Richard I. Hume - US grants

During neural development many more synapses are formed than are maintained. The long range goal of this research is to understand the mechanisms that determine which synaptic terminals will be maintained, and which eliminated. In this study an in vitro system, which allows for detailed examinations of the organizations and development of synaptic connections, will be used. The major experimental approach will be to use intracellular recording and staining to map the termination of presynaptic neurons onto the surface of dissociated chick sympathetic ganglion neurons, and then to examine in detail the properties of transmission at individual synaptic sites. Cultures will be studied at time points beginning soon after co-culture in order to determine hiw the distribution and the function of terminals change during development. The developmental sequence will be verified by folloiwng individual neurons in long term microcultures. Finally, the growth conditions will be manipulated in order to test possible rules governing synaptic organization. Issues that will be adddressed include the importance of competitive interactions and of pre- and postsynaptic impulse activity. This study will add to our understanding of the steps that lead to stable synaptic transmission. Knowledge of the basic mechanisms that regulate synaptic connections is likely to be relevant to understanding degenerative neurological diseases in which synaptic function is impaired or disrupted.

Adenosine Triphosphate (ATP) is present in many types of synaptic vesicles, but at most synapses it has not known function. The recent discovery that chick skeletal muscles fibers are excited by ATP (Kolb and Wakelam, 1983; Hume and Honig, 1986) provides an excellent opportunity for examining the role of ATP at synapses, since the neuromuscular junction is by far the best characterized synapse, both in maturity and during development. Muscle cells characterized synapse, both in maturity and during development. Muscle cells in culture are very accessible for electrophysiological and biochemical studies, so these observations also open the way for detailed studies of the cellular and molecular basis of ATP mediated excitation. The specific aims of this project are: 1. To resolve conflicting results regarding the nature of the single channels that underlie the ATP evoked depolarization. 2. To determine whether a second messenger system is involved in the ATP evoked depolarization. 3. To examine the time course and mechanism of the long term desensitization that this response displays. 4. To test the hypothesis that receptor activation involves an extracellular phosphorylation. 5. To determine any functional role that ATP receptors play in neuromuscular transmission or in muscle development. These experiments are designed to further understanding of the function of a receptor found on muscle precursor cells and young muscle cells. The early appearance of this receptor suggests that it may play a role in the development of muscle cells or in the formation of neuromuscular junctions. Knowledge of the cellular interactions that control normal development would provide a basis for understanding, and perhaps ameliorating, medically relevant deficits in nerve and muscle function.

This award provides funds for the establishment of a Research Training Group in Development of the Nervous System. The faculty group is a mixture of outstanding senior and junior investigators who come from a variety of disciplinary backgrounds and work with a variety of organisms, but share a common interest in development of the nervous system. The research programs in which trainees will participate are aimed at three problems central to the development of all nervous systems: neurogenesis, axonal navigation, and synaptogenesis. The funds will provide stipends for graduate students and postdoctoral fellows, will support research participation by undergraduate students, will defray part of the cost of the trainees' research and will enable the trainees to attend scientific meetings. In addition, funds will be used to purchase specialized research equipment to be used by trainees, and to bring experts from other research and academic institutions to aid in a summer laboratory and lecture course for trainees. In recent years, remarkable advances in the use of genetics, biochemistry, and microscopy have led to significant new knowledge about the development of multicellular organisms. Despite this, much remains to be learned about development and, in particular, about the mechanisms of key problems posed by the development of many different tissues and structures. These include the differentiation of cell types, the basis of cell-cell recognition, and the migration of cells during development. In this respect, some of the most exciting opportunities and, at the same time, most difficult challenges are presented by developmental studies of animal nervous systems, which typically contain a large variety of cell types and extend throughout the animal. Successful research in this area requires a mixture of diverse intellectual and experimental skills not often taught in traditional neuroscience programs. This award will provide funds for the training of young neuroscientists who have the multiple skill needed to attack one of the last great frontiers of modern biology.

Cell surface receptors for extracellular adenosine triphosphate (ATP) are found on developing chick skeletal muscle fibers, both in vitro and in vivo. These channels are coupled to two classes of ion channels. The goals of this study are to understand the cellular and molecular basis by which ATP activated channels operate, and to determine the function of these channels in developing skeletal muscle. Since ATP is present in cholinergic vesicles at high concentration, one possibility is that there may be purinergic, as well as cholinergic transmission at developing neuromuscular junctions. A second possibility is that ATP may be released from some myoblasts, and interact with receptors on other myoblasts to promote muscle differentiation. The specific aims for the current grant period are: 1) To identify the second messenger molecule that couples ATP receptors to the activation of a late potassium current. These experiments seem likely to result in the description of a new second messenger, or in the description of novel way that a known second messenger can affect ion channel function. 2) To compare the response of chick skeletal muscle to ATP with the responses of mammalian skeletal muscle and avian smooth muscle. If responses to ATP like those seen in chick skeletal muscle are found to be widespread, it will greatly increase the likelihood that the unusual properties of the ATP-activated channels endows the system with a special function. 3) To test the hypotheses that ATP receptors play an essential role in myoblast fusion or during synaptogenesis. These experiments will test the two most likely roles that ATP receptors might play in developing muscle. 4) To determine how cell-cell interactions regulate the expression of the ATP receptors. These experiments will allow us to determine whether two receptors that are expressed in the same tissue, ATP receptors and acetylcholine receptors, are regulated independently, or in a coordinated manner. The problem of the regulation of multiple receptors is faced by virtually all cells of the central nervous system. The issue can now be studied in an accessible system. 5) To initiate studies that will ultimately lead to the biochemical and molecular characterization of the ATP receptor. Knowledge of the cellular interactions that control normal muscle development will provide a basis for understanding, and perhaps ameliorating, medically relevant deficits in nerve and muscle function.

9413211 Oakley This award renews support of a joint training effort of 15 faculty from six departments in the Schools of Arts and Sciences, of Medicine and of Dentistry at the University of Michigan, Ann Arbor. The theme of this Research Training Group (RTG) is developmental neurobiology. Faculty and student research emphasize three aspects of the development of the nervous system: neurogenesis, axonal navigation, and synaptogenesis. The research addresses these issues in a variety of organisms, including invertebrates, fish, birds and mammals using a variety of techniques from cell and molecular biology. The RTG sponsors training at all three post-secondary levels. Besides opportunities for student research, RTG faculty have created new courses at both the undergraduate and graduate level. Graduate students are selected from students who apply to one of two departmental Ph.D. programs or an interdepartmental Neuroscience Ph.D. program, and receive support for up to three years. Postdoctoral fellows are selected for one year of support from among candidates proposed by participating faculty. Undergraduates are sponsored during both the academic year and summer term. Participants in the summer research program are recruited nationally. The level of involvement of undergraduates in the RTG is particularly noteworthy; over 80 such students carried out research under the auspices of the RTG during its first four years. ***

DESCRIPTION: The proposed experiments are aimed at establishing the role of polyamines in conferring rectification to certain types of glutamate receptors. The investigator presents three specific aims relating to the general problem of glutamate receptor function and polyamine action. The first aim will determine the role of specific polyamines in conferring the observed inward rectification observed for specific types of glutamate receptors. For this purpose the investigator will block the production of specific types of polyamines in Xenopus oocytes and mammalian cells which have been transfected with RNA coding for glutamate receptors. The block will be confirmed by HPLC analysis of the polyamine levels and the functional consequences will be determined by measurement of glutamate evoked outward currents. The information provided from these experiments will demonstrate that polyamines are the endogenous source of rectification as well as identifying the specific polyamine type responsible. In a second set of experiments the investigator will determine the sensitivity of different glutamate receptor types and mutant receptor types to different polyamines. Preliminary evidence suggests that GluR4 has a higher sensitivity to spermine than GluR1 in spite of the fact that the putative pore forming regions (TM2) are identical between these two subunits. Therefore, the investigator will use the information obtained from these experiments to design GluR1 and GluR4 chimeras to locate additional pore contributing regions. In a final set of experiments the investigator will search for the bases of current rectification which is observed for Glutamate receptors. In Specific Aim 2 the investigator will determine the residues responsible for differences in permeability of AMPA and Kainate receptor channels to calcium. The investigator proposes, on the bases of homology to voltage-dependent channels, a pore lining loop outside TM2. Point mutations will be made on GluR3 and GluR6 receptor subunits at locations shown to affect properties of voltage-dependent or ligand gated channels. The permeability to divalent cations and the sensitivity to polyamines will be assessed for each mutation. In Specific Aim 3 the investigator will perform single channel studies on wild type and mutant glutamate receptors in order to ascertain the bases for outward rectification. Three different potential sources of rectification will be directly tested; rectification at the single channel level, voltage-dependent gating, and multiple conductances which are voltage-dependent. The information obtained from these aims takes on additional significance given the recent disclosure that the Glutamate tertiary structure appears distinct from other members of the ligand gated receptor family.

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Hume

Nerve cells communicate information to each other at narrow zones of contact
referred to as synapses. The incoming presynaptic cell releases a chemical
signal (the neurotransmitter), which binds to receptors on the target cell,
eliciting electrical excitation or inhibition. The general goal of these
experiments is to understand the mechanisms that allow developing synapses
to reach their stable, mature state. The principal hypothesis to be explored
is that in addition to the two signals known to pass between nerve and
muscle to promote synapse maturation (agrin and ARIA), an additional
signaling system also transmits key information. This proposed signaling
system consists of ATP (adenosine 5' triphosphate) released from presynaptic
terminals interacting with two different types of ATP receptors on the
muscle cell surface. The hypothesis will be tested by using molecular
biological and pharmacological tools to alter the function of the ATP
receptors on the surface of chick muscle cells developing within the egg,
and in cell culture.

It is important that mature neuromuscular synapses be stable, so that they
can reliably control of the routine actions of the nervous system such as
commanding muscles to move in the pattern that allows us to breathe.
However, understanding the mechanisms that promote synaptic growth and
maturation are likely to also provide us with insight into the mechanisms
that bring about changes in synaptic function in other regions of the mature
nervous system where synapses are not always stable. Changes in the
amplitude of synaptic responses are thought to be the cellular basis by
which learning and forgetting occur.

DESCRIPTION (Adapted From The Abstract Provided By Applicant): This program will provide broad, early-stage training in the neurosciences to pre-doctoral students at the University of Michigan. It will support 8 Ph.D. students in the interdepartmental Neuroscience Program during their first two years in the program. Once students complete the initial training supported by this grant, they carry out doctoral research in the laboratory of one of 85 different faculty members. About half of the faculty hold primary appointments in basic science departments, and the other half hold primary appointments in clinical departments. Thus, there are opportunities to explore a wide range of possible topics in sensory, integrative, developmental and molecular neuroscience. Many students carry out their thesis research in institutes focused specifically on disorders of the brain or sensory systems (the Mental Health Research Institute, the Kresge Hearing Research Institute, the Kellogg Eye Center and the Michigan Alzheimer's Disease Research Center). Others carry out basic neuroscience research in the departments of Biology, Biological Chemistry, Cell and Developmental Biology, Human Genetics, Physiology, Pharmacology and Psychology. Among areas of particular expertise at the University of Michigan that are represented in these departments are cognitive neuroscience, axonal guidance and neuronal differentiation, synaptic function, movement disorders, neuronal signaling by kinases and phosphatases, and the neurobiology of drugs of abuse. The first two years of this program are designed to make sure that all graduates of the program are broadly trained. In the first year, students take an interdisciplinary, year-long course in the Principles of Neuroscience, two intensive laboratory courses, a course focused on the responsible conduct of research and carry out research rotations in at least two different laboratories. During the second year students take elective neuroscience courses, participate in an intensive neuroscience seminar course, complete their lab rotations and begin the initial work on the doctoral thesis.

The P2X proteins are ATP gated channels that depolarize cells and also allow calcium to enter. P2X receptors are expressed in virtually every tissue, including neurons and glia of the central and peripheral nervous system, smooth, skeletal and cardiac muscle, cochlear hair cells, platelets, most classes of white blood cells, hepatocytes, and endothelial cells in the lung and gastrointestinal tract. The importance of members of this gene family for normal physiology is apparent from the range of phenotypes that are seen in their absence, Mice in which specific P2X receptors are knocked out show dysfunction in pain perception, ability to void the bladder, gut motility, neuronal control of ejaculation, the ability of the nervous system to monitor the oxygen level in the blood, the ability to fight bacterial infection, and blood clotting. A major problem in the purinergic receptor field is the limited specificity of agonists and antagonists that can be used to alter ATP signaling in vivo. The goal of the experiments described here is to better characterize the molecular mechanisms that allow ATP and allosteric modulators to open P2X receptor channels. The results of these studies should facilitate the development of agents tha't act more specifically on particular receptors. We will use electrophysiological, biochemical, and molecular approaches to study receptors bearing complementary mutations in adjacent or non-adjacent subunits. The specific aims are: Goal 1- To test whether the zinc binding sites that modulate channel activity in P2X2, P2X3, and P2X4 receptors are within or between subunits, and to define residues that participate in these binding sites. We will also define our understanding about the mechanisms by which zinc promotes channel opening in P2X2 receptors. Goal 2 - To test whether the ATP binding site of P2X receptors is within or between subunits and to define additional residues that are exposed in the ATP binding pocket. These experiments will also test the number of molecules of ATP that must be bound in order to open a channel. Goal 3 - To define the molecular movements that are a consequence of zinc or ATP binding to P2X2 receptors. These experiments are of particular relevance to making progress in understanding and treating pain associated with tissue injury, as P2X2 and P2X3 receptors have been implicated as playing essential roles as sensing the damage and signaling the central nervous system. Having a better understanding of the structure of these receptors should allow the development of new treatments for this type of pain, and so greatly ease the suffering of individuals with burns and other injuries that produce persistent pain.

Gastric acid secretion from parietal cells is essential for food digestion and pathogen elimination in the stomach. Dysregulation of gastric acid homeostasis underlies a spectrum of acid-related diseases, including atrophic gastritis, gastric cancer, peptic and duodenal ulcers, and gastroesophageal reflux disease (GERD); GERD alone affects at least 20% of the US population. Histamine, the primary transmitter that ?switches on? H+ secretion into the stomach lumen, acts by relocating the H+-K+-ATPase proton pump from cytoplasmic tubulovesicles (TVs) onto the apical canalicular membranes via vesicular transport and fusion. However, the mechanisms by which histamine induces the exocytosis of TVs remain unclear. Most types of regulated exocytosis, including neurotransmitter release, are Ca2+-dependent, but it remains controversial whether Ca2+ is involved in histamine-triggered TV exocytosis. Human mutations of Transient Receptor Potential Mucolipin-1 (ML1), a Ca2+-permeable channel of intracellular membranes, cause achlorhydria (low acid secretion). Using super-resolution confocal imaging, and by developing a new patch-clamp method to record directly from canalicular and tubulovesicular membranes and a new organelle-targeted Ca2+ imaging method to detect Ca2+ release from TVs, we identified ML1 as a likely histamine-triggered Ca2+ release channel in TVs. The central goal of this proposal is to investigate the roles of ML1 in TV exocytosis and to use mouse models to explore the potential clinical use of ML1 agonists and inhibitors in controlling acid secretion in vivo. Using an integrative approach with Ca2+ imaging, electrophysiology, voltage imaging, electron microscopy, small molecule channel agonists and inhibitors, and transgenic mouse models, we will test the hypothesis that histamine-cAMP-PKA signaling activate ML1 and the TV K+ channel KCNQ1 to trigger TV exocytosis and acid secretion. Aim 1 is to test the roles of ML1 and PKA in histamine-induced secretion of gastric acids. Aim 2 is to investigate the roles of KCNQ1 channels in histamine-induced gastric acid secretion. Finally, Aim 3 is to determine the roles of ML1 and KCNQ1 channels in controlling gastric acid levels in vivo. Our long-term goal of the proposed research is to lay the groundwork necessary to develop new therapeutic strategies for acid-related diseases.