The role of molecular diffusion within dendritic spines in synaptic function

Spines are tiny nanoscale protrusions from dendrites of neurons. In the cortex and hippocampus, most of the excitatory postsynaptic sites reside in spines. The bulbous spine head is connected to the dendritic shaft by a thin membranous neck. Because the neck is narrow, spine heads are thought to function as biochemically independent signaling compartments. Thus, dynamic changes in the composition, distribution, mobility, conformations, and signaling properties of molecules contained within spines can account for much of the molecular basis of postsynaptic function and regulation. A major factor in controlling these changes is the diffusional properties of proteins within this small compartment. Advances in measurement techniques using fluorescence microscopy now make it possible to measure molecular diffusion within single dendritic spines directly. Here, we review the regulatory mechanisms of diffusion in spines by local intra-spine architecture and discuss their implications for neuronal signaling and synaptic plasticity.

IntroductionNeurons communicate with each other through synapses that organize to create functional circuits. Most excitatory synapses in the central nervous system are formed on dendritic spines, tiny protrusions that extend from dendrites (Bourne and Harris, 2008; Fig. 1 a). The spine typically has a head of 200–1,000-nm diameter, which is connected to the dendritic shaft via a neck of 100–200-nm width (Arellano et al., 2007; Fig. 1 b). The head contains postsynaptic density (PSD) proteins, the actin cytoskeleton, membrane structures, and organelles (Sheng and Hoogenraad, 2007; Fig. 1 c). The molecular composition of spine heads is different from that of the shaft. Because of its characteristic morphology, spines are thought to function as biochemically independent compartments by limiting molecular movement between the spine head and the rest of the dendrite (Adrian et al., 2014; Tønnesen and Nägerl, 2016). Clarifying this regulation is key to understanding how this unitary site of synaptic transmission is controlled. This is particularly crucial to our understanding about how changes in the postsynaptic site lead to synaptic plasticity.Open in a separate windowFigure 1.The shape and internal architecture of dendritic spines. (a) A super-resolution SIM image of a hippocampal neuron dendrite expressing GFP. (b) A surface image of a spine (arrow in a) reconstructed from a SIM image. (c) Schematic representation of a spine containing the PSD, actin cytoskeleton, recycling endosome, and SER.The control of spine architecture is critical at excitatory synapses in the brain (Alvarez and Sabatini, 2007; Forrest et al., 2018). Excitatory synapses exhibit synaptic plasticity, which changes the strength of synaptic transmission through mechanisms at both pre- and postsynaptic sides (Citri and Malenka, 2008). This process is generally thought to be a basis for changes in neural circuits controlled by experiences—i.e., learning and memory (Humeau and Choquet, 2019; Magee and Grienberger, 2020). Here, the size and shape of spines are strongly correlated with the strength of synaptic transmission (Kasai et al., 2010). Spine volume is proportional to PSD area (Harris and Stevens, 1989) and the number of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate–type glutamate receptors (AMPARs; Nusser et al., 1998; Matsuzaki et al., 2001). Recently, combinational analysis of electrophysiology and correlative light and EM (CLEM) revealed the linear relationship between PSD area and synaptic strength (Holler et al., 2021). Also, longer spine necks attenuate somatic potentials to a greater degree (Araya et al., 2006). Thus, structural and functional plasticity of spines is tightly regulated. Specifically, when synaptic transmission is strengthened (e.g., long-term potentiation [LTP]), spines grow (Matsuzaki et al., 2004). In turn, when synaptic transmission weakens (e.g., long-term depression), spines shrink (Zhou et al., 2004; Oh et al., 2013).While many molecules involved in the plasticity of spine synapses have been identified (Sala and Segal, 2014), their mechanisms and regulations can only be discovered by monitoring the regulated changes in the composition and signaling properties of these factors within the confined space of the spine’s cytoplasm. To this end, the development of local photolysis of caged-glutamate played an important role (Matsuzaki et al., 2001). This method made it possible to induce structural plasticity locally at a single spine. Spine enlargement is induced by uncaging of caged-glutamate in the absence of Mg²⁺ or with postsynaptic depolarization in the presence of Mg²⁺ to activate N-methyl-D-aspartate–type glutamate receptors (NMDARs; Matsuzaki et al., 2004). Conversely, spine shrinkage is induced by low-frequency uncaging of caged-glutamate in the absence of Mg²⁺ or with postsynaptic depolarization (Oh et al., 2013). Shrinkage can also be induced by glutamate uncaging temporally coupled with back propagation action potential and uncaging of caged–γ-aminobutyric acid (GABA; Hayama et al., 2013). Glutamate uncaging–induced structural plasticity has also been seen to occur in vivo (Noguchi et al., 2019).These methods have vastly improved our understanding of the molecular mechanisms of structural plasticity (Nishiyama and Yasuda, 2015). In particular, stimulus-dependent increases in spine size (structural LTP [sLTP]), which are thought to be associated with functional LTP, have been studied extensively as a model of LTP (Nakahata and Yasuda, 2018; Fig. 2). Strong synaptic input causes an influx of Ca²⁺ through NMDARs that activates Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and the downstream signaling cascades. This modification of signaling cascades can affect cytoskeletal organization and membrane trafficking, which are responsible for two subsequent cellular events. First, spine morphology is modulated through cytoskeletal changes (Borovac et al., 2018). Second, synaptic transmission is enhanced by increased AMPAR insertion into the plasma membrane and movement to the PSD (Huganir and Nicoll, 2013). However, the molecular mechanisms that link these two phenomena are not fully understood (Herring and Nicoll, 2016). As sLTP progresses, the molecular composition within spines changes (Bosch et al., 2014; Meyer et al., 2014). Specifically, immediately after sLTP induction, actin-related molecules such as cofilin and actin-related protein 2/3 (Arp2/3) complex accumulate within a stimulated spine. On the other hand, scaffold proteins such as PSD-95 slowly accumulate over tens of minutes. SynGAP, which is localized at the PSD through interaction with PSD-95, escapes from spines immediately after stimulation and contributes to the expression of sLTP (Araki et al., 2015). These changes in molecular compositions immediately after stimulation may be due not only to molecule-specific binding but also to the physical regulation of diffusion (Obashi et al., 2019).Open in a separate windowFigure 2.Molecular motion important in sLTP. Strong synaptic input causes an influx of Ca²⁺ through NMDARs that activates CaMKII and the downstream signaling cascades. This modification of signaling cascades can affect cytoskeletal organization and membrane trafficking, which regulate spine morphology. Spine morphology affects the molecular exchange between the spine head and the dendritic shaft and lateral diffusion of membrane proteins including AMPARs. Regulation of molecular movements through the spine neck affects the molecular composition within spines. This change affects signal propagation into nearby spines. For example, cofilin and Arp2/3 complex accumulate within spines. SynGAP and activated RhoA escape from spines. Reorganization of the actin cytoskeleton affects movement of large molecules and the formation of a large signaling complex containing CaMKII and Tiam1. Also, the structure of the PSD affects membrane protein diffusion and alters the synaptic trafficking of AMPARs.In addition to molecular localization, fluorescence lifetime imaging of FRET-based biosensors has made it possible to measure spatiotemporal changes in the activity of signaling molecules involved in sLTP (Yasuda, 2012). These studies have demonstrated a critical relationship between the time that a molecule spends within a spine and the rate of signal inactivation. This relationship determines whether an activated signaling molecule is confined within a single spine or escapes from the spine and interacts with effectors present in the adjacent dendritic shaft or nearby spines (Yasuda, 2017). The signal propagation into nearby spines is most likely related to heterosynaptic plasticity, where activated synapses influence neighbor synapses within the same dendritic segments (Oh et al., 2015; Colgan et al., 2018; Chater and Goda, 2021). Thus, diffusion is a central feature of the regulation of spine structural plasticity. However, because the size of spines is small relative to the spatial resolution of diffraction-limited fluorescence microscopy and measuring methods are limited, elucidation of the mechanism regulating diffusion within spines has been challenging.To address this gap in understanding, researchers advanced fluorescence microscopy techniques, which enabled us to measure changes in the nanoscale localization, diffusion, and signal activities inside spines. These studies allow us to directly understand how spine structures physically limit molecular diffusion and reveal fundamental mechanisms that control the localization and biochemical signal transduction pathways in neurons. Here, we summarize recent findings that have revealed physical barriers within spines using super-resolution microscopy (Sigal et al., 2018; Table 1) and molecular dynamics measurements (Fig. 3 and Table 2), and we discuss how these barriers serve as a fundamental feature controlling neuronal signaling and synaptic plasticity.Table 1.List of super-resolution microscopy techniques