell membrane and transport of secretory granules to bag-cell growth cones in culture. Treatment with the specific cAMP-dependent protein kinase (PKA) inhibitor RpcAMPS attenuated the amplitude and duration of ELH secretion, whereas the PKA stimulator Sp-cAMPS had no significant effects on secretion compared to untreated controls. These preliminary results suggest that: 1) PKA plays an important role in regulating ELH release; 2) Afterdischarge-induced PKA maximally stimulates ELH release such that further elevations in PKA do not enhance secretion.

There are multiple populations of gonadotropin releasing hormone (GnRH) neurons that have distinct physiological and behavioral functions. Teleost fish have a population of GnRH3 neurons located in the terminal nerve (TN) associated with the olfactory bulb that is thought to play a neuromodulatory role in multiple physiological systems, including olfactory, visual, and reproductive. We used transgenic zebrafish in which the GnRH3 promoter drives expression of a green fluorescent protein to identify GnRH3 neurons during development in live embryos. Unlike with hypophysiotropic GnRH neurons of zebrafish, TN-GnRH3 neurons are of neural crest origin and are one of the first populations of GnRH neurons to develop in the early embryo. Using a combination of optical imaging and electrophysiology, we showed that during the first three days post-fertilization, TN-GnRH3 neurons increase in number, extend neural projections, move in association with tissue expansion, and acquire an adult-pattern of spontaneous action potential firing. Early during development, about half of the neurons were quiescent/non-firing. Later, at three days post-fertilization, there was an increase in the proportion of neurons showing action potential firing and an increase in the number of neurons that showed an adult-like tonic or beating pattern of action potential firing with a firing frequency similar to that seen in adult TN-GnRH3 neurons. This study represents the first neurophysiological investigation of developing GnRH neurons in live embryos -- an important advance in understanding their potential non-reproductive roles during embryogenesis.

This study examined whether or not the reproductive response of female sheep to photoperiod varies with seasonal reproductive state. The specific objective was to test the hypothesis that the reproductive response to a long-day pattern of melatonin varies with the reproductive state of the ewe. The response examined was the synchronization of reproductive neuroendocrine induction (rise in serum luteinizing hormone, or LH) following nocturnal infusion of melatonin into pinealectomized ewes for 35 consecutive nights. This infusion restored a pattern of circulating melatonin similar to that in pineal-intact ewes maintained in a long photoperiod (LD 16:8). The ewes had been pinealectomized and without melatonin replacement for 16-25 months prior to the study. They were in differing reproductive states at the start of the infusion, as their endogenous reproductive rhythm had become desynchronized among individuals and with respect to time of year. Noninfused pinealectomized ewes served as controls. Regardless of the reproductive state at the start of the 35-day infusion of the long-day pattern of melatonin, all treated ewes exhibited the same reproductive neuroendocrine response after the infusion was ended. This consisted of a synchronized rise in LH some 6-8 weeks after the infusion was terminated, the maintenance of a high level of serum LH for some 15 weeks, and a subsequent precipitous fall in LH to a very low level. These results provide evidence that a long-day pattern of melatonin can synchronize reproductive neuroendocrine induction in the ewe, regardless of reproductive condition, and thus do not support the hypothesis that this response differs with seasonal reproductive state.

The primary purpose of these studies was to determine whether photoperiodic signals could influence seasonal egg-laying behavior in the marine mollusk, Aplysia californica. Egg-laying behavior was monitored from groups of animals that were collected at four times of year and maintained in different temperature and photoperiodic conditions in the laboratory. Animals that were obtained in autumn and kept in warm water laid eggs more frequently than those in cold water, regardless of photoperiod. Furthermore, animals maintained on short days and warm water laid eggs more frequently than those on long days and warm water. Animals in cold water showed little to no egg laying, and a photoperiodic response was not evident. Animals that were collected in either winter or spring and maintained in warm water showed little or no spontaneous egg laying throughout the study, regardless of photoperiod. As with the autumn animals, Aplysia individuals obtained in summer and kept on short days and warm water laid eggs more frequently than those kept on long days and warm water. These results provide the first evidence that the reproductive system of A. californica is responsive to photoperiod. Overall, the data suggest that warm water is permissive for egg laying, and that short days can further stimulate this behavior. However, there is a strong inhibition of spontaneous egg laying during the winter and spring, which neither warm water nor short photoperiod can overcome. The role of the eyes in mediating the photoperiodic response was also investigated. A control group of intact animals kept on short days laid eggs more frequently than those on long days, but this photoperiodic response was not evident in eyeless animals. These results suggest that the eyes play a role in mediating the effects of photoperiod on egg laying behavior.

Gonadotropin-releasing hormone (GnRH) is indispensable for reproductive activation in all vertebrates. Although several GnRH-like molecules have been isolated from non-chordates, the function of GnRH in these taxa remains unclear. We previously isolated the full-length cDNA sequence of a prohormone containing a GnRH-like molecule, termed ap-GnRH, from the gastropod mollusk, Aplysia californica. In this study, we characterized the distribution and quantity of ap-GnRH peptide in several central and peripheral tissues of A. californica. Further, we performed in vivo and in vitro studies to explore the function of ap-GnRH in these animals. Immunohistochemistry and radioimmunoassay using specific antisera against ap-GnRH showed that pedal ganglia contained the highest level of ap-GnRH peptide, followed by cerebral ganglia, abdominal ganglia, and then buccal ganglia. Ovotestis did not contain detectable levels of ap-GnRH peptide. Injection of sexually mature and immature animals with synthetic ap-GnRH over a course of 10-14 days had no effects on ovotestis mass, reproductive tract mass, egg-laying, and penile eversion. ap-GnRH also failed to alter oocyte growth and egg-laying hormone accumulation and secretion. Interestingly, ap-GnRH injection triggered acute behavioral responses including the stimulation of parapodial opening, inhibition of feeding, and promotion of substrate attachment. Our results showed that in A. californica, ap-GnRH could modulate a wide range of behavioral attributes. Most strikingly, ap-GnRH is not involved in the acute activation of reproduction in a fashion similar to vertebrate GnRH.

There are multiple populations of gonadotropin-releasing hormone (GnRH) neurons in the brains of vertebrates. The population located in the hypothalamus/preoptic area is the best studied and is known to ultimately control reproduction. Teleost fish have an additional population of GnRH neurons in the terminal nerve (TN) associated with the olfactory bulbs, the physiological function of which is still unclear. Anatomical and physiological studies provide evidence that TN-GnRH neurons have extensive projections in the brain and modulate neuronal activity. Although there is anatomical evidence that the TN receives olfactory and optic sensory inputs, it is not known if sensory information is transmitted to TN-GnRH neurons to modulate their activity. In the present study, we tested the hypothesis that social cues from conspecifics modulate electrical activity of TN-GnRH neurons from the intact brain of female medaka fish ( Oryzias latipes). We further investigated the potential roles of chemosensory and visual signals in mediating the social cue response. We used a transgenic line of medaka with TN-GnRH neurons genetically tagged with green fluorescent protein, allowing visualization of specific neurons for whole-cell current clamp electrophysiology. We demonstrated that 24-h exposure to male visual and chemosensory cues suppressed the electrical activity of female TN-GnRH neurons compared with exposure to other females. Chemosensory cues alone were insufficient to induce this social cue response. However, visual cues alone replicated the “combined” social cue response. These findings support our hypothesis that sensory signals—and specifically, visual social cues—modulate electrical activity of TN-GnRH neurons.

For over 20 years, the bag cell neurons of the marine mollusk Aplysia have been used to investigate second-messenger pathways that mediate effects of synaptic stimulation on ion currents and membrane excitability, presumably leading to exocytotic release of the neuropeptide egg-laying hormone (ELH). It is widely cited that a train of action potentials, called an afterdischarge, is necessary for activating cellular events leading to ELH secretion. Using a combination of electrophysiology, optical imaging of calcium signaling, and radioimmunoassay of ELH secretion, we show that an afterdischarge is not required for ELH secretion. Electrical stimulation that failed to produce afterdischarges but did lead to prolonged membrane depolarization and a rise in intracellular calcium concentration was sufficient to stimulate significant ELH release.

Local protein synthesis in neuronal processes is a common phenomenon and may play an important role in synaptic plasticity and hormonal regulation. We have used neuroendocrine bag cells of Aplysia californica as a model system to study local protein synthesis. In our previous work we found that bag cell neurites are capable of synthesizing and processing the prohormone of egg-laying hormone (pro-ELH). In the present study, we found that bag cell neurites are also capable of releasing locally synthesized pro-ELH and ELH-related products via both constitutive and regulated pathways. However, an electrical afterdischarge did not enhance local pro-ELH synthesis, as it does in the bag cell soma. This is the first evidence that isolated neurites are capable of secreting locally synthesized proteins.

Membrane depolarization plays a critical role in stimulating secretion of neuropeptides and can also be important in regulating transcriptional and translational events that control peptide biosynthesis. The purpose of this study was to test the hypothesis that persistent membrane depolarization after the end of an electrical afterdischarge plays an important role in stimulating both prolonged secretion of egg-laying hormone (ELH) and ELH synthesis from peptidergic bag cell neurons of Aplysia. Experimental preparations were treated with a low Na(+) solution to rapidly repolarize membrane potential (Vm). Compared with control preparations, the low Na(+) solution caused rapid membrane repolarization to resting levels, significant shortening of the duration of the afterdischarge, and significant decrease in the decay time constant of cytosolic Ca(2+) ([Ca(2+)](i)) concentrations, but no effect on peak [Ca(2+)](i), total [Ca(2+)](i) above baseline, or duration of elevated [Ca(2+)](i). Contrary to both theoretical expectations and findings in other cell types, low Na(+) treatment and the resulting premature repolarization of Vm did not inhibit ELH secretion. On the other hand, low Na(+) treatment that blocked prolonged depolarization, as well as inhibition of Ca(2+) influx, prevented the afterdischarge-induced stimulation of ELH synthesis. These findings provide support for membrane depolarization acting as a trigger mechanism, rather than a sustained driving force, for cellular events that control ELH secretion. It also demonstrates, for the first time, a critical role for postdischarge Vm in regulating an important aspect of neuroendocrine cell function-that of hormone synthesis.

The bag cell neurons (BCNs) of the mollusk Aplysia californica provide a simple model system for investigating cellular and molecular events regulating synthesis and secretion of a reproductive neuropeptide and their impact on physiology and behavior. The BCNs secrete a large amount of egg-laying hormone (ELH) in response to an electrical afterdischarge. The afterdischarge also triggers cellular and molecular events leading to upregulation of ELH biosynthesis to replenish the supply of releasable hormone that was lost because of secretion. In the present review, we discuss signal-transduction events that link membrane excitability to ELH biosynthesis. We present evidence that the afterdischarge stimulates ELH synthesis by upregulating translation of ELH mRNA rather than by activating ELH gene transcription. This increase in ELH synthesis is accompanied by a decrease in total protein synthesis, suggesting that the synthetic machinery is being funneled selectively toward ELH. We also discuss work showing that afterdischarge-induced ELH synthesis uses a novel mechanism of translation initiation, one involving a switch from cap-dependent to cap-independent translation initiation that activates an internal ribosome entry site (IRES) located in the 5'-untranslated region of ELH mRNA. The IRES-regulated translation provides a unique cellular mechanism to selectively upregulate synthesis of a critical reproductive hormone at the expense of nonessential proteins.

The purpose of this work was to test the hypothesis that an electrical afterdischarge (AD) causes prolonged elevation in cytosolic calcium levels that is associated with prolonged secretion of egg-laying hormone (ELH) from peptidergic neurons in intact nervous tissue of Aplysia. Using a combination of radioimmunoassay measurement of ELH secretion, electrophysiological measurement of membrane potential, and optical imaging of the concentration of intracellular free calcium ions ([Ca(2+)](i)), we verified that there was persistent secretion of ELH after the end of the AD; this was accompanied by prolonged post-AD membrane depolarization and prolonged post-AD elevation in [Ca(2+)](i). Extracellular treatment with the calcium chelator EGTA had no effect on the pattern or magnitude of ELH secretion or on the post-AD membrane potential (V(m)) and post-AD Ca(2+) signal, ruling out a role for extracellular calcium in the post-AD elevation of [Ca(2+)](i). BothV(m) and [Ca(2+)](i) returned to baseline well before ELH secretion, such that neither prolonged membrane depolarization nor prolonged Ca(2+) signaling can fully account for the extent of the persistent secretion of ELH. These findings suggest a unique relationship between membrane excitability, Ca(2+) signaling, and prolonged neuropeptide secretion.

The bilateral suprachiasmatic nuclei (SCN) of the hypothalamus is the location of the primary circadian clock in mammals. Humoral and/or neuronal outputs from the SCN drive rhythms in behavioral, physiological, and biochemical functions. Other chapters in this textbook have discussed circadian rhythmicity of hormone secretion, reflecting the output of the SCN to various neuroendocrine systems. The focus of this chapter will consider evidence that neurohormones can feed back on the SCN and modulate circadian clock function.

The purpose of this work was to investigate the effects of electrical afterdischarge on protein kinase C (PKC) activity from bag cell neurons (BCNs) of Aplysia. Bilateral clusters of BCNs were divided: one cluster was stimulated to afterdischarge, the other was a control. Clusters were processed for PKC activity assay 5-120 min after electrical stimulation. Afterdischarge triggered a rapid and persistent increase in both calcium-activated and calcium-independent PKC activity.

The role of Ca2+ from extracellular and intracellular sources in stimulating neurosecretion was investigated in four experiments using neuroendocrine bag cells of the marine mollusk Aplysia. (i) Bag cells were treated with either an extracellular calcium chelator (BAPTA) or Co(2+)-substitution within 30 s after onset of an electrical afterdischarge to prevent influx of Ca2+ from extracellular fluid. These treatments shortened the duration of the afterdischarge, but did not significantly affect the overall pattern or total amount of egg laying hormone (ELH) secretion, suggesting that extracellular Ca2+ is not required for maintenance of ELH release. (ii) Substitution of Ba2+ for Ca2+ has previously been shown to support bag cell afterdischarges that trigger transient elevations in intracellular Ca2+. We showed that this treatment also stimulates ELH secretion, suggesting that Ca2+ release from intracellular stores can stimulate ELH secretion. (iii) To raise intracellular Ca2+ levels in the absence of an afterdischarge, the calcium ionophore X537A was used to transport Ca2+ across plasma and organelle membranes. When this treatment was combined with extracellular calcium chelators so that the only source of Ca2+ was from intracellular compartments, ELH secretion was stimulated. Taken together, these findings are consistent with the hypothesis that release of Ca2+ from intracellular stores is sufficient to stimulate ELH secretion.

The insulin receptor is a tyrosine kinase receptor that is found in mammalian brain and at high concentrations in the bag cell neurons of Aplysia. We show here that insulin causes an acute rise in intracellular Ca2+ concentration ([Ca2+]i) in these neurons and triggers release of neuropeptide. The insulin-sensitive intracellular Ca2+ pool differs pharmacologically from previously described Ca2+ stores that are sensitive to inositol trisphosphate and from mitochondrial Ca2+ stores. Insulin, but not thapsigargin, stimulates Ca2+ release at the distal tips of neurites, the presumed site of neuropeptide secretion. The effects of insulin on intracellular Ca2+ release and neuropeptide secretion occur without triggering spontaneous action potentials. The insulin-sensitive rise in [Ca2+]i moves into the distal tips of neurites after exposure to a cyclic AMP analogue, a treatment that causes a similar translocation of neuronal vesicles. Our data indicate that Ca2+ release from a distinct intracellular pool associated with secretory vesicles may contribute to secretion of neuropeptide in the absence of neuronal discharge.

The neuroendocrine bag cells are an attractive model system for investigating cellular mechanisms mediating the effects of membrane excitability on hormone secretion because of the ability to easily monitor their electrical and secretory functions simultaneously. We investigated the role of extracellular calcium in regulating prolonged release of egg-laying hormone from bag cells following stimulation of an electrical afterdischarge. We report that calcium influx is required for initiation of the afterdischarge, but is not required for prolonged hormone secretion once the afterdischarge has been triggered.