methods-to-visualize-structural-and-functional-changes-at-synapses

Methods to Visualize Structural and Functional Changes at Synapses

ARTICLES DISCUSSED:
Pekarek, B. T., Hunt, P. J., Belfort, B. D. W., Liu, G., Arenkiel, B. R. Imaging and quantification of intact neuronal dendrites via ...

Duke University

Endocytosis of AMPA receptors and other postsynaptic cargo occurs at endocytic zones (EZs), stably positioned sites of clathrin adjacent to the postsynaptic density (PSD). The tight localization of postsynaptic endocytosis is thought to control spine composition and regulate synaptic transmission. However, the mechanisms that situate the EZ near the PSD and the role of spine endocytosis in synaptic transmission are unknown. Here, we report that a physical link between dynamin-3 and the postsynaptic adaptor Homer positions the EZ near the PSD. Disruption of dynamin-3 or its interaction with Homer uncouples the PSD from the EZ, resulting in synapses lacking postsynaptic clathrin. Loss of the EZ leads to a loss of synaptic AMPA receptors and reduced excitatory synaptic transmission that corresponds with impaired synaptic recycling. Thus, a physical link between the PSD and the EZ ensures localized endocytosis and recycling by recapturing and maintaining a proximate pool of cycling AMPA receptors.

Postsynaptic positioning of endocytic zones and AMPA receptor cycling by physical coupling of dynamin-3 to Homer.

Among diverse factors regulating excitatory synaptic transmission, the abundance of postsynaptic glutamate receptors figures prominently in molecular memory and learning-related synaptic plasticity. To allow for both long-term maintenance of synaptic transmission and acute changes in synaptic strength, the relative rates of glutamate receptor insertion and removal must be tightly regulated. Interactions with scaffolding proteins control the targeting and signaling properties of glutamate receptors within the postsynaptic membrane. In addition, extrasynaptic receptor populations control the equilibrium of receptor exchange at synapses and activate distinct signaling pathways involved in plasticity. Here, we review recent findings that have shaped our current understanding of receptor mobility between synaptic and extrasynaptic compartments at glutamatergic synapses, focusing on AMPA and NMDA receptors. We also examine the cooperative relationship between intracellular trafficking and surface diffusion of glutamate receptors that underlies the expression of learning-related synaptic plasticity.

Glutamate receptor dynamics in dendritic microdomains.

Changes in the molecular composition and signaling properties of excitatory glutamatergic synapses onto dendritic spines mediate learning-related plasticity in the mammalian brain. This molecular adaptation serves as the most celebrated cell biological model for learning and memory. Within their micron-sized dimensions, dendritic spines restrict the diffusion of signaling molecules and spatially confine the activation of signal transduction pathways. Much of this local regulation occurs by spatial compartmentalization of glutamate receptors. Here, we review recently identified cell biological mechanisms regulating glutamate receptor mobility within individual dendritic spines. We discuss the emerging functions of glutamate receptors residing within sub-spine microdomains and propose a model for distinct signaling platforms with specialized functions in synaptic plasticity.

Spine microdomains for postsynaptic signaling and plasticity.

We apply a wide-field quantitative phase microscopy technique based on parallel two-step phase-shifting on-axis interferometry to visualize live biological cells and microorganism dynamics. The parallel on-axis holographic approach is more efficient with camera spatial bandwidth consumption compared to previous off-axis approaches and thus can capture finer sample spatial details, given a limited spatial bandwidth of a specific digital camera. Additionally, due to the parallel acquisition mechanism, the approach is suitable for visualizing rapid dynamic processes, permitting an interferometric acquisition rate equal to the camera frame rate. The method is demonstrated experimentally through phase microscopy of neurons and unicellular microorganisms.

Parallel on-axis holographic phase microscopy of biological cells and unicellular microorganism dynamics.

Myosins-I are conserved proteins that bear an N-terminal motor head followed by a Tail Homology 1 (TH1) lipid-binding domain. Some myosins-I have an additional C-terminal extension (C(ext)) that promotes Arp2/3 complex-dependent actin polymerization. The head and the tail are separated by a neck that binds calmodulin or calmodulin-related light chains. Myosins-I are known to participate in actin-dependent membrane remodelling. However, the molecular mechanisms controlling their recruitment and their biochemical activities in vivo are far from being understood. In this study, we provided evidence suggesting the existence of an inhibitory interaction between the TH1 domain of the yeast myosin-I Myo5 and its C(ext). The TH1 domain prevented binding of the Myo5 C(ext) to the yeast WIP homologue Vrp1, Myo5 C(ext)-induced actin polymerization and recruitment of the Myo5 C(ext) to endocytic sites. Our data also indicated that calmodulin dissociation from Myo5 weakened the interaction between the neck and TH1 domains and the C(ext). Concomitantly, calmodulin dissociation triggered Myo5 binding to Vrp1, extended the myosin-I lifespan at endocytic sites and activated Myo5-induced actin polymerization.

Calmodulin dissociation regulates Myo5 recruitment and function at endocytic sites.

Dendritic spines are actin-rich, postsynaptic protrusions that contact presynaptic terminals to form excitatory chemical synapses. These synaptic contacts are widely believed to be the sites of memory formation and information storage, and changes in spine shape are thought to underlie several forms of learning-related plasticity. Both membrane trafficking pathways and the actin cytoskeleton drive activity-dependent structural and functional changes in dendritic spines. A key molecular player in regulating these processes is the activity-regulated cytoskeleton-associated protein (Arc), a protein that has diverse roles in expression of synaptic plasticity. In this review, we highlight important findings that have shaped our understanding of Arc's functions in structural and functional plasticity, as well as Arc's contributions to memory consolidation and disease.

Regulation of spine structural plasticity by Arc/Arg3.1.

Team-based learning (TBL) is a special form of collaborative learning that involves the use of permanent working teams throughout the semester. In this highly structured and interactive teaching method, students perform preparatory activities outside of class to gain factual knowledge and understand basic concepts. In class, students collaborate with peers to apply content, analyze findings, and synthesize new ideas. To better understand the learning outcomes specific to TBL courses, we analyzed end-of-semester course evaluations from an undergraduate neuroscience course taught using either a moderate structure active learning or TBL format. Our analysis reveals that the TBL taught classes had significantly higher levels of self-reported learning in the areas of gaining, understanding, and synthesizing knowledge. We propose that these gains are driven by the TBL readiness assurance process and peer evaluations. Both of these structural components are expected to increase student accountability, motivation, and engagement with course content.

Article	Year
Postsynaptic positioning of endocytic zones and AMPA receptor cycling by physical coupling of dynamin-3 to Homer. Neuron\| PubMed ID: 17880892	2007
Glutamate receptor dynamics in dendritic microdomains. Neuron\| PubMed ID: 18498731	2008
Spine microdomains for postsynaptic signaling and plasticity. Trends in cell biology\| PubMed ID: 19328694	2009
Parallel on-axis holographic phase microscopy of biological cells and unicellular microorganism dynamics. Applied optics\| PubMed ID: 20490249	2010
Calmodulin dissociation regulates Myo5 recruitment and function at endocytic sites. The EMBO journal\| PubMed ID: 20647997	2010
Regulation of spine structural plasticity by Arc/Arg3.1. Seminars in cell & developmental biology\| PubMed ID: 28943393	2018
Comparing Active Learning to Team-Based Learning in Undergraduate Neuroscience. Journal of undergraduate neuroscience education : JUNE : a publication of FUN, Faculty for Undergraduate Neuroscience\| PubMed ID: 32848518	2020

Thomas M. Newpher

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