Research Overview
Our research focuses on understanding neural circuits – the functionally connected neurons that give rise to thought and behavior. We are particularly interested in how neurons build appropriate synaptic connections and modulate their strength. Defects in synaptic development and plasticity are associated with a broad range of neurological disorders including developmental disorders such as autism; motor, cognitive and psychological impairment; and neurodegeneration. Thus, the identification and characterization of the molecules that regulate synapse formation and function is critical to our understanding of normal neural function and our ability to treat a variety of neurological disorders.
Communication between neurons occurs primarily at chemical synapses where action potentials trigger calcium-dependent release of neurotransmitter-filled synaptic vesicles at presynaptic active zones. This signal is received by neurotransmitter receptors that cluster opposite active zones in the postsynaptic cell. Synapse formation is a complex process that depends on intercellular communication between pre and postsynaptic cells – often at sites very distant from cell bodies. In fact, a single neuron can form thousands of synapses and must independently regulate the development of each. Synapses must also maintain the lifelong ability to modify their morphology and physiology in response to environmental stimuli and changes in activity levels – modifications that are thought to underlie learning and memory.
To understand the molecules and mechanisms underlying the formation, function, and plasticity of synapses, we conduct studies in Drosophila, where we employ the rich array of genetic tools and model synapses accessible to physiological, imaging, and behavioral studies. The remarkable conservation of synaptic genes enables us to gain generalizable insights into gene and synapse function. In parallel, we have focused on developing new genome engineering tools to overcome barriers to the study of synapses in vivo.
Identification of new regulators of synapse formation, function and plasticity: In our ongoing screens for new synaptic proteins, we are combining CRISPR with bioinformatic approaches for identifying evolutionarily conserved candidate synaptic genes. We are currently characterizing several recently identified genes that encode poorly understood, conserved neuronal proteins associated with human disease. We identified, Fife, the Drosophila homolog of Piccolo, in a behavioral screen, and found that Fife mutants exhibit locomotor deficits and severely decreased neurotransmitter release (Bruckner, Gratz et al., 2012). Prior to this identification, it was believed that Piccolo was a vertebrate-specific component of the otherwise highly conserved complex of presynaptic scaffolding proteins known as the active zone cytomatrix. Piccolo is linked to major depression and bipolar disorder, but has been difficult to study in mammals due to genetic redundancy. Through loss-of-function studies in Drosophila, we determined that Fife promotes high-probability neurotransmitter release by organizing active zone structure to spatially couple Ca2+ channel clusters and release-ready synaptic vesicles in nanometer proximity. In addition to its role in determining baseline release probability, Fife is essential for presynaptic homeostatic potentiation (Bruckner et al., 2017). These studies help explain how synaptic strength is established and modified locally at active zones to tune communication in neural circuits.
Understanding dynamic synapse structure-function relationships: To predictably control behavior while remaining responsive to an ever-changing environment, neural circuits must balance reliability and plasticity. Intrinsic variability in presynaptic neurotransmitter release properties may facilitate this balance. We are combining genetic and multi-level imaging approaches to gain a comprehensive view of synapse structure and its molecular determinants to advance understanding of how active zones are organized to achieve precise release properties – and reorganized to modulate circuit function in the face of changing inputs. To bridge the gap between synaptic function and structure, we are employing electron tomographic studies of synapse ultrastructure (Bruckner et al., 2017; Zhan et al., 2016), and combining CRISPR-mediated endogenous tagging of synaptic proteins with correlative light-electron microscopy, superresolution imaging, and functional imaging at the level of single synapses. We employed this approach in collaboration with the labs of Greg Macleod and Dion Dickman to investigate calcium channel regulation during presynaptic homeostasis – a form of synaptic plasticity that maintains stable circuit function in the face of perturbations. Surprisingly, different strategies are used to modulate calcium channels during opposing forms of plasticity. Live imaging revealed the recruitment of calcium channels to individual synapses within minutes to increase neurotransmitter release and stabilize communication (Gratz et al., 2019). Structural reorganization of active zones appears to be limited to presynaptic homeostatic potentiation as perturbations that trigger the homeostatic depression of neurotransmitter release to stabilize communication do not induce modulation of endogenous calcium channel levels. These findings provide insight into dynamic structure-function relationships at synapses, and suggest targets for actively manipulating synapse function.
Gene editing: Genetic and molecular techniques to manipulate the genomes of organisms are invaluable tools for understanding gene function. In collaboration with the labs of Jill Wildonger and Melissa Harrison at the University of Wisconsin-Madison, we demonstrated that the bacterially derived CRISPR RNA-guided Cas9 nuclease can be employed to delete and replace genes in Drosophila, and that these genome modifications can be efficiently transmitted through the germline. We developed an online tool for identifying highly specific CRISPR target sites and high-throughput approaches for following endogenous synaptic proteins in vivo, including in subsets of their endogenous patterns, and for identifying, labeling, and controlling specific subpopulations of neurons in the intact brain. The ability to follow endogenous synaptic proteins in sparse subsets of neurons will greatly facilitate in vivo studies in the synapse-dense central nervous system. For more information visit the flyCRISPR website.