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The basic interests of the laboratory centre around the mechanisms by which neurotransmitter release is regulated at central nervous system (CNS) synapses.
1) Presynaptic receptors, through ionotropic and metabotropic mechanisms, represent a fundamental means for regulating neurotransmitter release. One of our interests is to identify and characterize presynaptic receptors that modulate the release of the neurotransmitters glutamate and GABA. The model system we use for these studies is the isolated nerve terminal preparation (synaptosomes). Nerve terminal depolarization leads to Ca2+-influx and exocytosis, followed by endocytosis and recycling of transmitter containing small synaptic vesicles (SSVs). To delineate the loci at which presynaptic receptor activation impinge, we use membrane potential-sensitive dyes to assay nerve terminal excitability and depolarization, fura-2 to monitor Ca2+-influx and on-line enzymatic assays or HPLC to determine the release of glutamate and GABA by the exocytosis of small synaptic vesicles (SSVs).
Post-translational modification of the proteins involved in the cascade of events leading to neurotransmitter release offers a powerful means of mediating presynaptic plasticity. Thus, one way that presynaptic receptor activation can potentially modulate the properties of proteins involved in neurotransmitter release is through the stimulation of second messenger cascades that lead to protein phosphorylation or dephosphorylation. Using synaptosomes labelled with 32P-orthophosphate, we can ascertain presynaptic receptor-mediated activation of specific protein and lipid kinases and phosphatases employing identified intraterminal substrates for these enzymes. Currently, we are characterising the nerve terminal modulatory roles of mitogen-activated protein kinases and lipid kinases leading to the production of polyphosphoinositides.
2) The second major focus of the laboratory is to determine the role of specific protein kinases or phosphotases in the cascade of events leading to SSV exocytosis and endocytosis. For these studies, we have taken the approach of altering enzyme expression in neuronal cell lines and primary cell cultures that are amenable to molecular biological procedures. Currently, we are evaluating the effects of altered expression of the major Ca2+-dependent protein phosphatase, protein phosphatase 2B (calcineurin, CN) and its associated proteins. We wish to characterize of the role of CN in: (a) controlling voltage-dependent Ca2+-influx and, (b) the exocytotic/endocytotic cycling of SSVs and control thereof by the CN substrates, synapsin I and dynamin. We examine VDCC activity using whole-cell patch-clamping and Ca2+-influx, either spectrophometrically or by Ca2+-imaging of single cells, using Ca2+-sensitive fluorophors. Effects of phosphorylation/dephosphorylation on SSV-associated proteins are elucidated using styryl SV-probes (e.g. FM1-43, FM2-10) to image endocytosis/exocytosis.
The basic interests of the laboratory centre around the mechanisms by which neurotransmitter release is regulated at central nervous system (CNS) synapses.
1) Presynaptic receptors, through ionotropic and metabotropic mechanisms, represent a fundamental means for regulating neurotransmitter release. One of our interests is to identify and characterize presynaptic receptors that modulate the release of the neurotransmitters glutamate and GABA. The model system we use for these studies is the isolated nerve terminal preparation (synaptosomes). Nerve terminal depolarization leads to Ca2+-influx and exocytosis, followed by endocytosis and recycling of transmitter containing small synaptic vesicles (SSVs). To delineate the loci at which presynaptic receptor activation impinge, we use membrane potential-sensitive dyes to assay nerve terminal excitability and depolarization, fura-2 to monitor Ca2+-influx and on-line enzymatic assays or HPLC to determine the release of glutamate and GABA by the exocytosis of small synaptic vesicles (SSVs).
Post-translational modification of the proteins involved in the cascade of events leading to neurotransmitter release offers a powerful means of mediating presynaptic plasticity. Thus, one way that presynaptic receptor activation can potentially modulate the properties of proteins involved in neurotransmitter release is through the stimulation of second messenger cascades that lead to protein phosphorylation or dephosphorylation. Using synaptosomes labelled with 32P-orthophosphate, we can ascertain presynaptic receptor-mediated activation of specific protein and lipid kinases and phosphatases employing identified intraterminal substrates for these enzymes. Currently, we are characterising the nerve terminal modulatory roles of mitogen-activated protein kinases and lipid kinases leading to the production of polyphosphoinositides.
2) The second major focus of the laboratory is to determine the role of specific protein kinases or phosphotases in the cascade of events leading to SSV exocytosis and endocytosis. For these studies, we have taken the approach of altering enzyme expression in neuronal cell lines and primary cell cultures that are amenable to molecular biological procedures. Currently, we are evaluating the effects of altered expression of the major Ca2+-dependent protein phosphatase, protein phosphatase 2B (calcineurin, CN) and its associated proteins. We wish to characterize of the role of CN in: (a) controlling voltage-dependent Ca2+-influx and, (b) the exocytotic/endocytotic cycling of SSVs and control thereof by the CN substrates, synapsin I and dynamin. We examine VDCC activity using whole-cell patch-clamping and Ca2+-influx, either spectrophometrically or by Ca2+-imaging of single cells, using Ca2+-sensitive fluorophors. Effects of phosphorylation/dephosphorylation on SSV-associated proteins are elucidated using styryl SV-probes (e.g. FM1-43, FM2-10) to image endocytosis/exocytosis.
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Frontiers in molecular neuroscience (2018): 195-195
G. Flores,J. V. Negrete-Diaz, M. Carrion,Y. Andrade-Talavera, S. A. Bello,T. S. Sihra,A. Rodriguez-Moreno
AMINO ACIDS IN HUMAN NUTRITION AND HEALTHpp.427-453, (2012)
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作者统计
#Papers: 55
#Citation: 3698
H-Index: 32
G-Index: 55
Sociability: 5
Diversity: 1
Activity: 0
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