Kathleen Dunlap - US grants

The goal of this research is to understand the control of bioluminescence in the hydrozoan coelenterate, Obelia, as part of a more general understanding of how epithelial action potentials control effector responses in lower invertebrate species. In this animal all of the behavioral responses are under the control of action potentials in electrically coupled epithelial cells, rather than under neural control as in higher animals. Luminescence in these organisms results from the activation of a calcium-dependent photoprotein which provides an endogenous intracellular calcium indicator. Previous experiments using a combination of patch clamp analysis and video image analysis have shown that calcium entering nonluminescent epithelial cells during an action potential results in the passage of a chemical signal into the luminescent cells via gap junctions. This signal (hypothesized to be calcium, itself) then results in activation of the intracellular photoprotein. Experiments are proposed which will establish the identity of the chemical signal. In addition to the demonstration that luminescence is triggered as the result of a chemical signal passing across gap junctions, other experiments have disclosed the existance of a novel type of voltage independent calcium channel within the luminescent cell itself. Like the initiation of luminescence, the activation of this current is also dependent on chemical signalling through the gap junction. In further studies, whole cell and single channel recording techniques will be used to characterize this channel which represents a novel mechanism of calcium entry across the plasma membrane. This research on the physiological mechanism controlling light emission by a luminescent marine organism has already produced important insights into the function of junctions that mediate communication between cells. "Gap junctions" are structurally specialized appositions between cells and are found in many tissues of animals at all levels of complexity. Much indirect evidence has led to the belief that these junctions serve in transmission of chemical signals between cells. The results of this research have provided the first clear demonstration that chemical signal transmission across gap junctions is involved in an important physiological function. In addition, a novel type of channel for calcium entry into cells has been discovered. A continuation of the study of this interesting system will surely expand our understanding of the control of light emission by these small marine organisms, but this study is also likely to provide further fundamental insights into the general mechanisms of cellular communication.

In recent years a unique form of neurotransmitter action has been described which does not result from a transmitter-induced opening of a membrane channel. Rather, it results from a modulation by the transmitter of a channel opened by voltage. A role for this form of neurotransmitter action was first implicated in the positive inotropic and chronotropic effects of nor-adrenaline on cardiac muscle. Since that time, neurotransmitter modulation of voltage-dependent channels has been described for many tissues. In particular, this type of transmitter effect may be involved in presynaptic inhibition of sensory neurotransmission in the spinal cord. This proposal investigates this phenomenon in embryonic chick sensory neurons in vivo and in vitro. Specifically, the mechanism by which neurotransmitters (e.g., GABA and nor-epinephrine) decrease the voltage-dependant Ca current in the cell bodies of the neurons will be investigated using voltage clamp and single channel recording techniques. The experiments are designed to test the involvement of cyclic nucleotides and intracellular Ca in channel modulation and to gain information about their roles in the regulation of neurotransmitter release from sensory neurons. Tests of hypotheses concerning the mechanism of pre-synaptic inhibition will also be performed both in hemisected spinal cord preparations and at identified synapses between sensory neurons and spinal cord cells grown together in culture. These studies will make use of the observation that two pharmacologically and functionally distinct GABA receptors exist on embryonic chick sensory neurons. The specific agonists for these receptors will allow a discrimination between two proposed hypotheses for presynaptic inhibition in the spinal cord.

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

The importance of voltage-dependent calcium channels in cellular processes is indisputable. Their pivotal role in controlling such functions as muscle contraction, neurosecretion, and membrane permeability has made calcium channels a natural target for both extracellular and intracellular modulatory agents. Despite the variety of modulators that have been identified and the prevalence of the modulation, the fundamental mechanisms responsible for controlling calcium channel function have not been adequately described. Impediments to our understanding have derived, at least in part, from the inadequacy of early classification schemes to effectively discriminate between the different calcium channel subtypes and a reluctance to treat calcium channel modulation as a potentially inhomogeneous process. Experiments in this application will investigate the mechanisms underlying transmitter-induced inhibition of one type of high voltage-activated calcium channels (N-type) in embryonic chick sensory neurons. Preliminary results presented in the proposal demonstrate that the modulation produced by GABA can be separated into two components (not previously recognized in the calcium channel field). These can be distinguished from one another on the basis of their transmitter concentration-dependence, time course, and voltage-dependence. We will test the hypothesis that the two components are produced by separate mechanisms. Experiments are proposed to dissect and characterize the components biophysically (Aim 1) and to investigate whether they involve the activation of different GTP-binding proteins (Aim 2) and/or protein kinase C (Aim 3). Differences in the underlying mechanisms and/or the relative contributions of these two distinct components may explain a number of discrepancies surrounding the modulation that have been reported in the calcium channel literature. Experiments in Aim 4 will determine whether this is, in fact, the case comparing selected aspects of the modulation in two different, commonly-studied preparations.. In Aim 5, we will test the extent of conservation of the mechanisms that underlie N-type calcium channel modulation by determining whether they also characterize the modulation of other high voltage-activated calcium channel types. In these experiments, we will examine the inhibition of non-N-type channels in chromaffin cells. As specific calcium-dependent cellular processes are likely to be triggered by particular calcium channel subtypes, an in-depth understanding of the varied mechanisms by which the subtypes are controlled win lead to the development of methods that can differentially target (and alter) individual cellular functions. Such an achievement may, ultimately, allow sophisticated clinical interventions into a wide variety of pathological conditions, particularly in the cardiovascular, neurological, and endocrine systems.

This study proposes to investigate sleep patterns in patients with chronic pelvic pain in order to document and quantitate sleep disorders. The patient with chronic pelvic pain presents a difficult dilemna to the practicing gynecologist. A significant relationship between the complaint of chronic pelvic pain and abnormal sleep patterns has been described. If sleep patterns are abnormal, then pharmacological interventions such as low dose tricyclics or serotonin reuptake inhibitors may be useful.

Estrogen protects the cardiovascular system from injury by complex mechanisms that have both short- and long-term components. Longer- term estrogen actions occurs following hours of estrogen exposure and is mediated by estrogen receptors acting in the nucleus as transcription factors for growth-related genes. The rapid effects of estrogen, by contrast, occurs in a seconds-to-minutes time frame and do not require gene transcription. Project 5 of the SCOR application addresses the molecular mechanisms underlying one of the most important of these rapid effects-vasodilation. The signaling pathways underlying this non- genomic action of estrogen are incompletely understood but new data support they involve the action of nitric oxide synthase in vascular cells, elevation of nitric oxide, stimulation of vascular smooth muscle cell soluble guanylyl cyclase, and substrate phosphorylation by cGMP- dependent protein kinase (PKG). Although the key substrate(s) for PKG is unknown, new data support one likely possibility is the smooth muscle large conductance Ca2+-activated K+ (BKCa) channel. Blockade of BKCa channels in vascular rings significantly reduces estrogen-induced vasodilation, demonstrating the central role that BKCa channels in vascular rings significantly reduces estrogen-induced vasodilation, demonstrating the central role that BKCa channels play in the acute physiological response to estrogen. Experiments in Aim 1 will use BKCa channels in vascular rings significantly reduces estrogen-induced vasodilation, demonstrating the central role that BKCa channels play in the acute physiological response to estrogen. Experiments in Aim 1 will use BKCa channel activity recorded from single smooth muscle cells dissociated from mouse aorta as an assay for characterizing upstream elements in the estrogen signaling pathway that targets BKCa channels. Experiments will assess the relative contributions of endothelial and smooth muscle cells, the types of estrogen receptor involved, and the requirement for nitric oxide synthase activation. Experiments in Aim 2 will focus on the molecular mechanism by which BKCa channel activity is regulated by estrogen. We will identify the molecular variants of BKCa channel activity is regulated by estrogen. We will identify the molecular variants. variants of BKCa channels that are expressed in smooth muscles cells from mouse aorta and study the recombinant channels expressed heterologously in order to test the hypothesis that estrogen brings about BKCa channel activation through a direct PKG-dependent phosphorylation of the channel protein. Such information is essential to further understand the protective effects of estrogen on vascular tissue and may form the basis for development of new therapies for cardiovascular disease.

DESCRIPTION:(adapted from applicant's abstract) Naturally-occurring mutations in the gene encoding class A (or P/Q-type) calcium channels are associated with multiple abnormalities, ranging from migraine headache to motor ataxias to absence epileptic seizures. These heterogeneous neurological phenotypes underscore the central importance of P/Q-type calcium channels-the dominant exocytotic channels in central nervous system. P/Q is not, however, the only type of calcium channel controlling synaptic transmission in the CNS. N-type (or class B) calcium channels usually co-exist with P/Q and, together, they jointly govern the release of many, if not all, transmitters. Whether P/Q and N channels play unique functional roles at the synapse is unclear. Experiments with one P/Q channel mutant mouse, tottering, suggest, however, that the two channels are not functionally redundant and that tottering offers an opportunity to explore their different roles in exocytosis. Homozygous tottering animals display a dramatic neurological phenotype, characterized by ataxia and frequent absence seizures. Our preliminary experiments on tottering demonstrate that a primary consequence of the P/Q channel mutation is a shift in the ratio of P/Q:N channels in some (but not all) nerve terminals. For example, release of the excitatory transmitter glutamate and glutamatergic synaptic transmission at the parallel fiber-Purkinje cell synapse in cerebellum are controlled largely by N-type calcium channels in the mutant, rather than P/Q-type as they are in wild-type animals. As a consequence of these changes in the presynaptic calcium channel complement, excitatory transmission is reduced and G protein-dependent inhibition is enhanced at mutant synapses. In contrast, GABA release from inhibitory nerve terminals appears to be unaffected in tottering animals. On the basis of these observations, we hypothesize that the selective effect of the tottering allele on excitatory transmission leads to an overall decreased excitation of Purkinje cells. Three interacting factors contribute: 1) glutamate release from excitatory inputs is impaired due to the decreased involvement of P/Q channels; 2) the relative increase in N channel-mediated release further enhances susceptibility of these inputs to presynaptic inhibition (because N channels are more effectively modulated by G proteins than are P/Q channels); and 3) unimpaired inhibitory, GABAergic inputs are relatively more efficacious in the face of reduced excitation. As Purkinje cells control cerebellar output via GABAergic inhibitory transmission onto output neurons in deep cerebellar nuclei, we predict that a reduction in Purkinje cell activity will enhance net cerebellar output. Ultimately, such changes would excite thalamus and motor cortex, providing a plausible mechanism for the ataxia and seizures observed in these animals. Experiments proposed here will stringently test this hypothesis through in-depth cellular and synaptic exploration of calcium channels and calcium-dependent exocytosis in tottering cerebellum. Results will provide essential information for understanding the consequences of the mutation on cerebellar circuit behavior and may, in the long term, offer suggestions for new therapeutic interventions into ataxia and other motor disorders.

DESCRIPTION (provided by applicant): The failure of pancreatic islets of Langerhans to supply insulin in sufficient quantities to maintain blood glucose within physiological limits underlies Type 1 (insulin-dependent) and Type 2 (insulin-independent) diabetes mellitus. Although great strides have been made in the treatment of diabetes since the discoveries of insulin and the insulin receptor, the clinical management of these disorders remains a challenge even under the best of circumstances. As most forms of diabetes are precipitated and/or exacerbated by a reduction in numbers of insulin-secreting beta cells, developing methods that promote beta cell proliferation may lead to the development of new therapies for the estimated 200 million individuals world-wide who live with diabetes. A number of islet growth factors have been reported; many of these are known islet secretagogues as well (e.g., glucose, amino acids), suggesting the possibility that factors released from beta cells act as feedback regulators of islet function. Our preliminary studies suggest that ?-aminobutyric acid (GAGA), long known to be synthesized, stored, and secreted by beta cells, is such a factor. We demonstrate that GABA's effects on islets are mediated through metabotropic GABAB receptors. In contrast to GABA's reputation as a fast inhibitory neurotransmitter, its proliferative action on islets is rather slow (requiring hours); further, GABA appears to work in concert with insulin to stimulate islet proliferation, as its effects are blocked by inhibitors of insulin receptor signaling. Such studies predict that, in the long term, GABA enhances the insulin-releasing capacity of the pancreas. We further demonstrate that, paradoxically, GABA also acts rapidly (seconds to minutes) on islets to inhibit insulin secretion from beta cells. Thus, we suggest the hypothesis that GABA is an endogenous biphasic regulator of islet function. A putative physiological rationale for such dual regulation is that GABA may serve to limit islet proliferation during short exposure to glucose and to encourage it under conditions of frequent or persistent elevations in glucose. Experiments outlined in three Aims will explore this novel hypothesis by defining which GABAB receptor isoforms are expressed in islets and characterizing the signaling pathways underlying their two actions. Understanding the natural biological mechanisms by which islets are regulated is prerequisite to harnessing them for the development of new treatment strategies for diabetes.

This Core is not based on an existing Core and represents a novel concept that is likely to increase the research capabilities of a large number of Neuroscience Investigators as well as promote collaborations. The goal of this Core is to provide equipment for electrophysiological experiments as well as the technical capability for performing the experiments. This will allow Neuroscience investigators to carry out preliminary experiments that will test the feasibility of applying electrophysiological approaches to ongoing projects. In addition, the Core will provide a well-defined venue for Neuroscience Investigators to obtain expert advice regarding establishing electrophysiological methods in their own laboratories. This Core will be situated adjacent to the laboratories of the Director and Co-Directors (Dunlap; Blaustein & Cox). A key component of the Core that will be critical for its success is the hiring of an expert electrophysiologist. The equipment available will allow a variety of recording techniques including whole cell current and voltage clamp, single channel recording, two electrode voltage clamp, tissue slice single cell current and voltage clamp and extracellular field recording. As currently proposed, the Core would eventually have 2 rigs, the 2nd one being purchased in the 2nd year and dedicated primarily for slice electrophysiology. While the idea of having an electrophysiology core is well regarded, it is important to have realistic goals for the core. The nuances associated with slice recording that are unique to each preparation raise questions about the feasibility of incorporating slice electrophysiology into the Core. The goals of the 1st rig seem more realistic and feasible to achieve with the hiring of a well trained electrophysiologist.

DESCRIPTION (provided by applicant): Multidisciplinary approaches drive progress in the neurosciences perhaps more than in most other areas of biomedical research. The next generation of research neuroscientists, therefore, must be equipped with a multi-faceted skill set if they are to transition to successful independent careers and contribute meaningfully to the field. To meet this challenge, we propose a new predoctoral Synapse Neurobiology Training Program (SNTP) at Tufts University School of Medicine that will provide in-depth, multidisciplinary research education of 4 trainees in the area of synaptic function-a particular strength of the Tufts neuroscience faculty. The synapse forms the foundation of nervous system function, and research on synapses is, arguably, one of the most interdisciplinary areas in modern neurobiology. The SNTP training plan, thus, includes several mechanisms that position trainees to become leading neuroscientists, working at the interface between traditional disciplines: Each trainee will be co-mentored by two SNTP faculty members who provide training in distinct yet complementary areas. Through subsidized, one-on-one training in imaging, bioinformatics, electrophysiology, and animal behavior methods (provided via the core facilities and PhD-level Managers in the NINDS-funded Center for Neuroscience Research at Tufts), SNTP trainees will acquire the state-of-the-art tools and training required for an effective and influential multidisciplinary approach. Innovative quantitative skills and techniques courses and individualized training plans will further aid each SNTP trainee in mastering the necessary skills to accomplish his/her research career goals. Frequent opportunities for individual interactions with visiting speakers will provide SNTP trainees with further exposure to new methodologies and ideas as well as advice and guidance.