Faculty Bibliography

The use of fluorescence-based calcium indicators has, over the years, unraveled important calcium-dependent mechanisms underlying neuronal function and development. However, difficulties associated with the loading of calcium indicators have limited their widespread use, particularly for the study of neuronal processing in the adult nervous system. Here, we show that in the central and peripheral nervous systems, populations of neurons and their processes, including dendritic spines and filopodia, can be labeled rapidly and efficiently by delivering calcium indicator-coated particles using a 'gene gun'. Importantly, neuronal labeling occurred both in vitro and in vivo, and across a wide range of ages and preparations. The labeled cells demonstrate spontaneous and evoked calcium transients, indicating that particle-mediated delivery is not deleterious to neuronal function. Furthermore, unlike loading with patch pipettes, cytoplasmic content is preserved following ballistic loading. This enables the study of calcium-dependent second messenger pathways without loss of signaling components. The ballistic delivery of calcium indicators thus opens up many new avenues for further exploration of the structure and function of the nervous system from single spines to neuronal networks

In developing muscle, synapse elimination reduces the number of motor axons that innervate each postsynaptic cell. This loss of connections is thought to be a consequence of axon branch trimming. However, branch retraction has not been observed directly, and many questions remain, such as: do all motor axons retract branches, are eliminated branches withdrawn synchronously, and are withdrawing branches localized to particular regions? To address these questions, we used transgenic mice that express fluorescent proteins in small subsets of motor axons, providing a unique opportunity to reconstruct complete axonal arbors and identify all the postsynaptic targets. We found that, during early postnatal development, each motor axon loses terminal branches, but retracting branches withdraw asynchronously and without obvious spatial bias, suggesting that local interactions at each neuromuscular junction regulate synapse elimination

We describe a technique for rapid labeling of a large number of cells in the nervous system with many different colors. By delivering lipophilic dye-coated particles to neuronal preparations with a 'gene gun,' individual neurons and glia whose membranes are contacted by the particles are quickly labeled. Using particles that are each coated with different combinations of various lipophilic dyes, many cells within a complex neuronal network can be simultaneously labeled with a wide variety of colors. This approach is most effective in living material but also labels previously fixed material. In living material, labeled neurons continue to show normal synaptic responses and undergo dendritic remodeling. This technique is thus useful for studying structural plasticity of neuronal circuits in living preparations. In addition, the Golgi-like labeling of neurons with many different colors provides a novel way to study neuronal connectivity

We describe a method for in vivo confocal fluorescence imaging of synaptic terminals and subsequent electron microscopic reconstructions of the same terminals. By iontophoretically applying lipophilic dye to nerve terminals at a single neuromuscular junction with a sharp microelectrode in living neonatal mice, we were able to quickly label other synaptic terminals of the same motor unit. This vital labeling technique allows the same synapses to be imaged in living animals for several days. By using two dyes applied to separate junctions we could visualize competing axons converging at the same site. We also show that similar approaches can be used to study synaptic inputs to neurons. Following photoconversion, the dye labeled axons and synapses were easily identified and distinguished from unlabeled synapses of other axons ultrastructurally. This new labeling technique thus provides a useful means to study reorganization of synaptic structure at high temporal and spatial resolution

Throughout the developing nervous system, competition between axons causes the permanent removal of some synaptic connections. In mouse neuromuscular junctions at birth, terminal branches of different axons are intermingled. However, during the several weeks after birth, these branches progressively segregated into nonoverlapping compartments before the complete withdrawal of all but one axon. Segregation was caused by selective branch atrophy, detachment, and withdrawal; the axon branches that were nearest to the competitor's branches were removed before the more distant branches were removed. This progression suggests that the signals that mediate the competitive removal of synapses must decrease in potency over short distances