The complex nanostructure and spatiotemporal dynamics of central synapses remain among the fundamental mysteries of neurobiology. The resolution of traditional microscopy techniques-constrained by the intrinsic limits of light diffraction-is largely insufficient to study central synapses effectively. Conventional imaging can resolve areas roughly the size of a synapse's active zone, that is, severalfold larger than the size of synaptic vesicles. Recent advances have generated several super-resolution imaging modalities that overcome or bypass the light diffraction limit to support studies of synaptic nanostructure. In this chapter, we present the principles, features, and limitations of the most common super-resolution imaging tools. Though these advancements have greatly improved our understanding of synaptic architecture and dynamics, significant challenges remain. Difficulties of translating the existing tools to in vivo applications, and the inherent trade-off between spatial and temporal resolution, continue to limit studies of the function of central synapses in native tissue.
Dissecting the mechanisms of synaptic transmission touches on nearly all fields of neuroscience. Of particular recent importance is the discovery that protein distribution within single synapses is highly organized across multiple spatial scales, ranging from the nanoscale accumulation of just a few protein molecules to larger domains with unique multiprotein compositions. Here, we address recent data regarding postsynaptic molecular organization. We argue that the complexity of synaptic nanostructure generates functional capabilities that can fine-tune synaptic strength and that far exceed the classical limits of quantal synaptic transmission. We focus first on the critical scaffold protein PSD-95 as a case study for how to approach the emergent problem of describing and classifying forms of protein organization, including trans-synaptic "nanocolumn" relationships. Then, we discuss recent work identifying new features of NMDA receptor subsynaptic organization that appear likely to regulate the patterns of neural activity that can induce synaptic plasticity. Overall, we assert that these mechanisms of molecular coordination at scales of 20-150 nm enhance the synapse's ability to tune synaptic transmission, carry out detailed biochemical signaling, and allow more complex impacts on the cell.
Glia, which are nonneuronal cells in the central nervous system, include astrocytes, microglia, and oligodendrocyte lineage cells. Historically, their passive roles in maintaining central nervous system function-such as supplying substrates for neuronal energy, buffering neurotransmitters, and improving neural conductance-have been primarily highlighted. Importantly, recent research has revealed that glial cells express genes directly related to controlling synapse nano-organization. In particular, astrocytes have the ability to secrete molecules that induce synapse formation and maturation. They also modulate the structure of synapses by expressing proteins that make direct contact with the synaptic membrane. Moreover, astrocytes can actively eliminate synapses through their phagocytic machinery during development and adulthood, thereby establishing circuit homeostasis. Microglia can also assist in the integration of synapses into the neural circuit and regulate synapse formation, maintenance, and elimination. Here, we review key findings on the mechanisms of glial contributions to synapse nano-organization. We will also discuss how different glial cells contribute to the development and homeostasis of synapses through distinct cellular and molecular pathways in both health and disease.
Electron tomography (ET) has emerged as a critical tool for visualizing the three-dimensional ultrastructure of biological specimens at nanometer resolution. This chapter focuses on the application of ET in studying synaptic ultrastructure, in particular postsynaptic density, providing a detailed overview of the techniques and methodologies used to achieve high-resolution three-dimensional reconstruction of synapses. Beginning with an introduction to electron tomography, this chapter delves into the principles of electron tomography, including sample preparation, data collection, and image processing. Special emphasis is placed on the application of cryogenic electron tomography, which allows for the visualization of biological samples in their near-native state. This chapter also reviews the historical context of postsynaptic ultrastructure studies using conventional electron microscopy and explores the significant insights gained using electron tomography, particularly in understanding the nanoscale organization and structural complexity of excitatory synapses. This chapter concludes by discussing the future potential of electron tomography in advancing our knowledge of synaptic biology and its implications for neuroscience.
