Johns Hopkins School of Medicine

Revealing ultrastructural plasticity of neurons with a millisecond resolution

Our research program aims to uncover the cellular, molecular, and biophysical principles that underlie neuronal and synaptic activity. Synapses are the fundamental processing units of the brain, mediating neuronal communication through regulated release of signaling molecules, neurotransmitters, and display remarkable plasticity for information transfer and storage. Their malfunction has been linked to many neurological disorders and neurodegenerative diseases. Thus, mechanistic descriptions of these processes are essential in understanding the physiology of neurons in health and disease. 

At its heart, synaptic transmission involves exocytosis of vesicles that contain neurotransmitters and subsequent retrieval of vesicles through endocytosis. Many synaptic proteins have been identified, but how these proteins are organized and regulated in space and time for these membrane trafficking events is not well understood. Our research program aims to bridge this knowledge gap by developing innovative electron microscopy techniques* to probe ultrastructural changes of neurons and molecular organization during synaptic transmission with millisecond resolution. Our observations so far have led to five major findings:

1. Exocytic sites and neurotransmitter receptors are organized trans-synaptically to efficiently activate NMDA-type glutamate receptors, which act as a switch for plasticity. 
2. Synaptic signaling is transiently amplified by accelerated docking of new vesicles at exocytic sites – a process we have termed ‘transient docking’.
3. The mechanical force generated by exocytosis induces vesicle recovery by an “ultrafast” endocytosis cycle.
4. Pre-recruitment of endocytic proteins enables completion of ultrafast endocytosis within 100ms.
5. Clathrin-dependent endosomal sorting completes the synaptic vesicle cycle.

We are now uniquely positioned to build on these advances to dissect the mechanisms that underlie synaptic transmission and plasticity. Our goal is to arrive at a complete molecular and physical description of 1) synaptic vesicle exocytosis, 2) synaptic vesicle endocytosis, 3) axonal plasticity, and 4) glial contribution to synaptic transmission and plasticity.

 *Novel approaches

Membrane dynamics. We pioneered the “flash-and-freeze” approach that adds temporal information to electron micrographs (Fig.1).  Electron microscopy traditionally only captures a static image of cells. To visualize membrane dynamics, we combined optogenetic stimulation of neurons with high-pressure freezing. By varying the intervals between stimulation and freezing, we can essentially make a “flip-book” of cellular dynamics at a millisecond temporal resolution. 

Molecular topology and dynamics. We developed a correlative super-resolution fluorescence microscopy and electron microscopy approach that visualizes proteins in electron micrographs (Fig. 2). We found a method that preserves fluorescence through harsh fixation and plastic embedding. We perform super-resolution and electron microscopy imaging on the same ultrathin sections of tissues and map the molecular topology onto the subcellular structures. This technique can be used to pinpoint the locations of proteins within their subcellular context.