Functional and ultrastructural investigation of mouse and human dendritic spines

Abstract

Dendritic spines are the physical site of the majority of excitatory synaptic connections in the mammalian brain. Their complex morphological attributes and protein composition have inspired decades of theoretical and experimental work on how classes of dendritic spines differentially sculpt input processing and plasticity. The technical challenge of tethering physiological measurements of synaptic strength to spine morphology and protein expression in specific cell types leaves many open questions for the field. Here, we combine super-resolution protein imaging and patch-clamp electrophysiology to investigate the formation of nascent connections in the adult mouse cortex, and we developed a new method to apply these techniques to human neurons. In the first project presented in Chapter 2, we used super-resolution protein imaging and patch-clamp electrophysiology to identify filopodia as the structural substrate for silent synapses in adult neocortex. Of 2,234 spiny synapses from adult mouse layer 5 pyramidal neurons, a surprisingly large fraction (~25%) lacked AMPA receptors. These putative silent synapses were located at the tips of thin dendritic protrusions that lack the distinct head of conventional spines, known as filopodia, which were more abundant in adult cortex by an order of magnitude than previously believed (compromising ~30% of all dendritic protrusions). Physiological experiments revealed that filopodia do indeed lack AMPAR-mediated transmission, but they exhibit NMDAR-mediated synaptic transmission. We further showed that functionally silent synapses on filopodia can be unsilenced via Hebbian plasticity, recruiting new active connections into a neuron’s input matrix. In the second project presented in Chapter 3, we developed Patch2MAP to perform super-resolution imaging of proteins localized in the 3D morphology of any cell type in human tissue (or in any other species) without the need for exogenous protein expression. Our method, which combines patch-clamp electrophysiology with epitope-preserving magnified analysis of proteome (eMAP), further allows for correlation of physiological properties with subcellular protein expression. We applied Patch2MAP to individual spiny synapses in human cortical pyramidal neurons and demonstrated that electrophysiological AMPA-to-NMDA receptor ratios correspond tightly to respective protein expression levels. Taken together, the combination of protein imaging and physiological measurements expand our understanding on how the interplay of structure and protein content of spiny synapses shape synaptic input and open new avenues for a comprehensive investigation of synaptic function in humans.