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.
Cell adhesion molecules (CAMs) play critical roles in mediating intercellular interactions in the context of the nervous system, such as guiding neuronal development, synapse formation and maturation, and synaptic plasticity. In addition to their extracellular adhesive roles, most CAMs induce intracellular signaling events and scaffold large protein complexes through intracellular domains. The molecular biology of how CAMs regulate synaptic development and function has been hugely advanced by decades of structural biology. These structures have illuminated multiple modes of CAM regulation, including how alternative splicing regulates CAM homotypic and heterotypic interactions. CAMs are diverse in size and contain a variety of adhesion domain classes such as immunoglobulin (Ig), leucine-rich repeats (LRR), and laminin G/neurexin/sex hormone (LNS). In this chapter, we detail structures of key synaptic adhesion complexes, including a mechanistic explanation of how these structures have informed functional work. Detailing the structural basis of synaptic adhesion provides a foundation for deciphering the complex interactions underlying neuronal connectivity and function in health and disease.
Synapses are the fundamental units of communication and information processing in the nervous system. They show remarkable functional autonomy, as well as speed of signaling, bidirectional plasticity, and diversity. Synapses shape complex network functions, such as learning, memory, pattern separation, pattern completion, and many others. Synaptic dysfunction is responsible for a wide range of neurological and psychiatric diseases. Many of these diseases are emerging as synaptopathies, but the precise disease mechanisms are unknown. Given their micron-scale small size and seemingly compact structure, synapses were generally thought to be functionally indivisible structures. However, with the advent of high-resolution electrophysiology and imaging techniques, it is increasingly clear that synapses harbor functionally specialized nanomodules. This expanding nanobiology of the synapse provides a new perspective on synaptic signaling. The novel insight gained from this work is critical to understand disease mechanisms and to guide the development of appropriate therapeutic strategies for major brain diseases ranging from neurodegenerative and neurodevelopmental disorders to neuropsychiatric disorders such as depression and schizophrenia.
Synaptic transmission between pre- and postsynaptic neurons occurs when the presynaptic neuron terminal is temporarily depolarized upon action potential arrival, opening voltage-gated Ca2+ channels at synapses. Ca2+ will flow into the presynapse, and it will trigger the fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane in less than a millisecond. Neurotransmitter molecules are then released into the synaptic cleft and bind to receptors in the postsynaptic membrane. Understanding the mechanisms that underlie this complex cellular process requires detailed knowledge of the spatial and structural organization of the macromolecular components of the synapse. This chapter focuses on recent structural insights into the presynaptic machinery and the spatial relationship between synaptic vesicles, presynaptic factors, and postsynaptic receptors.
Synaptic vesicles are small trafficking organelles that store neurotransmitters. Proper communication between neurons depends on the tight temporal and spatial regulation of synaptic vesicle fusion and recycling. Synapses evolved to have different pools of synaptic vesicles, defined both by their functional properties and molecular composition, allowing them to adapt the kinetics and type of neurotransmitter released to the functional demands of the neuron circuit. In this chapter, we will discuss the life cycle of synaptic vesicles, from biogenesis to fusion and regeneration at synapses, integrating our knowledge of their molecular identity, functional properties, and spatial organization at presynaptic axon terminals.
Synaptic cell adhesion molecules are critical components of the molecular programs underlying synapse formation and neural circuit assembly. Here, we discuss our current understanding of the functional roles of several of these synaptic adhesion molecules in the mammalian central nervous system. Emerging evidence, driven by advances in super-resolution approaches, supports that pre- and postsynaptic machinery are highly organized at the nanoscale level and that these precise sub-synaptic positions are important for synaptic transmission. We subsequently describe the nano-organization of several synaptic cell adhesion molecules and how these trans-synaptic complexes align release machinery to shape the synaptic function. Collectively, an understanding of the mechanistic roles of synaptic cell adhesion complexes will provide insights into how neural circuits assemble by vast numbers of diverse synaptic connections in the brain.
The presynaptic active zone (AZ) is a precisely organized nanoscale domain where synaptic vesicle exocytosis and neurotransmitter release are governed by tightly regulated protein networks. This review synthesizes recent insights from electron microscopy (EM) and super-resolution fluorescence microscopy that have deepened our understanding of active zone architecture in mammalian central nervous system synapses and at the Drosophila neuromuscular junction (NMJ). These imaging techniques have elucidated the spatial organization of key active zone proteins relative to one another and to the plasma membrane, which is notably well-ordered at the Drosophila NMJ. Here, we present a detailed overview of the nanometer-scale positioning of AZ proteins across the two types of synapses. In parallel, the idea that active zone nanostructures may form through liquid-liquid phase separation has emerged as a potential organizing principle. The transient and dynamic interactions characteristic of phase-separated protein condensates contrast with models that attribute nanodomain organization to specific, stable protein-protein interactions, raising the question of how the active zone's stable core architecture is reconciled with its capacity for dynamic plasticity.
Within the single micron of the synapse, three distinct modes of neurotransmission, driven by synchronous, asynchronous, and spontaneous neurotransmitter release, occur concurrently. In this chapter, we discuss the synaptic nano-organization comprised of neurotransmitter release machinery, molecular platforms, scaffolding proteins, and liquid complexes that support the discrete signaling of these three modes of neurotransmission. This robust nano-organization supports unique functional roles for each discrete mode at both excitatory glutamatergic and inhibitory GABAergic synapses. Modular nanocolumn organization of excitatory synapses and largely single-domain organization of inhibitory synapses maintain homeostatic plasticity within neural circuits. These recent findings support a basic design principle where the single synapse is a highly ordered and compartmentalized unit that by the functional nano-segregation of distinct forms of neurotransmission shapes synaptic efficacy, determines neurotransmission reliability, and tunes plasticity. The development of novel tools will be instrumental in further elucidating the nano-environment of the synapse, essential to both uncovering mechanisms underlying neurological disorders as well as their treatment.
Monoamine transmission is critical for regulating numerous physiological processes, including stress, learning, motor activity, and reward. Over the past few decades, the adoption of fast scan cyclic voltammetry has unveiled an intricate interplay between monoamine release and uptake dynamics, particularly concerning monoamine transporter involvement in reward and reinforcement processes for drugs of abuse. This review discusses how fast scan cyclic voltammetry has revolutionized our understanding of the processes that govern monoamine release and uptake, emphasizing the heterogeneity in transporter function across terminal regions, the influence of autoreceptors on monoamine transmission, and the complex interactions between drugs of abuse and monoamine transporters. While much of the review focuses on what is known about dopamine transporters-due to the wealth of evidence on dopamine transmission-we also emphasize significant gaps in knowledge regarding the serotonin and norepinephrine transporters. Finally, we highlight remaining questions about the dynamic nature of monoaminergic transporter efficiency and suggest new areas of investigation to gain a more comprehensive understanding of the biochemical mechanisms through which monoamine transporters regulate behavior.
The clinical efficacy of psychostimulant drugs, which target monoamine transporters, in treating attention-deficit/hyperactivity disorders (ADHDs) has stimulated interest on the role of transporter proteins like the dopamine (DA) transporter (DAT) in neurotransmission as well as the potential utility of DAT knockout organisms as models for neuropsychiatric disorders. Indeed, the study of DAT-deficient worms, flies, fish, mice, and rats has revealed a conserved role for DAT in the control of motor behavior as well as repetitive behavior, threat aversion, social behavior, and cognition in mammals. However, the disconnect between phenotypes observed in DAT-deficient model organisms and humans, which exhibit an early-onset syndrome characterized by Parkinsonism/dystonia and premature death, challenges the construct validity of DAT knockout models with respect to modeling neurobehavioral disorders. As an alternate approach, several groups have utilized coding variants in the SLC6A3 gene linked to psychiatric conditions, which display divergent molecular phenotypes. This chapter reviews the development and characterization of models of DAT gene deletion and mutation with a particular emphasis on comparing/contrasting the functional impact of DAT deficiency to DAT dysregulation triggered by neuropsychiatric disorder-linked DAT mutants in vivo. Ultimately, the study of DAT knockout and mutant models has revealed novel functions for DA in the mammalian brain, uncovered a dynamic interplay between the monoaminergic systems, highlighted sex differences in the DA system that determine the behavioral trajectory of DAT deregulation, and allowed for the screening of potential leads for therapeutics to treat disorders linked to aberrant dopaminergic neurotransmission.
The dopamine transporter (DAT) is a plasma membrane protein expressed in dopamine (DA) neurons of the central nervous system and is critical for regulating DA neurotransmission. The DAT is responsible for the reuptake of released DA back into the presynaptic neuron, resulting in the termination of DA transmission. This process also recycles the DA back into the dopaminergic neuron for subsequent release. DAT is the target of psychostimulants including cocaine and amphetamines and has been associated with several neuropsychiatric disorders. Only DAT proteins located on the plasma membrane can remove DA from the extracellular space, and the number of DAT proteins on the cell-surface therefore determines the efficiency of DA clearance. As a result, regulating DAT surface expression is a critical means to regulating the magnitude and duration of DA neurotransmission. This chapter will discuss the different processes and proteins that have been shown to affect DAT surface expression and discuss the relevance to normal DA physiology and diseases that involve aberrant DA signaling.
Some monoamine-uptake inhibitors are among first-line treatments for specific chronic pain conditions. It concerns tricyclic antidepressant drugs, such as amitriptyline or nortriptyline, and the more selective serotonergic and noradrenergic reuptake inhibitors, such as duloxetine. They are recommended for treating neuropathic pain, which is pain caused by a lesion or disease of the somatosensory nervous system, and fibromyalgia, which is a chronic widespread pain. Clinically, their action on pain was proposed to be independent from the one on depression. Research in animal models provided some understanding of the pain-relieving mechanism. The noradrenergic component of monoamine-uptake inhibitors appears to be critical, with the therapeutic effect likely involving targets at peripheral, spinal and supraspinal levels. At least two independent mechanisms would contribute to pain relief. One is spinal, relying on the recruitment of aminergic descending controls of pain, with a downstream key role of the α2 adrenergic receptors; the other may require more sustained treatment and relies on the noradrenergic recruitment of β2 adrenergic receptors and a downstream anti-neuroimmune action. Both mechanisms require a functional endogenous opioid system. These insights, however, focused on the sensory component of pain, and the contribution of the supraspinal action of antidepressant drugs needs to be explored in detail.
Monoamine transporters are essential proteins located at presynaptic terminals that play a crucial role in regulating neurotransmission of serotonin, dopamine, and norepinephrine by rapid reuptake of released amines from the synapse. Clinically used antidepressants and widely abused psychostimulants exhibit a high affinity for amine transporters. Function and expression of biogenic amine transporter are altered in subjects suffering from psychiatric diseases such as depression and in psychostimulant use disorder. Therefore, proper functional regulation of monoamine transporters is critical in maintaining normal amine homeostasis. Monoamine transporters possess several potential phosphorylation sites/motifs and exist in a phosphorylated state. Various cellular protein kinases and phosphatases are known to regulate the phosphorylation dynamics of amine transporters, which in turn influences subcellular expression and trafficking, microdomain-specific protein-protein interactions, transporter protein degradation, and overall transport capacity. Dysfunctional amine transporter function, phosphorylation, and association with interacting proteins are evident in neuropsychiatric disease states, including psychostimulant use disorder. However, the neurobiological consequences of in vivo amine transporter phosphorylation and its regulation remain unclear. Recent studies utilizing intact animal models are beginning to connect these molecular mechanisms with observed animal behaviors. This review summarizes current knowledge on the causal role of amine transporter phosphorylation in regulating amine transport and its relevance to animal behavior. Further understanding of phosphorylation-dependent molecular mechanisms governing amine transporter regulation potentially identifies regulatory motif(s) as potential therapeutic targets for treating neuropsychiatric disorders.
Solute carrier 6 family members comprise of neurotransmitter sodium symporters that are the transporters primarily involved in the reuptake of released neurotransmitters from the synaptic space. The family includes structurally related transporters involved in biogenic amine and amino acid neurotransmitter uptake and is a well-known therapeutic target for several classes of inhibitors for the treatment of ailments ranging from depression, pain, addiction, seizures and anxiety. Inhibition of NSS transporters can work either through competitive inhibition or through allosteric inhibition at diverse sites that target multiple conformational states of the transporters. This chapter explores the diverse inhibition strategies observed with numerous inhibitors that target this class of transporters and potential for improvements in inhibition strategies.
Drosophila melanogaster, commonly referred to as the "fruit fly," has been a long-utilized animal model in multiple areas of biological research. It is estimated that 75% of human genes, which are associated with disease, have homologues in Drosophila. The conservation of biological systems, the genetic tractability, short generation time, and a broad array of available behavioral assessments make Drosophila an especially robust model organism for neuroscience investigations. The dopamine (DA) system, in particular, is highly conserved between mammals and Drosophila. Mutations of the DA transporter (DAT), a negative regulator of DA neurotransmission, have been associated with multiple different neuropsychiatric and neurodegenerative disorders, including autism spectrum disorders (ASDs), attention deficit hyperactivity disorder (ADHD), and Parkinson's disease (PD). Utilization of Drosophila models demonstrates specific structural and functional alterations in mutated DAT that manifest as unique behavioral phenotypes. Ultimately, combining techniques ranging from biochemistry, electrochemistry, and complex behavioral analyses facilitated a deeper understanding of how transporter function and dysfunction can translate to neurological and neuropsychiatric disorders.
In this chapter we will show how electrophysiological recordings were used to gain insights into the transport kinetics and pharmacology of monoamine transporters (MATs). We will discuss data obtained from whole cell patch clamp recordings that allow for real time monitoring of MAT function. A notable property of MATs is that they carry so-called uncoupled currents. We will begin this chapter by reviewing the experimental evidence that has led to the conclusion that the currents carried by MATs are largely uncoupled and, therefore, not directly related to substrate transport. We will discuss how this has made it difficult to understand the operation of MATs. We will also explain why the existence of these currents has led to the proposition that MATs do not operate by alternate access but rather by a single file diffusion mechanism. However, we will show that ultimately the uncoupled currents carried by MATs can be most parsimoniously explained within the framework of the alternate access mechanism. We will review the existing evidence that MATs, like most other transporters, undergo a cycle during which they visit outward and inward-facing conformations (i.e., the transport cycle). We will outline what we have learned about the transport cycle of MATs from electrophysiological recordings. Thereafter, we will describe how electrophysiological recordings can be utilized to understand how drugs that target MATs affect their operation. To this end, we will discuss the binding modes of three different MAT ligands: (i) amphetamines, (ii) ibogaine, and (iii) zinc.
