Mental health and well-being of unpaid caregivers: a cross-sectional survey protocol.

INTRODUCTION: Unpaid caregiving, care provided by family/friends, is a public health issue of increasing importance. COVID-19 worsened the mental health conditions of unpaid caregivers, increasing substance/drug use and early development of chronic disease. The impact of the intersections of race and ethnicity, sex, age and gender along with unpaid care work and caregivers' health and well-being is unknown. The aim of this study is to describe the inequities of caregiver well-being across the intersections of race and ethnicity, sex, age and gender using a cross-sectional survey design. METHODS AND ANALYSIS: We are collaborating with unpaid caregivers and community organisations to recruit a non-probability sample of unpaid caregivers over 18 years of age (n=525). Recruitment will focus on a target sample of 305 South Asian, Chinese and Black people living in Canada, who represent 60% of the Canadian racial and ethnic populations. The following surveys will be combined into one survey: Participant Demographic Form, Caregiver Well-Being Index, interRAI Self-report of Carer Needs and the GENESIS (GENdEr and Sex DetermInantS of Cardiovascular Disease: From Bench to Beyond-Premature Acute Coronary Syndrome) PRAXY Questionnaire. Sample characteristics will be summarised using descriptive statistics. The scores from the Caregiver Well-Being Index will be dichotomised into fair/poor and good/excellent. A two-stage analytical strategy will be undertaken using logistic regression to model fair/poor well-being and good/excellent well-being according to the following axes of difference set a priori: sex, race and ethnicity, gender identity, age, gender relations, gender roles and institutionalised gender. The first stage of analysis will model the main effects of each factor and in the second stage of analysis, interaction terms will be added to each model. ETHICS AND DISSEMINATION: The University of Toronto's Health Sciences Research Ethics Board granted approval on 9 August 2022 (protocol number: 42609). Knowledge will be disseminated in pamphlets/infographics/email listservs/newsletters and journal articles, conference presentation and public forums, social media and through the study website. TRIAL REGISTRATION NUMBER: This is registered in the Open Sciences Framework with a Registration DOI as follows: https://doi.org/10.17605/OSF.IO/PB9TD.

Impact of gender, organized athletics, and video gaming on driving skills in novice drivers.

Given that novice drivers tend to be young, and teenagers and young adult drivers are involved in the greatest number of accidents, it is important that we understand what factors impact the driving skills of this population of drivers. The primary aim of the present study was to understand the impact of gender, organized athletics, and video gaming on driving skills of novice drivers under real-world driving conditions. Novice driving students having less than five hours driving experience previous to a normal driving lesson were evaluated on their self-confidence (self-reported) prior to the lesson and driving skill evaluated by their instructor during the course of the lesson. Information was collected about gender, age, whether or not the students were involved in organized athletics, and the extent of their video game playing. There was no impact of gender or extent of video game playing on driving skills. Females were significantly less self-confident with driving than males, but this did not translate to gender differences in driving skills. Being involved in organized athletics-either currently or in the past-significantly enhanced driving skills in both females and males. Finally, novice drivers' age was negatively correlated with driving skills. That is, younger novice drivers (especially males) had better driving skills than older novice drivers. This is counter to popular belief that young drivers lack technical driving skills because they have less experience behind the wheel. Based on the results of the current study, we hypothesize that the relatively high accident rate of younger drivers (especially male drivers) is most likely due to inattention to safety considerations rather than lack of technical driving ability.

Morphological and Physiological Interactions Between GnRH3 and Hypocretin/Orexin Neuronal Systems in Zebrafish (Danio rerio).

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.

Actions of Bisphenol A and Bisphenol S on the Reproductive Neuroendocrine System During Early Development in Zebrafish.

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, lhß, fshß, 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.

Kisspeptins modulate the biology of multiple populations of gonadotropin-releasing hormone neurons during embryogenesis and adulthood in zebrafish (Danio rerio).

Kisspeptin1 (product of the Kiss1 gene) is the key neuropeptide that gates puberty and maintains fertility by regulating the gonadotropin-releasing hormone (GnRH) neuronal system in mammals. Inactivating mutations in Kiss1 and the kisspeptin receptor (GPR54/Kiss1r) are associated with pubertal failure and infertility. Kiss2, a paralogous gene for kiss1, has been recently identified in several vertebrates including zebrafish. Using our transgenic zebrafish model system in which the GnRH3 promoter drives expression of emerald green fluorescent protein, we investigated the effects of kisspeptins on development of the GnRH neuronal system during embryogenesis and on electrical activity during adulthood. Quantitative PCR showed detectable levels of kiss1 and kiss2 mRNA by 1 day post fertilization, increasing throughout embryonic and larval development. Early treatment with Kiss1 or Kiss2 showed that both kisspeptins stimulated proliferation of trigeminal GnRH3 neurons located in the peripheral nervous system. However, only Kiss1, but not Kiss2, stimulated proliferation of terminal nerve and hypothalamic populations of GnRH3 neurons in the central nervous system. Immunohistochemical analysis of synaptic vesicle protein 2 suggested that Kiss1, but not Kiss2, increased synaptic contacts on the cell body and along the terminal nerve-GnRH3 neuronal processes during embryogenesis. In intact brain of adult zebrafish, whole-cell patch clamp recordings of GnRH3 neurons from the preoptic area and hypothalamus revealed opposite effects of Kiss1 and Kiss2 on spontaneous action potential firing frequency and membrane potential. Kiss1 increased spike frequency and depolarized membrane potential, whereas Kiss2 suppressed spike frequency and hyperpolarized membrane potential. We conclude that in zebrafish, Kiss1 is the primary stimulator of GnRH3 neuronal development in the embryo and an activator of stimulating hypophysiotropic neuron activities in the adult, while Kiss2 plays an additional role in stimulating embryonic development of the trigeminal neuronal population, but is an RFamide that inhibits electrical activity of hypophysiotropic GnRH3 neurons in the adult.

Early development of the gonadotropin-releasing hormone neuronal network in transgenic zebrafish.

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 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 h 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.

Recording electrical activity from identified neurons in the intact brain of transgenic fish.

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).

Effects of kisspeptin1 on electrical activity of an extrahypothalamic population of gonadotropin-releasing hormone neurons in medaka (Oryzias latipes).

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 Ca(2+)/high Mg(2+) 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.

Gender differences in leadership amongst first-year medical students in the small-group setting.

PURPOSE: 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. METHOD: 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. RESULTS: 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. CONCLUSIONS: 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.

Acquisition of spontaneous electrical activity during embryonic development of gonadotropin-releasing hormone-3 neurons located in the terminal nerve of transgenic zebrafish (Danio rerio).

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 3 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 3 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 advancement in understanding their potential non-reproductive roles during embryogenesis.

Gonadotropin-releasing hormone-like molecule is not an acute reproductive activator in the gastropod, Aplysia californica.

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.

Social cues from conspecifics alter electrical activity of gonadotropin-releasing hormone neurons in the terminal nerve via visual signals.

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.

Impact of bisphenol-A on early embryonic development and reproductive maturation.

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 24h 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.

Beta-endorphin alters electrical activity of gonadotropin releasing hormone neurons located in the terminal nerve of the teleost medaka (Oryzias latipes).

Endogenous opioid peptides (EOPs) are an important class of modulators of the hypothalamo-pituitary axis; treatment with opiates leads to inhibition of GnRH and LH secretion and suppression of reproductive functions. However, little work has been done to investigate the effect of opiates on the electrical activity of GnRH neurons, which ultimately controls GnRH secretion. The purpose of the present study was to investigate the effects of the EOP beta-endorphin on electrical activity of GnRH neurons located in the terminal nerve (TN) associated with the olfactory bulb. We used an excised intact brain preparation from transgenic medaka in which green fluorescent protein (GFP) is genetically expressed in TN-GnRH neurons. These GFP-expressing neurons were then targeted for whole-cell current clamp recordings. Treatment with beta-endorphin led to changes in several characteristics of electrical activity, including depolarization of membrane potential and a decrease in spike amplitude--similar to that observed in response to depolarizing high K(+) treatment. This finding suggests a model in which beta-endorphin depolarizes membrane potential leading to Na(+)-channel inactivation, and subsequent suppression of action-potential amplitude. On the other hand, beta-endorphin had no effect on membrane potential in synaptically isolated GnRH neurons. These results suggest that beta-endorphin is acting indirectly on TN-GnRH neurons to inhibit action potential firing.

Whole-cell electrophysiology of gonadotropin-releasing hormone neurons that express green fluorescent protein in the terminal nerve of transgenic medaka (Oryzias latipes).

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-GnRH3-GFP 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+ 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.

Synaptic stimulation of Aplysia peptidergic neurons can activate hormone secretion in the absence of an afterdischarge.

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.

Secretion of locally synthesized neurohormone from neurites of peptidergic neurons.

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.

Activity-dependent regulation of neurohormone synthesis and its impact on reproductive behavior in aplysia.

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.

Post-afterdischarge depolarization does not stimulate prolonged neurohormone secretion but is required for activity-dependent stimulation of neurohormone biosynthesis from peptidergic neurons.

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.