Darwin K Berg
University Of California San Diego
Project start date: 1979-05-01
Project end date: 2016-12-31
Sponsored Links Excellgen http://Excellgen.com
NEURONAL DEVELOPMENT AND SYNAPSE FORMATION IN VITRO
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 2R01NS012601-12 from National Institute Of Neurological Disorders And Stroke IRG: NEUC
Abstract: Two components have been identified in extracts prepared from embryonic eye tissue that influence the long-term fate of chick ciliary ganglion neurons in dissociated cell culture. One of the components, with an apparent molecular weight of about 2x104, stimulates overall growth of the neurons but does not stimulate development of choline acetyltransferase activity. The other component, with an apparent molecular weight of about 5x104, stimulates development of choline acetyltransferase levels but has no effect on neuronal growth. The components will be purified from eye extract and examined both in culture and in vivo to determine whether they serve as target-derived trophic factors for ciliary ganglion neurons. To aid in these studies, monoclonal antibodies will be prepared against the partially purified components and used to purify the components further, manipulate component levels in vivo, and construct quantitative radioimmune assays for the components. Conditions will also be sought which induce the expression of adrenergic properties by the normally cholinergic ciliary ganglion neurons in culture as a way of identifying components which influence the differentiation of neural crest derivatives. Studies will be carried out to determine whether the neurons form chemical and electrical synapses on each other in cell culture, and how such connections are affected by input from neurons of the accessory oculomotor nucleus, the normal preganglionic input to ciliary ganglion neurons. Bgt 3.1, a protein neurotoxin present as a minor component in B. multicinctus venom, reversibly blocks acetylcholine (ACh) sensitivity on ciliary ganglion neurons and, in a separate action, induces the internalization of Alpha-bungarotoxin bound to the neurons. Alpha-Bungarotoxin does neither of these. Bgt 3.1 will be radiolabeled and examined as a possible ligand for the neuronal ACh receptor. If suitable, it will be used to study regulation of the neuronal receptor. Also, ultrastructural studies will be carried out to determine the distribution of Alpha-bungarotoxin binding sites during development since they do not appear to be associated with synaptic regions at early times, and to follow the path of bound Alpha-bungarotoxin that is internalized by Bgt 3.1 treatment.
Keywords: NERVOUS SYSTEM AUTONOMIC, PARASYMPATHETIC NERVOUS SYSTEM, NERVOUS SYSTEM, NERVE ENDINGS, SYNAPSES, NERVOUS SYSTEM, NERVES, INNERVATION (GENERAL), NEUROLOGY A STUDY SECTION, NEUROLOGY, DEVELOPMENTAL, NEUROGENESIS, BRAIN, MESENCEPHALON, OCULOMOTOR NUCLEI, CHOLINE, ACETYLCHOLINE, INFORMATION PROCESSING AND CONTROL (NEURAL), NERVOUS SYSTEM AUTONOMIC, CILIARY GANGLION, NEUROPHYSIOLOGY, NEURAL TRANSMISSION, NEUROTOXINS, NEUROTRANSMITTERS RECEPTORS, CHOLINERGIC RECEPTORS, TOXICOLOGY, ANIMAL POISONS, REPTILE, BUNGAROTOXIN, TRANSACYLASES, CHOLINE TRANSACYLASE, neuroanatomy, BIOLOGICAL PREPARATIONS AND STANDARDIZATION, NATURAL PRODUCTS PRODUCTION BY GENETIC MANIPULATION, BIRDS, CHICK EMBRYO, IMMUNOLOGICAL PREPARATIONS, MONOCLONAL ANTIBODIES, IMMUNOLOGICAL TESTS AND IMMUNOASSAY, RADIOIMMUNOASSAY, TISSUE (CELL) CULTURE, EMBRYONIC-FETAL CELL LINES
Project start date: 1979-05-01
Project end date: 1994-04-30
Nicotinic Synapse Formation On Neurons
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 2R01NS012601-27 from National Institute Of Neurological Disorders And Stroke IRG: MDCN
Abstract: This application will examine the formation of nicotinic synapses on neurons. Despite much information about the vertebrate neuromuscular junction and a variety of synapses in the CNS, almost nothing is known about postsynaptic components at nicotinic synapses on neurons, other than the identify of the nicotinic acetylcholine receptor (nAChR) itself. Given the prevalence of nicotinic signaling and its implication in a variety of neural functions and disorders, it is becoming increasingly important to understand the assembly and regulation of the molecular structures involved. Nicotinic receptors containing alpha7 subunits (a7-nAChRs) are among the most abundant in the nervous system and regulate calcium-dependent events, due in part to their having a high relative calcium permeability. Recent studies on ciliary ganglion neurons show the receptors are concentrated on somatic spines where they utilize calcium influx to control events ranging from immediate receptor modulation to long-term transcriptional control. Preliminary results suggest that both cadherin-related neuronal receptors (CNRs) and PDZ-containing proteins are associated with nicotinic synapses on the neurons and may play important organizational and regulatory roles. The receptors also form clusters on hippocampal neurons in culture but are often associated with presumptive non-nicotimc synapses where they are regulated by neural activity. These findings provide the basis for the following aims (1) Complete 3D tomographic EM analysis of a complete nicotinic calyx synapse to elucidate structure-function relationships, (2) Identify molecular components driving nicotinic synapse formation on neurons, beginning with CNRs and PDZ-containing proteins, (3) Examine postsynaptic determinants of a7-nAChR function that control their contributions to synaptic signaling, and (4) Use rat hippocampal cultures to examine the regulation and significance of synaptic a7-nAChRs on mammalian CNS neurons. The experimental approaches include confocal and EM imaging, gene cloning and manipulation of dominant negative constructs, immunocytochemistry and immunopurification, and patch clamp recording in culture and in situ. The expected results should identify postsynaptic components that link nAChRs to regulatory and signal transduction elements and tether them to the cytoskeleton. This information will provide insight into the organization of nicotinic synapses and suggest mechanisms controlling their development and function. The biomedical relevance stems in part from the role of nicotinic signaling in fundamental brain function such as learning and memory, and in related disorders such as Alzheimers disease and nicotine addiction.
Keywords: cadherin, ciliary ganglion, developmental neurobiology, nicotinic receptor, protein structure function, synaptogenesis, transcription factor, biological signal transduction, embryogenesis, hippocampus, neuron, protein localization, receptor expression, synapse, chick embryo, confocal scanning microscopy, immunocytochemistry, laboratory rat, molecular cloning, scanning electron microscopy, tissue /cell culture, voltage /patch clamp
Project start date: 1979-05-01
Project end date: 2006-04-30
2R01NS012601-27 (2002): $324900
NEURONAL DEVELOPMENT AND SYNAPSE FORMATION IN VITRO
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 2R01NS012601-19 from National Institute Of Neurological Disorders And Stroke IRG: NEUC
Abstract: The long-term goal of this project is to understand the function and regulation of synaptic components-on neurons since this is essential for understanding how the nervous system develops and generates specific behaviors. One component of critical importance at the synapse is the neurotransmitter receptor. Families of receptors exist for each major transmitter and a single neuron can express multiple members of the family, but the significance of this for neuronal function is unknown. The central purpose of the current project is to determine the composition, function, and regulation of native acetylcholine receptors (AChRs) on a population of primary neurons with the goal of understanding the contribution each major AChR subtype makes to cell-cell signaling. The model system being used for most of these studies is the chick ciliary ganglion. The neurons contain receptors that are primarily synaptic in location and bind the monoclonal antibody mAb 35 (mAb 35- AChRs). They also have large numbers of receptors primarily synaptic in location that bind alpha- bungarotoxin (alphaBgt-AChRs). The subunit composition of mAb 35-AChRs on the neurons will be examined with subunit- specific mAbs in conjunction with solid phase immunoprecipitations and immunoblots. Individual receptor subtypes in the population will be identified, their contribution to the pool of AChRs on the cell surface determined, and the stoichiometry of subunits in the receptor subtype measured. The physiological properties of alphaBgt-AChRs and mAb 35- AChRs will be compared using whole cell voltage clamp and rapid application of agonist to determine their dose-response curves, desensitization profiles, and ion selectivities. The receptors will be tested for their contributions to ganglionic transmission, and will be compared for their abilities to regulate calcium-dependent events in the cells including calcium-activated currents and release of calcium from internal stores. Immunocytochemistry and immunoblots with subunit- specific mbs will be used to determine which AChR subtypes are destined for unique sites on the neurons such as a presynaptic location on axon terminals or a postsynaptic location on the soma. The source of AChRs containing the neuronal alpha7 subunit in embryonic muscle tissue will be identified and their composition determined. Second messenger regulation of neuronal AChRs will be examined including those of cyclic AMP and arachidonic acid, distinguishing effects on receptor number and function. The role of the postsynaptic target tissue in regulating AChRs on the neurons will be studied, including the contribution of a fibroblast growth factor-like component in the tissue. The results will provide new information about the molecular mechanisms responsible for generating and controlling cell-cell signalling in the nervous system. The health relevance of this research derives from the increase in basic knowledge that it will provide about the nervous system. The findings may hake medical benefits in identifying critical elements of synaptic development and function, and may suggest molecular mechanisms underlying neurodegenerative disorders much as Alzheimer s disease where a neuronal AChR deficit has been reported.
Keywords: cholinergic receptor, ciliary ganglion, developmental neurobiology, receptor expression, synaptogenesis, acetylcholine, arachidonate, bungarotoxin, calcium flux, cyclic AMP, fibroblast growth factor, gene induction /repression, neural transmission, neuromuscular junction, protein structure function, chick embryo, immunocytochemistry, monoclonal antibody
Project start date: 1979-05-01
Project end date: 1997-04-30
2R01NS012601-19 (1994): $186657
2R01NS012601-22 (1997): $213232
Signal Transduction At Neuronal Nicotinic Synapses
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 2R01NS012601-31 from National Institute Of Neurological Disorders And Stroke IRG: MNPS
Abstract: Nicotinic acetylcholine receptors (nAChRs) are widely expressed in the nervous system and have been implicated in a variety of behaviors and neuropathologies. In most cases the receptors reach their highest levels during late embryogenesis and early postnatal life. Almost nothing is known, however, about the developmental roles of neuronal nAChRs, other than that they are cation-selective and can regulate calcium- dependent events. We have preliminary evidence that during development nAChRs utilize signal transduction to regulate synaptic capabilities, including that of adjacent non-cholinergic pathways, in both the CNS and autonomic nervous systems. This proposal will test specific hypotheses about the roles of nicotinic input in shaping the development of nicotinic and GABAergic signaling, and will examine candidate molecules that may facilitate or transduce nicotinic effects. In Aim I the chick ciliary ganglion is used to test hypotheses about the significance of newly discovered GABAergic pathways in this cholinergic ganglion, the role of nicotinic input in controlling GABAergic maturation from excitatory to inhibitory mode, and the consequences this has for ganglionic throughput. In Aims II and III, the mammalian hippocampus is used to test hypotheses about the role of nicotinic signaling in regulating filopodia fate, induction of dendritic spines, maturation of GABAergic signaling from excitatory to inhibitory, and acute modulation of GABA responses. Long-lasting nicotine sensitization is also examined. In Aims IV and V three candidate proteins that appear to specifically interact with neuronal nAChRs are examined for their roles in determining the number, location, function, and downstream signaling capabilities of the receptors. Each of these aims is based on preliminary data supporting the central hypotheses being tested. The experimental approaches include electrophysiology, imaging, molecular biology, and biochemistry, and are carried out on neurons in cell culture and slice preparations from wildtype and mutant sources. Together these approaches and systems should provide mechanistic insight into novel roles of nicotinic signaling in the developing nervous system. The findings should also have biomedical relevance in revealing the consequences of nicotine exposure during these formative periods.
Keywords: cadherin, ciliary ganglion, developmental neurobiology, nicotinic receptor, protein structure function, synaptogenesis, transcription factor, biological signal transduction, embryogenesis, hippocampus, neuron, protein localization, receptor expression, synapse, chick embryo, confocal scanning microscopy, immunocytochemistry, laboratory rat, molecular cloning, scanning electron microscopy, tissue /cell culture, voltage /patch clamp
Project start date: 1979-05-01
Project end date: 2011-04-30
2R01NS012601-31 (2006): $341453
NEURONAL DEVELOPMENT AND SYNAPSE FORMATION IN VITRO
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 2R01NS012601-04 from National Institute Of Neurological Disorders And Stroke IRG: NEUA
Abstract: Five related aspects of neuronal development and synapse formation will be examined in associated cell cultures prepared with embryonic chick ciliary ganglion (CG) neurons. These include (1) the normal determinants of neuronal survival, (2) the specification of neurotransmitter synthesis, (3) the regulation of the neuronal alpha-bungarotoxin binding component and its relation to the neuronal acetylcholine (ACh) receptor, (4) the formation and maintenance of nerve-nerve and nerve-muscle synapses, and (5) the neuronal regulation of myotube ACh receptors. When grown alone in cell culture with muscle- or heart-conditioned medium, DCG neurons exhibit quantitative long-term survival, including those neurons that were destined to die in ovo as part of normal ganglionic development. The factor(s) present in conditioned medium that are responsible for the survival will be purified and their mode of action characterized. Neurotransmitter synthesis will be examined to identify the events that commit CG neurons to a cholinergic fate. An early developmental stage will be sought in which the neurons retain the capacity to express other differentiated properties characteristic of neuronal crest derivatives such as catecholamine biosynthesis. The binding of alpha-bungarotoxin to the neurons will be studied to distinguish the different classes of binding and to determine their relationship to the neuronal ACh receptor. Synapse formation in CG neuron-myotube cultures will be examined (1) to assess the stability of individual points of nerve-muscle synaptic contact, (b) to determine whether synaptic activity can prevent the formation of additional synapses on an innervated myotube, and (c) to determine whether CG neurons can innervate each other in cell culture. The neuronal regulation of myotube ACh receptors will be examined to identify the mechanisms underlying the induction of receptors and to determine whether the neurons also promote a re-distribution of the receptors in the myotube membrane. Fluorescent- and radiolabeled-toxins together with iontophoretic application of ACh will be used for the receptor analysis.
Keywords: NERVOUS SYSTEM AUTONOMIC, PARASYMPATHETIC NERVOUS SYSTEM, NERVOUS SYSTEM, NERVE ENDINGS, SYNAPSES, NERVOUS SYSTEM, NERVES, INNERVATION (GENERAL), NERVOUS SYSTEM, NEURONS, NEUROLOGY STUDY SECTION, NEUROLOGY, DEVELOPMENTAL, NEUROGENESIS, CELL COMPONENTS, MICROTUBULES (GENERAL), CHOLINE, ACETYLCHOLINE, INFORMATION PROCESSING AND CONTROL (NEURAL), MEMBRANE, MEMBRANE (BIOLOGICAL) STRUCTURE, MUSCLE CELLS, NEUROPHYSIOLOGY, NEURAL TRANSMISSION, NEUROTRANSMITTERS BIOSYNTHESIS, NEUROTRANSMITTERS RECEPTORS, CHOLINERGIC RECEPTORS, TOXICOLOGY, ANIMAL POISONS, REPTILE, BUNGAROTOXIN, neuroanatomy, BIRDS, CHICK EMBRYO, ELECTROIONIZATION (IONTOPHORETIC) METHODS, NONTHERAPEUTIC, RADIOASSAY (RADIOMETRY), TISSUE (CELL) CULTURE, EMBRYONIC-FETAL CELL LINES
Project start date: 1979-05-01
Project end date: 1982-04-30
Grants awarded to Darwin K Berg
CELLULAR And MOLECULAR NEUROBIOLOGY: SYNAPSE FORMATION And MODULATION
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5P41RR004050-100043 from National Center For Research Resources
Abstract: We are using 3D electron microscopic techniques to examine membrane fine structure in the vicinity of nicotinic receptor clusters previously described as being "perisynaptic" on chick ciliary ganglion neurons. The receptors are composed of alpha 7 subunits and bind alpha-bungarotoxin with high affinity. The hypothesis being tested is whether the receptor clusters may in large part represent receptors distributed over pseudodendrites compressed against the cell surface in patches. Neurons both freshly dissociated and in whole ganglia will be labeled with either HRP conjugated or biotinylated alpha-bungarotoxin and either examined straightaway with high voltage EM or will be examined first by confocal fluorescence microscopy to locate receptor clusters and them examined by EM to identify related fine structure. The biotinylated bungarotoxin will be localized by 10 nm colloidal gold. Work on this project is underway. Several thick sections of HRP-labeled ciliary ganglion cells have been examined which indicated that these neurons possess somatic spines that are heavily labeled with bungarotoxin. The pattern of bungarotoxin binding sites is being examined further using electron tomography and colloidal gold labeled specimens. In the tomographic reconstructions, we can easily resolve the complex tangle of somatic spines as well as quantify the labeling over spiny and non-spiny regions of membrane.
Keywords: biomedical resource, growth /development, microscopy, model design /development, nervous system, protein, animal tissue
NEURONAL DEVELOPMENT AND SYNAPSE FORMATION IN VITRO
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01NS012601-18 from National Institute Of Neurological Disorders And Stroke IRG: NEUC
Abstract: Two components have been identified in extracts prepared from embryonic eye tissue that influence the long-term fate of chick ciliary ganglion neurons in dissociated cell culture. One of the components, with an apparent molecular weight of about 2x104, stimulates overall growth of the neurons but does not stimulate development of choline acetyltransferase activity. The other component, with an apparent molecular weight of about 5x104, stimulates development of choline acetyltransferase levels but has no effect on neuronal growth. The components will be purified from eye extract and examined both in culture and in vivo to determine whether they serve as target-derived trophic factors for ciliary ganglion neurons. To aid in these studies, monoclonal antibodies will be prepared against the partially purified components and used to purify the components further, manipulate component levels in vivo, and construct quantitative radioimmune assays for the components. Conditions will also be sought which induce the expression of adrenergic properties by the normally cholinergic ciliary ganglion neurons in culture as a way of identifying components which influence the differentiation of neural crest derivatives. Studies will be carried out to determine whether the neurons form chemical and electrical synapses on each other in cell culture, and how such connections are affected by input from neurons of the accessory oculomotor nucleus, the normal preganglionic input to ciliary ganglion neurons. Bgt 3.1, a protein neurotoxin present as a minor component in B. multicinctus venom, reversibly blocks acetylcholine (ACh) sensitivity on ciliary ganglion neurons and, in a separate action, induces the internalization of Alpha-bungarotoxin bound to the neurons. Alpha-Bungarotoxin does neither of these. Bgt 3.1 will be radiolabeled and examined as a possible ligand for the neuronal ACh receptor. If suitable, it will be used to study regulation of the neuronal receptor. Also, ultrastructural studies will be carried out to determine the distribution of Alpha-bungarotoxin binding sites during development since they do not appear to be associated with synaptic regions at early times, and to follow the path of bound Alpha-bungarotoxin that is internalized by Bgt 3.1 treatment.
Keywords: innervation, neurogenesis, parasympathetic nervous system, synapse, acetylcholine, bungarotoxin, choline acetyltransferase, ciliary ganglion, neural information processing, neural transmission, neuroanatomy, neurotoxin, nicotinic receptor, oculomotor nuclei, biological product, chick embryo, embryo /fetus cell culture, genetic manipulation, monoclonal antibody, radioimmunoassay
Project start date: 1979-05-01
Project end date: 1994-04-30
5R01NS012601-18 (1993): $200003
5R01NS012601-17 (1992): $191051
Sponsored Links Excellgen http://Excellgen.com
Nicotinic Synapse Formation On Neurons
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01NS012601-28 from National Institute Of Neurological Disorders And Stroke IRG: MDCN
Abstract: This application will examine the formation of nicotinic synapses on neurons. Despite much information about the vertebrate neuromuscular junction and a variety of synapses in the CNS, almost nothing is known about postsynaptic components at nicotinic synapses on neurons, other than the identify of the nicotinic acetylcholine receptor (nAChR) itself. Given the prevalence of nicotinic signaling and its implication in a variety of neural functions and disorders, it is becoming increasingly important to understand the assembly and regulation of the molecular structures involved. Nicotinic receptors containing alpha7 subunits (a7-nAChRs) are among the most abundant in the nervous system and regulate calcium-dependent events, due in part to their having a high relative calcium permeability. Recent studies on ciliary ganglion neurons show the receptors are concentrated on somatic spines where they utilize calcium influx to control events ranging from immediate receptor modulation to long-term transcriptional control. Preliminary results suggest that both cadherin-related neuronal receptors (CNRs) and PDZ-containing proteins are associated with nicotinic synapses on the neurons and may play important organizational and regulatory roles. The receptors also form clusters on hippocampal neurons in culture but are often associated with presumptive non-nicotimc synapses where they are regulated by neural activity. These findings provide the basis for the following aims (1) Complete 3D tomographic EM analysis of a complete nicotinic calyx synapse to elucidate structure-function relationships, (2) Identify molecular components driving nicotinic synapse formation on neurons, beginning with CNRs and PDZ-containing proteins, (3) Examine postsynaptic determinants of a7-nAChR function that control their contributions to synaptic signaling, and (4) Use rat hippocampal cultures to examine the regulation and significance of synaptic a7-nAChRs on mammalian CNS neurons. The experimental approaches include confocal and EM imaging, gene cloning and manipulation of dominant negative constructs, immunocytochemistry and immunopurification, and patch clamp recording in culture and in situ. The expected results should identify postsynaptic components that link nAChRs to regulatory and signal transduction elements and tether them to the cytoskeleton. This information will provide insight into the organization of nicotinic synapses and suggest mechanisms controlling their development and function. The biomedical relevance stems in part from the role of nicotinic signaling in fundamental brain function such as learning and memory, and in related disorders such as Alzheimers disease and nicotine addiction.
Keywords: cadherin, ciliary ganglion, developmental neurobiology, nicotinic receptor, protein structure function, synaptogenesis, transcription factor, biological signal transduction, embryogenesis, hippocampus, neuron, protein localization, receptor expression, synapse, chick embryo, confocal scanning microscopy, immunocytochemistry, laboratory rat, molecular cloning, scanning electron microscopy, tissue /cell culture, voltage /patch clamp
Project start date: 1979-05-01
Project end date: 2006-04-30
5R01NS012601-28 (2003): $324900
5R01NS012601-30 (2005): $324900
5R01NS012601-29 (2004): $324900
NEURONAL DEVELOPMENT AND SYNAPSE FORMATION IN VITRO
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01NS012601-06 from National Institute Of Neurological Disorders And Stroke IRG: NEUA
Abstract: The survival and development of embryonic chick ciliary ganglion neurons are being examined in dissociated cell culture. Factors are being isolated and characterized that influence the development of cholinergic properties by the neurons. Cell culture conditions are also being sought that permit the normally cholinergic neurons to express novel differentiated properties characteristic of other neural crest derivatives such as the synthesis of catecholamines. Experiments are also being carried out on the specificity of synapse formation by ciliary ganglion neurons in cell culture. Studies with protein neurotoxins are being done to develop a specific ligand for studying regulation of the neuronal acetylcholine receptor and for studying the internalization of membrane components by neurons.
Keywords: NERVOUS SYSTEM AUTONOMIC, PARASYMPATHETIC NERVOUS SYSTEM, NERVOUS SYSTEM, NERVE ENDINGS, SYNAPSES, NERVOUS SYSTEM, NERVES, INNERVATION (GENERAL), NERVOUS SYSTEM, NEURONS, NEUROLOGY A STUDY SECTION, NEUROLOGY, DEVELOPMENTAL, NEUROGENESIS, CELL COMPONENTS, MICROTUBULES (GENERAL), CHOLINE, ACETYLCHOLINE, INFORMATION PROCESSING AND CONTROL (NEURAL), MEMBRANE, MEMBRANE (BIOLOGICAL) STRUCTURE, MUSCLE CELLS, NEUROPHYSIOLOGY, NEURAL TRANSMISSION, NEUROTRANSMITTERS BIOSYNTHESIS, NEUROTRANSMITTERS RECEPTORS, CHOLINERGIC RECEPTORS, TOXICOLOGY, ANIMAL POISONS, REPTILE, BUNGAROTOXIN, neuroanatomy, BIRDS, CHICK EMBRYO, ELECTROIONIZATION (IONTOPHORETIC) METHODS, NONTHERAPEUTIC, RADIOASSAY (RADIOMETRY), TISSUE (CELL) CULTURE, EMBRYONIC-FETAL CELL LINES
Project start date: 1979-05-01
Project end date: 1982-04-30
5R01NS012601-20 (1995): $195194
5R01NS012601-26 (2001): $240132
5R01NS012601-25 (2000): $233140
5R01NS012601-24 (1999): $226347
5R01NS012601-23 (1998): $219752
CELLULAR And MOLECULAR NEUROBIOLOGY SYNAPSE FORMATION And MODULATION
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5P41RR004050-140034 from National Center For Research Resources
Abstract: The distribution of voltage sensitive sodium channels on axons in the dorsal and ventral spinal roots of the dystrophic mouse 129/ReJ-Lama2dy was determined using immunocytochemistry. In these nerves there are regions in which Schwann cells fail to proliferate and myelinate axons in a normal fashion, leaving bundles of closely packed large diameter amyelinated axons. We have identified discrete and focal concentrations of sodium channel immunoreactivity on these axons by confocal immunofluorescence, immunoelectron microscopy and Intermediate Voltage Electron Microscopy (IVEM) using a peptide-derived polyclonal antibody. In addition, simultaneous labeling with an antibody recognizing neuronal-specific ankyrinG revealed a distinct colocalization with the sodium channels on both normal and amyelinated axons. The presence of patches of sodium channels along with their anchoring protein on amyelinated axons in the absence of intervening Schwann cells demonstrates that axons can independently form and maintain these initial aggregations. This confirms that direct contact between Schwann cell and axon is not required for the formation of sodium channel patches of nodal dimensions and density. Furthermore, this strongly suggests that local transfer of sodium channels from Schwann cells to axons is not required for this process. This work was published in the Journal of Neuroscience (Deerinck et al., J. Neurosci., 17 5080-5088, 1997).
Keywords: Mammalia, biomedical resource, microscopy, nervous system, rehabilitation, human tissue
Sponsored Links Excellgen http://Excellgen.com
CELLULAR & MOLECULAR NEUROBIOLOGY SYNAPSE FORMATION & MODULATION
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 3P41RR004050-13S10034 from National Center For Research Resources
Abstract: The distribution of voltage sensitive sodium channels on axons in the dorsal and ventral spinal roots of the dystrophic mouse 129/ReJ-Lama2dy was determined using immunocytochemistry. In these nerves there are regions in which Schwann cells fail to proliferate and myelinate axons in a normal fashion, leaving bundles of closely packed large diameter amyelinated axons. We have identified discrete and focal concentrations of sodium channel immunoreactivity on these axons by confocal immunofluorescence, immunoelectron microscopy and Intermediate Voltage Electron Microscopy (IVEM) using a peptide-derived polyclonal antibody. In addition, simultaneous labeling with an antibody recognizing neuronal-specific ankyrinG revealed a distinct colocalization with the sodium channels on both normal and amyelinated axons. The presence of patches of sodium channels along with their anchoring protein on amyelinated axons in the absence of intervening Schwann cells demonstrates that axons can independently form and maintain these initial aggregations. This confirms that direct contact between Schwann cell and axon is not required for the formation of sodium channel patches of nodal dimensions and density. Furthermore, this strongly suggests that local transfer of sodium channels from Schwann cells to axons is not required for this process. This work was published in the Journal of Neuroscience (Deerinck et al., J. Neurosci., 17 5080-5088, 1997).
Keywords: Mammalia, biomedical resource, microscopy, nervous system, rehabilitation, human tissue
NEURONAL DEVELOPMENT AND SYNAPSE FORMATION IN VITRO
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01NS012601-02 from National Institute Of Neurological Disorders And Stroke IRG: NEUA
Abstract: The development and regulation of neurochemical mechanisms underlying synaptic transmission will be examined to gain information about processes governing formation and long-term modulation of synapses. These events will be studied in dissociated cell cultures prepared from embryonic chick spinal cord and muscle. The major focus of the proposal will be to examine the development of cholinergic neurons with respect to their capacity to synthesize, store, and release ACh, and to determine how these properties depend on muscle influence. Neuronal influences on muscle ACh receptor distribution and number as a consequence of synapse formation will also be examined. High affinity choline uptake by spinal cord cells in culture will be studied to assess whether it is restricted to cholinergic neurons under these conditions, and if so, whether it can be used to identify cholinergic neurons and follow their development. BIBLIOGRAPHIC REFERENCES Berg, D. K. and Hall, Z.W. (1975) Increased extrajunctional acetylcholine sensitivity produced by chronic prostsynaptic neuromuscular blockage. J. Physiol. 244 659-676. Berg, D.K. and Hall, Z.W. (1975) Loss of alpha-bungarotoxin from junctional and extrajunctional acetylcholine receptors in rat diaphragm muscle in vivo and in organ culture. J. Physiol. 255 771-789.
Keywords: CELL TYPES, NERVOUS SYSTEM AUTONOMIC, PARASYMPATHETIC NERVOUS SYSTEM, NERVOUS SYSTEM, NERVE ENDINGS, SYNAPSES, NERVOUS SYSTEM, NERVES, INNERVATION (GENERAL), NERVOUS SYSTEM, NEUROGENESIS, NERVOUS SYSTEM, NEURONS, NEUROLOGY STUDY SECTION, CHOLINE, ACETYLCHOLINE, INFORMATION PROCESSING AND CONTROL (NEURAL), MEMBRANE, MEMBRANE (BIOLOGICAL) STRUCTURE, MUSCLE CELLS, NERVOUS SYSTEM CENTRAL, SPINAL CORD, NEUROPHYSIOLOGY, NEURAL TRANSMISSION, NEUROTRANSMITTERS RECEPTORS, ADRENERGIC RECEPTORS, DOPAMINERGIC RECEPTORS, neuroanatomy, BIRDS, CHICK EMBRYO*, PHYSICAL SEPARATION, ELECTROPHORESIS, GEL*, RADIOASSAY (RADIOMETRY), TISSUE (CELL) CULTURE*
Project start date: 1976-05-01
Project end date: 1979-04-30
Neuronal Nicotinic Receptors- Expression And Regulation
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01NS035469-12 from National Institute Of Neurological Disorders And Stroke IRG: SYN
Abstract: Nicotinic signaling is widespread in the nervous system and influences numerous behaviors and neuropathologies. The signaling depends on nicotinic receptors which often promote calcium influx and regulate calcium-dependent events. The consequence of signaling, therefore, can depend critically both on receptor location and on the identity of associated intracellular components. Recently it has been shown that members of the PSD-95/SAP90 family form a postsynaptic PDZ-scaffold associated with nicotinic receptors on neurons. The scaffold not only helps mediates downstream signaling but also plays an important but unknown role in supporting synaptic input to the neuron. Preliminary results suggest that postsynaptic neuroligin, EphB2 receptors, and rapid activity-driven SNARE-dependent receptor trafficking also converge in the postsynaptic neuron to regulate nicotinic signaling. Four specific aims are proposed to pursue these findings (1) Determine how postsynaptic PDZ-scaffolds influence synaptic input and identify possible intermediate components exerting the effect. (2) Test the hypothesis that postsynaptic neuroligin supports nicotinic input independently from PDZ-scaffolds and determine how it interacts with nicotinic receptors. (3) Examine the mechanisms promoting rapid trafficking of nicotinic receptors on neurons and assess their physiological significance for synaptic signaling. (4) Test the hypothesis that EphB2 receptors enhance nicotinic effects and examine the interdependence of convergent pathways controlling nicotinic signaling in neurons. Fluorescence imaging will be used to visualize synaptic components; transfections will be used to manipulate interacting partners; electrophysiological analysis will be used to study functional consequences; and biochemical and molecular biological techniques will be used to probe interactions. Experiments will focus mainly on chick ciliary ganglion neurons but will also employ transfected cell lines to define molecular interactions, and rat hippocampal neurons to assess the generality of the findings. These results will provide new insight into regulatory mechanisms that shape nicotinic signaling, important because the signaling is pervasive and has profound biomedical consequences.
Keywords: biological signal transduction, neuron, nicotinic receptor, receptor expression, ciliary ganglion, electrophysiology, hippocampus, membrane protein, membrane structure, nerve /myelin protein, neurophysiology, protein localization, protein protein interaction, protein transport, protein tyrosine kinase, synapse, chicken, fluorescent dye /probe, laboratory rabbit, laboratory rat, tissue /cell culture, transfection
Project start date: 1996-05-01
Project end date: 2009-02-28
5R01NS035469-12 (2007): $410365
5R01NS035469-11 (2006): $420361
2R01NS035469-10 (2005): $355739
NICOTINIC CONTROL OF NON-NICOTINIC SYNAPSES IN THE HIPPOCAMPUS
Darwin K Berg, Professor
University Of California San Diego, 9500 Gilman Dr, Dept 0934, La Jolla, Ca 92093-0934
Grant 2R01NS035469-14A1 from National Institute Of Neurological Disorders And Stroke
Abstract: Nicotinic cholinergic signaling uses the transmitter acetylcholine to activate ligand-gated ion channels that are cation-selective in mammals. This form of signaling is widespread in the nervous system, reaches peak levels during early postnatal life, and continues throughout adulthood. It contributes to a variety of behaviors including arousal and cognition, participates in a number of neurodegenerative disorders including Alzheimer´s and Parkinson´s diseases, and is responsible for nicotine addiction. Despite intensive effort, little is understood about the role of nicotinic signaling during development when it drives spontaneous waves of excitation across the nervous system, and little is understood about the nicotinic mechanisms that subsequently exert global effects across networks in the adult brain. This proposal tests two novel hypotheses fundamental to these issues. The first is that nicotinic signaling during development promotes the formation of glutamatergic synapses both on early postnatal and adult born neurons (Aims I & II). Since glutamatergic pathways provide the principal form of excitation in brain, this effect of nicotinic input is likely to have lasting consequences for nervous system function. The second hypothesis is that nicotinic activity in the adult brain can acutely and reversibly alter GABAergic signaling such that it stops being inhibitory and transiently becomes excitatory (Aim III). This could exert far-reaching effects across networks radically altering output. Preliminary studies performed on the hippocampus strongly support these ideas. The two major nicotinic acetylcholine receptors, homopentameric ?7-nAChRs and heteropentameric ?2-containing nAChRs, appear to complement each other in promoting glutamate synapse formation. One appears to act in cell-autonomous fashion to drive postsynaptic spine formation while the other may act indirectly to recruit components required for synaptic function. Preliminary results also support the second hypothesis low levels of nicotine experienced by tobacco users may be sufficient to transiently invert the chloride gradient in adult neurons, thereby rendering GABA temporarily depolarizing. This could dramatically change the excitability of networks housing those neurons. The hypotheses will be tested by pharmacological and genetic manipulation, including loss-of-function and rescue experiments, performed on hippocampal slices and in vivo. The underlying molecular mechanisms will be analyzed and their consequences evaluated for system function. Imaging and electrophysiological approaches will be combined in this analysis. The experiments proposed here test pivotal ideas about the purpose of nicotinic cholinergic signaling in the nervous system. The results are likely to change how we think about fundamental processes guiding development and regulation of function in neural networks. The vulnerability of these processes to exploitation by tobacco-derived nicotine gives this work compelling health-related significance. This project will test new ideas about the roles of endogenous nicotinic cholinergic signaling in shaping the developing nervous system and controlling function subsequently in the adult. Because these are the same pathways hijacked by nicotine from tobacco consumption, the studies will also provide important information about the systems at risk and consequences likely to ensue. In addition to addressing fundamental aspects of nervous system function, the studies may identify targets for therapeutic intervention both to reverse or to correct biological deficiencies and to mitigate the impact of habitual tobacco use
Keywords: 2-(Acetyloxy)-N, N, N-trimethylethanaminium; 21+ years old; 4-Aminobutanoic Acid; 4-Aminobutyric Acid; AMPA Receptors; Acetylcholine; Acute; Address; Adult; Affect; Alzheimer; Alzheimer disease; Alzheimer sclerosis; Alzheimer syndrome; Alzheimer`s; Alzheimer`s Disease; Alzheimers Dementia; Alzheimers disease; Aminalon; Aminalone; Ammon Horn; Appearance; Arousal; Behavior; Biological; Biological Neural Networks; Brain; Butanoic acid, 4-amino-; Cations; Cell Communication and Signaling; Cell Signaling; Cells; Chloride; Chloride Ion; Chlorides; Cl- element; Cognition; Complement; Complement Proteins; Cornu Ammonis; Degenerative Diseases, Nervous System; Degenerative Neurologic Disorders; Dementia, Alzheimer Type; Dementia, Primary Senile Degenerative; Dementia, Senile; Dendrites; Dendritic Spines; Dentate Fascia; Dentate Gyrus; Dependence, Nicotine; Deposit; Deposition; Development; Effects, Longterm; Embryo Development; Embryogenesis; Embryonic Development; Encephalon; Encephalons; Environment; Ethanaminium, 2-(acetyloxy)-N, N, N-trimethyl-; Fascia Dentata; GABA; Gated Ion Channel; Glutamates; Gyrus Dentatus; Hippocampus; Hippocampus (Brain); Housing; Human, Adult; Idiopathic Parkinson Disease; Image; Intracellular Communication and Signaling; Ion Channel; Ionic Channels; Knockout Mice; Knowledge; L-Glutamate; Lewy Body Parkinson Disease; Life; Ligands; Long-Term Effects; Mammalia; Mammals; Mammals, General; Membrane Channels; Mice, Knock-out; Mice, Knockout; Molecular; NRVS-SYS; Nerve Cells; Nerve Unit; Nervous System; Nervous System Physiology; Nervous System, Brain; Nervous system structure; Neural Cell; Neural Growth; Neurocyte; Neurodegenerative Diseases; Neurodegenerative Disorders; Neurologic Body System; Neurologic Degenerative Conditions; Neurologic Diseases, Degenerative; Neurologic Organ System; Neurologic function; Neurological function; Neuronal Growth; Neurons; Nicotine; Nicotine Dependence; Nicotinic Acetylcholine Receptors; Nicotinic Receptors; Null Mouse; Outcome; Output; Paralysis Agitans; Parkinson; Parkinson Disease; Parkinson`s; Parkinson`s disease; Parkinsons disease; Pathway interactions; Phase; Play; Primary Parkinsonism; Primary Senile Degenerative Dementia; Process; Pyramidal neuron; Pyridine, 3-(1-methyl-2-pyrrolidinyl)-, (S)-; Receptor Protein; Receptors, AMPA; Recruitment Activity; Regulation; Risk; Role; Shapes; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Slice; Spinal Column; Spine; Structure of dentate gyrus; Surface; Synapses; Synaptic; System; System, LOINC Axis 4; Testing; Therapeutic Intervention; Tobacco; Tobacco Consumption; Tobacco use; Vertebral column; Work; adult human (21+); backbone; biological signal transduction; cell type; cholinergic; critical period; dementia of the Alzheimer type; dendrite spine; dentate gyrus; design; designing; experience; experiment; experimental research; experimental study; gamma-Aminobutyric Acid; genetic manipulation; hippocampal; hippocampal pyramidal neuron; imaging; in vivo; innervation; intervention therapy; loss of function; nerve supply; nervous system development; nervous system function; neural network; neurodegenerative illness; neurogenesis; neuronal; newborn neuron; nicotine addiction; novel; pathway; postnatal; postsynaptic; presynaptic; primary degenerative dementia; public health relevance; receptor; recruit; research study; senile dementia of the Alzheimer type; social role; synapse formation; synapse function; synaptic function; synaptogenesis
Relevance: This project will test new ideas about the roles of endogenous nicotinic cholinergic signaling in shaping the developing nervous system and controlling function subsequently in the adult. Because these are the same pathways hijacked by nicotine from tobacco consumption, the studies will also provide important information about the systems at risk and consequences likely to ensue. In addition to addressing fundamental aspects of nervous system function, the studies may identify targets for therapeutic intervention both to reverse or correct biological deficiencies and to mitigate the impact of habitual tobacco use
Project start date: 1996-05-01
Project end date: 2014-04-30
Budget start date: 1-MAY-2009
Budget end date: 30-APR-2010
PFA/PA: PA-07-070
2R01NS035469-14A1 (2009): $337969
SIGNAL TRANSDUCTION AT NEURONAL NICOTINIC SYNAPSES
Darwin K Berg, Professor
University Of California San Diego, 9500 Gilman Dr, Dept 0934, La Jolla, Ca 92093-0934
Grant 5R01NS012601-34 from National Institute Of Neurological Disorders And Stroke
Abstract: Nicotinic acetylcholine receptors (nAChRs) are widely expressed in the nervous system and have been implicated in a variety of behaviors and neuropathologies; In most cases the receptors reach their highest levels during late embryogenesis and early postnatal life. Almost nothing is known, however, about the developmental roles of neuronal nAChRs, other than that they are cation-selective and can regulate calcium- dependent events. We have preliminary evidence that during development nAChRs utilize signal transduction to regulate synaptic capabilities, including that of adjacent non-cholinergic pathways, in both the CNS and autonomic nervous systems. This proposal will test specific hypotheses about the roles of nicotinic input in shaping the development of nicotinic and GABAergic signaling, and will examine candidate molecules that may facilitate or transduce nicotinic effects. In Aim I the chick ciliary ganglion is used to test hypotheses about the significance of newly discovered GABAergic pathways in this cholinergic ganglion, the role of nicotinic input in controlling GABAergic maturation from excitatory to inhibitory mode, and the consequences this has for ganglionic throughput. In Aims II and III, the mammalian hippocampus is used to test hypotheses about the role of nicotinic signaling in regulating filopodia fate, induction of dendritic spines, maturation of GABAergic signaling from excitatory to inhibitory, and acute modulation of GABA responses. Long-lasting nicotine sensitization is also examined. In Aims IV and V three candidate proteins that appear to specifically interact with neuronal nAChRs are examined for their roles in determining the number, location, function, and downstream signaling capabilities of the receptors. Each of these aims is based on preliminary data supporting the central hypotheses being tested. The experimental approaches include electrophysiology, imaging, molecular biology, and biochemistry, and are carried out on neurons in cell culture and slice preparations from wildtype and mutant sources. Together these approaches and systems should provide mechanistic insight into novel roles of nicotinic signaling in the developing nervous system. The findings should also have biomedical relevance in revealing the consequences of nicotine exposure during these formative periods
Keywords: 21+ years old; 4-Aminobutanoic Acid; 4-Aminobutyric Acid; Acute; Adult; Affect; Aminalon; Aminalone; Ammon Horn; Autonomic nervous system; Behavior; Biochemistry; Blood Coagulation Factor IV; Butanoic acid, 4-amino-; Ca++ element; Calcium; Calcium Ion Signaling; Calcium Signaling; Cations; Cell Communication and Signaling; Cell Culture Techniques; Cell Signaling; Chemistry, Biological; Chronic; Coagulation Factor IV; Competence; Cornu Ammonis; DNA Molecular Biology; Data; Dendritic Spines; Development; Effectiveness; Electrophysiology; Electrophysiology (science); Embryo Development; Embryogenesis; Embryonic Development; Event; Factor IV; Filopodia; GABA; Ganglia; Ganglion Cysts; Ganglionic Cysts; Ganglions; Gene Expression; Hippocampus; Hippocampus (Brain); Human, Adult; Image; Intracellular Communication and Signaling; Life; Link; Location; Mediating; Mediation; Molecular Biology; Myxoid cyst; NRVS-SYS; Negotiating; Negotiation; Nerve Cells; Nerve Unit; Nervous System; Nervous system structure; Neural Cell; Neural Ganglion; Neurocyte; Neurologic Body System; Neurologic Organ System; Neurons; Neurophysiology / Electrophysiology; Nicotine; Nicotinic Acetylcholine Receptors; Nicotinic Receptors; Output; Pathway interactions; Position; Positioning Attribute; Preparation; Proteins; Pyramidal neuron; Pyridine, 3-(1-methyl-2-pyrrolidinyl)-, (S)-; Receptor Protein; Regulation; Role; Shapes; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Slice; Source; Specific qualifier value; Specified; Spinal Column; Spine; Structure; Structure of ciliary ganglion; Synapses; Synaptic; System; System, LOINC Axis 4; Testing; Transmission; Vertebral column; adult human (21+); backbone; base; biological signal transduction; cholinergic; ciliary ganglion; dendrite spine; gamma-Aminobutyric Acid; gene product; hippocampal; hippocampal pyramidal neuron; imaging; innervation; insight; mutant; nerve supply; neuronal; neuropathology; novel; pathway; postnatal; postsynaptic; presynaptic; receptor; response; scaffold; scaffolding; social role; trafficking; transmission process
Project start date: 1979-05-01
Project end date: 2011-04-30
Budget start date: 1-MAY-2009
Budget end date: 30-APR-2010
5R01NS012601-34 (2009): $330705
Sponsored Links Excellgen http://Excellgen.com
5R01NS012601-32 (2007): $331343
NEURONAL DEVELOPMENT AND SYNAPSE FORMATION IN VITRO
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01NS012601-03 from National Institute Of Neurological Disorders And Stroke IRG: NEUA
Abstract: Neuronal development and synapse formation will be examined in dissociated cell cultures prepared from embryonic chick tissue. The major cell type to be studied will be cholinergic neurons from spinal cord tissue and from ciliary ganglia. Skeletal myotubes will provide the postsynaptic target cells for the cholinergic neurons. Individual neuron cell types in heterogeneous cultures will be distinguished by electrophysiological methods and by autoradiographic labeling with neurotransmitter precursors. Neurotransmitter metabolism will be examined as a marker for the development of the presynaptic neurons. Electrophysiological techniques will be used to characterize functional synaptic transmission. The number and distribution of actylcholine receptors in the myotubes will be monitored to follow postsynaptic events during synapse formation. The correlation of these approaches should provide information about how synapses are formed between appropriate cell types.
Keywords: CELL TYPES, NERVOUS SYSTEM AUTONOMIC, PARASYMPATHETIC NERVOUS SYSTEM, NERVOUS SYSTEM, NERVE ENDINGS, SYNAPSES, NERVOUS SYSTEM, NERVES, INNERVATION (GENERAL), NERVOUS SYSTEM, NEUROGENESIS, NERVOUS SYSTEM, NEURONS, NEUROLOGY STUDY SECTION, CHOLINE, ACETYLCHOLINE, INFORMATION PROCESSING AND CONTROL (NEURAL), MEMBRANE, MEMBRANE (BIOLOGICAL) STRUCTURE, MUSCLE CELLS, NERVOUS SYSTEM CENTRAL, SPINAL CORD, NEUROPHYSIOLOGY, NEURAL TRANSMISSION, NEUROTRANSMITTERS RECEPTORS, ADRENERGIC RECEPTORS, DOPAMINERGIC RECEPTORS, neuroanatomy, BIRDS, CHICK EMBRYO, PHYSICAL SEPARATION, ELECTROPHORESIS, GEL, RADIOASSAY (RADIOMETRY), TISSUE (CELL) CULTURE
Project start date: 1976-05-01
Project end date: 1979-04-30
NEURONAL NICOTINIC RECEPTORS-EXPRESSION AND REGULATION
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01NS035469-09 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Abstract: From applicant s ) Nicotinic acetylcholine receptors containing the alpha7 gene product (alpha7-nACHRs) are among the most abundant in the nervous system and have been implicated in a wide variety of higher brain functions and neurodegenerative diseases. Because of their high calcium permeability and diverse locations, the receptors can influence many cellular events. The present renewal application is based on three new discoveries the receptors can be concentrated on somatic spines by cytoskeletal interactions for synaptic signaling; receptor function may be constrained substantially by negative regulation in vivo; and the receptors can influence gene expression in novel ways. These findings shape the four specific aims proposed. (1) Identify components that tether alpha7-nAChRs at synaptic sites. (2) Analyze mechanisms controlling the responsiveness of alpha7-nAChRs. (3) Assess the significance of alpha7-nAChR signaling for gene expression. (4) Test CNS models of postsynaptic alpha7-nAChRs. The initial work will be carried out with chick ciliary ganglion neurons because they provide the richest known source of the receptors. The neurons will be examined both in vivo and in cell culture to identify cell-cell interactions and receptor-associated molecules that govern somatic spine formation and alpha7-nAChR clustering. Exogenous components that dramatically increase the whole-cell alpha7-nAChR response will be examined for molecular mechanism and for in vivo relevance. The conditions and mechanisms enabling alpha7-nAChR receptors to influence gene expression will also be examined, and the gene families affected will be identified in order to test hypotheses about the physiological relevance of the effect. CNS neurons enriched in alpha7-nAChRs will then be used to determine which of the regulatory principles governing ciliary ganglion receptors can be extended broadly across systems having different physiological assignments. The experimental approaches will utilize fluorescence imaging, 3-D tomographic EM reconstruction, and whole-cell patch clamp recording in situ and in cell culture. These will be combined with biochemical and molecular biological approaches to identify and examine molecules influencing receptor function and location, and to determine which gene families are most affected by the receptors. The information obtained will indicate how a major nicotinic receptor in brain is controlled and how it, in turn, exerts long-term control through gene regulation. The biomedical relevance is substantial because of the enormous health-related consequences of tobacco usage and nicotine addiction and because of growing evidence that alpha7-nAChRs in particular are involved in neurodegenerative diseases such as Alzheimer s with a devastating toll on the public.
Keywords: biological signal transduction, gene induction /repression, intermolecular interaction, neuron, nicotinic receptor, receptor expression, age difference, cell cell interaction, cell component structure /function, central nervous system, ciliary ganglion, cytoskeleton, neurophysiology, posttranslational modification, protein localization, synapse, chicken, confocal scanning microscopy, electron microscopy, fluorescence, laboratory rabbit, laboratory rat, tissue /cell culture, voltage /patch clamp, western blotting
Project start date: 1996-05-01
Project end date: 2005-02-28
5R01NS035469-09 (2004): $337551
5R01NS035469-08 (2003): $337763
5R01NS035469-07 (2002): $337964
2R01NS035469-06 (2001): $341625
NEURONAL DEVELOPMENT AND SYNAPSE FORMATION IN VITRO
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01NS012601-05 from National Institute Of Neurological Disorders And Stroke IRG: NEUA
Abstract: Five related aspects of neuronal development and synapse formation will be examined in associated cell cultures prepared with embryonic chick ciliary ganglion (CG) neurons. These include (1) the normal determinants of neuronal survival, (2) the specification of neurotransmitter synthesis, (3) the regulation of the neuronal alpha-bungarotoxin binding component and its relation to the neuronal acetylcholine (ACh) receptor, (4) the formation and maintenance of nerve-nerve and nerve-muscle synapses, and (5) the neuronal regulation of myotube ACh receptors. When grown alone in cell culture with muscle- or heart-conditioned medium, CG neurons exhibit quantitative long-term survival, including those neurons that were destined to die in ovo as part of normal ganglionic development. The factor(s) present in conditioned medium that are responsible for the survival will be purified and their mode of action characterized. Neurotransmitter synthesis will be examined to identify the events that commit CG neurons to a cholinergic fate. An early developmental stage will be sought in which the neurons retain the capacity to express other differentiated properties characteristic of neuronal crest derivatives such as catecholamine biosynthesis. The binding of alpha-bungarotoxin to the neurons will be studied to distinguish the different classes of binding and to determine their relationship to the neuronal ACh receptor. Synapse formation in CG neuron-myotube cultures will be examined (1) to assess the stability of individual points of nerve-muscle synaptic contact, (b) to determine whether synaptic activity can prevent the formation of additional synapses on an innervated myotube, and (c) to determine whether CG neurons can innervate each other in cell culture. The neuronal regulation of myotube ACh receptors will be examined to identify the mechanisms underlying the induction of receptors and to determine whether the neurons also promote a re-distribution of the receptors in the myotube membrane. Fluorescent- and radiolabeled-toxins together with iontophoretic application of ACh will be used for the receptor analysis.
Keywords: NERVOUS SYSTEM AUTONOMIC, PARASYMPATHETIC NERVOUS SYSTEM, NERVOUS SYSTEM, NERVE ENDINGS, SYNAPSES, NERVOUS SYSTEM, NERVES, INNERVATION (GENERAL), NERVOUS SYSTEM, NEURONS, NEUROLOGY A STUDY SECTION, NEUROLOGY, DEVELOPMENTAL, NEUROGENESIS, CELL COMPONENTS, MICROTUBULES (GENERAL), CHOLINE, ACETYLCHOLINE, INFORMATION PROCESSING AND CONTROL (NEURAL), MEMBRANE, MEMBRANE (BIOLOGICAL) STRUCTURE, MUSCLE CELLS, NEUROPHYSIOLOGY, NEURAL TRANSMISSION, NEUROTRANSMITTERS BIOSYNTHESIS, NEUROTRANSMITTERS RECEPTORS, CHOLINERGIC RECEPTORS, TOXICOLOGY, ANIMAL POISONS, REPTILE, BUNGAROTOXIN, neuroanatomy, BIRDS, CHICK EMBRYO, ELECTROIONIZATION (IONTOPHORETIC) METHODS, NONTHERAPEUTIC, RADIOASSAY (RADIOMETRY), TISSUE (CELL) CULTURE, EMBRYONIC-FETAL CELL LINES
Project start date: 1979-05-01
Project end date: 1982-04-30
Beta-Amyloid Blockade Of Hippocampal Nicotinic Alpha7-R*
Darwin K Berg, Professor
University Of California San Diego
9500 Gilman Dr, Dept 0934
la Jolla, Ca 920930934
Grant 1R03AG019400-01 from National Institute On Aging IRG: ZAG1
Abstract: Alzheimer´s Disease Drug Discovery. Alzheimer´s disease is a physiologically devastating condition that is threatening to reach epidemic proportions as the population ages. Cholinergic deficit is a widely recognized aspect of Alzheimer´s disease, and is now thought to include nicotinic signaling as a critical component. One of the most interesting nicotinic receptors is the subtype composed of alpha7 subunits because it is widely expressed in the nervous system and has a high relative permeability to calcium which enables it to control diverse cellular functions. Recently it has been shown that alpha7-containing receptors bind the beta-amyloid peptide (amino acids 1-42) in the pM- nM range. The mammalian hippocampus is an interesting system for studying such interactions because alpha7-containing receptors are relatively abundant there, and the hippocampus is centrally involved in memory formation which is a major target of Alzheimer´s disease. In preliminary studies we have found that the beta-amyloid peptide in the nM range reversibly inhibits alpha7-containing nicotinic receptors on hippocampal neurons in culture. Surprisingly, certain peptides applied exogenously can quickly and reversibly potentiate the alpha7- containing receptor response by an order of magnitude. This raises the real possibility that drugs capable of accessing the same site may be able to compensate for beta-amyloid inhibition of the receptors. This pilot proposal will examine the interactive of beta-amyloid peptide with hippocampal alpha7-containing receptors both functionally and physically. In addition, it will test the hypothesis that exogenous application of appropriate regulatory molecules will overcome the inhibition mediated by beta-amyloid peptide. The approach will use whole-cell patch clamp recording (usually in perforated patch mode) from the neurons while applying test compounds in desired sequences from a multi-bore rapid applicator. Competition binding experiments with radiolabeled probes will also be used to assess direct interactions. Functional characterization will include modulation of spontaneous synaptic events in the cultures by alpha7-containing receptors, and determining the impact of beta-amyloid peptide on the modulation in the presence of absence of the candidate potentiators. If this pilot study proves successful (as the preliminary experiments encourage us to think it will), the results will provide the basis for a subsequent full-scale follow-up to analyze beta-amyloid effects on CNS alpha7-containing nicotinic receptors, to analyze Alzheimer´s brain tissue for specific involvement of alpha7-receptors in the disease, and to evaluate compounds capable of crossing the blood-brain barrier and potentiating the receptors in situ as a possible therapeutic strategy
Keywords: Alzheimer`s disease, amyloid protein, hippocampus, nicotinic receptor neuron, receptor sensitivity laboratory rat, tissue /cell culture, voltage /patch clamp
Project start date: 2001-07-03
Project end date: 2003-06-30
1R03AG019400-01 (2001): $76000
NEURONAL NICOTINIC RECEPTORS--EXPRESSION AND REGULATION
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01NS035469-05 from National Institute Of Neurological Disorders And Stroke IRG: NLS
Abstract: Adapted from applicant s ) Neuronal nicotinic acetylcholine receptors (AChRs) will be studied using stably transfected QT-6 quail cell lines to express epitope-tagged chick neuronal AChR gene constructs under inducible promoters. a7 AChRs will be given special emphasis because of their unusual properties (they function as homomers, have high calcium permeability and are located extrasynaptically in ciliary ganglia). Also to be studied are combinations of a3, b4, a5 + b2 subunits (because AChRs of these types are found postsynaptically in ciliary ganglia). The cell lines will be used to analyze subunit interactions, assembly pathways, and functional properties. Surface AChRs will be quantitated using toxins and mAbs to the epitope tags. Function will be assayed by whole cell patch clamp recording. Assembly intermediates will be identified by pulse labeling and immune precipitation. Subunit composition will be analyzed by immunoprecipitation and immunoblot analysis using mAbs to subunits and tags. Distribution within the cells will be evaluated by confocal microscopy. Neuronal cell lines which express a7 will also be used for comparison. These studies should reveal important aspects of the cell and molecular biology of neuronal nicotinic receptors and are relevant to the role of nicotinic AChRs in nicotine addiction.
Keywords: ciliary ganglion, neuron, nicotinic receptor, receptor expression, calcium flux, gene induction /repression, genetic promoter element, intermolecular interaction, confocal scanning microscopy, immunoprecipitation, tissue /cell culture, voltage /patch clamp
Project start date: 1996-05-01
Project end date: 2001-02-28
5R01NS035469-05 (2000): $236776
5R01NS035469-04 (1999): $227669
Sponsored Links Excellgen http://Excellgen.com
5R01NS035469-03 (1998): $218911
5R01NS035469-02 (1997): $210488
3R01NS035469-04S1 (1999): $50000
Darwin K Berg
University Of California San Diego
Project start date: 1996-05-01
Project end date: 2014-04-30
Neuronal Nicotinic Receptors- Expression And Regulation
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 3R01NS035469-10S1 from National Institute Of Neurological Disorders And Stroke IRG: SYN
Project start date: 1996-05-01
Project end date: 2009-02-28
3R01NS035469-10S1 (2006): $12046
CELLULAR And MOLECULAR NEUROBIOLOGY: SYNAPSE FORMATION And MODULATION
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 2P41RR008605-110045 from National Center For Research Resources IRG: ZRG1
Keywords: biomedical resource, developmental neurobiology, synapse, synaptogenesis
Project start date: 2004-06-18
Project end date: 2005-04-30
NEURONAL DEVELOPMENT AND SYNAPSE FORMATION IN VITRO
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 5R01NS012601-21 from National Institute Of Neurological Disorders And Stroke IRG: NEUC
Project start date: 1979-05-01
Project end date: 1997-04-30
5R01NS012601-21 (1996): $240589
NEURONAL NICOTINIC RECEPTORS--EXPRESSION AND REGULATION
Darwin K Berg, Professor
University Of California San Diego 9500 Gilman Dr, Dept 0934 La Jolla, Ca 920930934
Grant 1R01NS035469-01 from National Institute Of Neurological Disorders And Stroke IRG: NLS
Project start date: 1996-05-01
Project end date: 2001-04-30
1R01NS035469-01 (1996): $194410