This project will examine the cellular and molecular mechanisms that govern the establishment and refinement of synaptic connections in a model system, the Drosophila neuromuscular junction. Recent studies in this system indicate that axon guidance and synaptogenesis involve multiple repulsive and attractive signals that act on the growth cones. The mechanisms of efferent outgrowth and neuromuscular taget selection will be examined in developing embryos and larvae using new Drosophila lines with endogenous Green Fluorescent Protein (GFP) expression. Detailed time-lapse morphometric studies of the growth cones will be made, testing the resonses to several cellular, genetic, and molecular manipulations. There is good evidence for morphological plasticity in the neuromuscular system, in response to alterations of the degree of innervation and synaptic activity. The mechanisms of motoneuron arbor refinement and plasticity will be tested by vital imaging of the developing synapses in both embros and lavae, in response to situations where neuromuscular activity is altered pharmacologically or genetically, as well as in response to denervation or altered innervation protocols. Also, candidate genes whose functions are needed to mediate neuromuscular development and synaptic plasticity will be identified in a F1 mutagenesis screen. Finally, as part of a revers genetic study, 4200 P-element enhancer detector inserts were screened for expression patterns in the developing neuromuscular system. 20 Drosophila lines were identified with specificity of expression in subsets of muscle fibers and/or neurons during embryonic synaptogenesis. We will complete an ongoing molecular characterization of these enhancer detector lines, including a detailed analysis of mutant phenotypes that result from excision of the P-element inserts.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS031651-06
Application #
2750869
Study Section
Neurology B Subcommittee 2 (NEUB)
Program Officer
Nichols, Paul L
Project Start
1993-05-01
Project End
2001-07-31
Budget Start
1998-08-01
Budget End
1999-07-31
Support Year
6
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Yale University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Vonhoff, Fernando; Keshishian, Haig (2017) In Vivo Calcium Signaling during Synaptic Refinement at the Drosophila Neuromuscular Junction. J Neurosci 37:5511-5526
Vonhoff, Fernando; Keshishian, Haig (2017) Cyclic nucleotide signaling is required during synaptic refinement at the Drosophila neuromuscular junction. Dev Neurobiol 77:39-60
Berke, Brett; Wittnam, Jessica; McNeill, Elizabeth et al. (2013) Retrograde BMP signaling at the synapse: a permissive signal for synapse maturation and activity-dependent plasticity. J Neurosci 33:17937-50
Olsen, Douglas P; Keshishian, Haig (2012) Experimental methods for examining synaptic plasticity in Drosophila. Cold Spring Harb Protoc 2012:162-73
Berke, Brett; Keshishian, Haig (2011) Cracking the combinatorial semaphorin code. Neuron 70:175-7
Leiserson, William M; Keshishian, Haig (2011) Maintenance and regulation of extracellular volume and the ion environment in Drosophila larval nerves. Glia 59:1312-21
Leiserson, William M; Forbush, Biff; Keshishian, Haig (2011) Drosophila glia use a conserved cotransporter mechanism to regulate extracellular volume. Glia 59:320-32
Carrillo, Robert A; Olsen, Douglas P; Yoon, Kenneth S et al. (2010) Presynaptic activity and CaMKII modulate retrograde semaphorin signaling and synaptic refinement. Neuron 68:32-44
Nicholson, Louise; Singh, Gunisha K; Osterwalder, Thomas et al. (2008) Spatial and temporal control of gene expression in Drosophila using the inducible GeneSwitch GAL4 system. I. Screen for larval nervous system drivers. Genetics 178:215-34
Fernandes, Joyce J; Keshishian, Haig (2005) Motoneurons regulate myoblast proliferation and patterning in Drosophila. Dev Biol 277:493-505

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