George J. Augustine - US grants

The goal of this research is to further elucidate the physiological properties of chemical synapses between nerve cells. In particular, this project considers the entry of calcium (Ca) ions into presynaptic nerve terminals and the ability of Ca ions within nerve terminals to trigger transmitter secretion. Experiments will be performed upon squid 'giant' presynaptic terminals, one of the few nerve terminals large enough to permit direct, electrical measurements of Ca entry and accumulation. This project considers several aspects of the role of Ca in presynaptic function. Entry of Ca ions into presynaptic terminals will be assessed by measuring Ca currents with the 3-microelectrode voltage clamp method. The hypothesis that the neuropeptide enkephalin decreases synaptic transmission by decreasing entry of Ca ions into presynaptic terminals will be tested by examining the effect of enkephalin upon voltage-gated Ca currents. The ability of Ca ions within presynaptic terminals to trigger transmitter release will be examined by measuring the relationship between external Ca concentration, Ca entry and transmitter release. These experiments will also use Ca-sensitive microelectrodes to ask whether presynaptic neurotoxins trigger transmitter release by increasing the concentration of Ca ions within nerve terminals. Consideration of these specific roles of Ca ions in the function of presynaptic terminals will help define the physiological processes underlying synaptic transmission, and may ultimately clarify the action of numerous neurological disorders which result from abnormal synaptic function.

[unreadable] DESCRIPTION (provided by applicant): The release of neurotransmitters at synapses is a very important process that is responsible for information flow through brain circuits. Modifications in neurotransmitter release also are likely to be involved in changing neuronal function during development, learning, disease states, and other forms of brain plasticity. Neurotransmitter release is known to result from the orderly conduit of synaptic vesicles through a series of membrane trafficking reactions. The general goal of this project is to understand the molecular basis for these reactions, in particular the exocytotic release of neurotransmitters and the endocytotic recycling of vesicle components. In recent years, many presynaptic proteins have been identified and a large fraction of these now have been implicated in neurotransmitter release. However, because so many proteins are involved, it is difficult to sort out the specific role that each plays in synaptic vesicle trafficking. We will address this problem by defining the temporal order of protein action, specifically by determining when several key proteins act relative to each other and relative to the time at which synaptic vesicles fuse or are endocytosed. For this purpose, we will use high-resolution optical methods to determine the timing of protein action. The proteins to be considered include SNARE proteins, clathrin, and clathrin-associated proteins, such as AP180, Eps15, epsin, and auxilin. Flash photolysis of caged binding-site peptides will be used to examine several interactions involving SNARE proteins, while Fluorescence resonance energy transfer will be used to look at interactions of clathrin with its banding partners during endocytosis. The results of these experiments will discriminate among many existing molecular models of synaptic vesicle trafficking reactions and lead to more refined, quantitative models. By clarifying several important aspects of the molecular basis of synaptic communication in the brain, this work will ultimately yield insights into the etiology of numerous neurological disorders that result from defects in synaptic transmission. [unreadable] [unreadable]

neural transmission; synapses; epilepsy; hippocampus; granule cell; kindling; glutamate receptor; receptor expression; evoked potentials; glutamates; pyramidal cells; calcium flux; synapsins; neuropharmacology; calmodulin dependent protein kinase; tissue /cell culture; flash photolysis; laboratory rat; voltage /patch clamp;

Simultaneous activity in the parallel fiber (PF) and climbing fiber (CF) synapses innervating cerebellar Purkinje cells causes a long-lasting depression (LTD) of transmission at the PF synapse. This LTD is a form of synaptic plasticity that may underlie certain forms of learning that occur in the cerebellum and is widely studied as an experimentally tractable example of long-lasting synaptic plasticity. The general goal of this project is to understand the molecular events that occur in postsynaptic Purkinje cells to produce LTD. The specific focus of the work is to study the roles of intracellular second messengers, such as IP3 and Ca ions, in inducing LTD and to understand how such messengers cause long-lasting changes in postsynaptic glutamate receptors. The proposed experiments combine electrical and optical methods to study these signaling pathways. Light-induced lease of Ca and IP3 from inert "caged" molecules will be used to elevate the concentration of these messengers very rapidly within small regions of the Purkinje cell. Simultaneous use of a high-speed confocal laser-scanning microscope will permit measurement of the consequences of these manipulations upon Ca concentration within the Purkinje cell and patch-clamp measurements will determine whether these messengers cause a LTD or PF synaptic transmission. Such experiments will define the roles of IP3 and Ca in LTD. Localized light-induced release of glutamate will also define, for the first time, the changes that occur in Purkinje cell glutamate receptors during LTD. This information will provide a molecular description of the final expression of LTD and give further clues about the coupling of second messenger pathways to glutamate receptors. Although this work is primarily basic research, it will yield new insights into basic signaling processes of neurons and synapses and will be useful for understanding the roles of such processes in the various neurological diseases that arise from defective neuronal signaling.

While many methods are available for functional imaging of excitatory processes in the brain, until now there has been no practical way to image synaptic inhibition in the brain. The goal of this project is to adopt clomeleon, a genetically-encoded indicator protein, for imaging Cl-dependent synaptic inhibition in the brain. This indicator was produced by fusing the chloride-sensitive yellow fluorescent protein with the chloride-insensitive cyan fluorescent protein; the ratio of fluorescence resonance energy transfer (FRET) dependent emission of these two fluorophores varies in proportion to the intracellular concentration of chloride ions ([Cl-]i). The proposed experiments will use a genetic strategy to target expression of clomeleon to subsets of neurons in the mouse brain. This should allow many novel experimental analyses of the physiological functions of Cl-. During the Phase I experiments proposed here, fluorescence imaging methods will be use to look for neuron-specific variations in resting [Cl-]i and changes in [Cl-]i associated with activation of inhibitory synaptic pathways in neurons of hippocampal, cerebellar, and cortical tissue slices. In Phase II, these experimental procedures will be extended to in vivo conditions to image the temporal and spatial patterns of synaptic inhibition in neural networks of the intact brain. This new technology should provide the first experimental views of the dynamics of synaptic inhibition in the brain and offers the promise of elucidating many important features of brain activity during normal function and as a consequence of drug abuse.

DESCRIPTION (provided by applicant): The release of neurotransmitters at chemical synapses is a process that is central to information transmission and storage within the brain. In this project, molecular genetic approaches will be used to define the functions of synapsins, a family of proteins thought to be important for neurotransmitter release. Mice deficient in the three mammalian synapsin genes will be generated by targeted gene disruption, with the expectation that removal of synapsins will impair any synaptic functions that rely upon these proteins. Synaptic transmission will be assessed by performing electrical and optical measurements on neurons from these synapsin-deficient mice. The functions of the three synapsin genes will then be assessed by transfecting them individually into synapsin-deficient neurons and then comparing the ability of each synapsin to rescue the synaptic defects observed in the mutant neurons. Using a similar technical approach, the roles of phosphorylation in regulating the function of synapsins will be assessed by transfecting synapsins with mutations that prevent phosphorylation or with mutations that mimic permanent phosphorylation. Likewise, the role of the reversible association of synapsins with the synaptic vesicle will be addressed by imaging this process in living neurons and by determining how alterations in the association of synapsins with vesicles alters neurotransmitter release properties. These experiments should clarify important aspects of the molecular basis of communication in the brain and, ultimately, will field insights into neurological and psychiatric disorders that result from defects in synaptic transmission.

DESCRIPTION (provided by applicant): While many methods are available for functional imaging of excitatory processes in the brain, until now there has been no practical way to image synaptic inhibition in the brain. The goal of this project is to use Clomeleon, a genetically-encoded indicator protein, to image chloride-dependent synaptic inhibition in the brain. This indicator was produced by fusing the chloride-sensitive yellow fluorescent protein with the chloride-insensitive cyan fluorescent protein; the ratio of fluorescence resonance energy transfer dependent emission of these two fluorophores varies in proportion to the intracellular concentration of chloride ions ([Cl]i). The proposed experiments are designed to optimize the Clomeleon technique for imaging the spatial and temporal dynamics of inhibitory circuits in living brain tissue. First, we will use mutagenesis to change the chloride binding properties of Clomeleon. This will enhance the ability of Clomeleon to report changes in [Cl]i in the range relevant for synaptic inhibition. Second, we will use two genetic strategies to target expression of Clomeleon to subsets of neurons in the mouse brain. Third, we will use fluorescence imaging methods to measure changes in [Cl]i associated with activation of inhibitory synaptic pathways in neurons of slices of the cerebellum, amygdala, and superior colliculus from these Clomeleon-expressing mice. While these experimental procedures will be used in vitro, it is hoped that our improvements in Clomeleon technology eventually will allow imaging of the temporal and spatial patterns of synaptic inhibition in neural networks of the intact brain. This new technology should provide the first spatially-resolved views of the dynamics of synaptic inhibition in the brain and offers the promise of elucidating many important features of brain activity during normal function, during psychiatric and neurological disorders, and as a consequence of drug abuse.