The refinement of synaptic connections, through selective elimination and consolidation of early-formed synapses, is crucial for generating functional synaptic circuitry in many regions of the nervous system. Defects in this synapse elimination are thought to underlie some types of developmental neurological disorders in humans. Our long-term objective is to understand mechanisms determining the retention of some synapses (winners) versus the elimination of others (losers) in the developing central nervous system (CNS). Ultimately, we aim to identify signaling molecules that selectively strengthen winners and/or weaken losers;such molecules offer the potential of manipulating synapse elimination in order to treat neurological disorders that currently have no effective clinical intervention. The goal of this project is to reveal how winners and losers are determined during competitive elimination of supernumerary climbing fiber (CF) axons in the developing cerebellum, a classic example of synapse elimination in the CNS. We have developed three innovative methods that overcome some of the fundamental technical limitations of studying CNS synapse elimination and promise mechanistic insight: 1) multi-color in vivo imaging that allows time-lapse vital observation of axonal competition for the first time in the CNS, 2) selective photo-ablation of target CFs combined with the multi-color in vivo imaging, and 3) electrophysiological analysis of two competing CFs with clear morphological identities in acute cerebellar slices. Preliminary studies using these methods strongly suggest that CF terminals have distinct structural and functional properties depending on their postsynaptic location (soma versus dendrites), providing competitive vigor to the CFs with dendritic synapses. Pursuing these studies is of great significance because it is expected to reveal previously unrecognized mechanisms of synapse elimination in the CNS: developing synapses are regulated differently among different postsynaptic domains and the difference plays a crucial role in winner/loser determination. In the proposed research, we will use the methods listed above and reveal how structural stability (Aim 1) and functional maturity (Aim 2) of competing CFs are regulated differently between soma and dendrites. We will also examine how the decision of winner/loser is determined by selective photo-ablation of winning CFs.
In Aim 3, we will determine how protein kinase C? (PKC?), a known signaling molecule involved in CF elimination, functions in this subcellular location- specific regulation of CFs. To do so, we will compare structural stabiliy and functional maturity of CF terminals between soma and dendrites in PKC? deficient mice. The completion of this project will provide substantial new insight into this classic example of synapse elimination in the CNS and explore novel roles of neuronal subcellular domains in determining winners versus losers during developmental refinement of synaptic circuitry.
The proposed research is relevant to public health because emerging evidence strongly suggests that defects in developmental synapse elimination underlie human neurological disorders, including autism spectrum disorders, episodic ataxia type 2, and spinocerebellar ataxia type 14. Deeper understanding of the mechanisms of winner/loser determination is expected to provide substantial new insights into normal CNS development as well as guide the design of future therapies for these neurological disorders. Thus, this project is directly relevant to the part of NIH's mission that pertains to reducing the burden of neurological diseases through basic research.
