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Abstract Long-term potentiation (LTP) induces presynaptic bouton enlargement and a reduction in the number of synaptic vesicles. To understand the relationship between these events, we performed 3D analysis of serial section electron micrographs in rat hippocampal area CA1, 2 hours after LTP induction. We observed a high vesicle packing density in control boutons, contrasting with a lower density in most LTP boutons. Notably, the summed membrane area of the vesicles lost in low-density LTP boutons is comparable to the surface membrane required for the observed bouton enlargement when compared to high-density control boutons. These novel findings suggest that presynaptic vesicle density provides a new structural indicator of LTP that supports a local mechanism of bouton enlargement.
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Immunohistochemistry and Morphometric Analysis of Drosophila Larval Body Wall Neuromuscular Junction Preparations
TheDrosophilaneuromuscular junction (NMJ) is an excellent model for studying vertebrate glutamatergic synapses. Researchers have uncovered fundamental mechanisms at the fly NMJ that are conserved in higher-order organisms. To gain molecular and structural insight into these and other structures, immunolabeling is invaluable. In this protocol, we describe how to use immunolabeling to visualize embryonic/larval presynaptic and postsynaptic structures at the NMJ. We also include details about amplification of weak immunohistochemistry signals and how to use these signals to quantify synaptic growth via bouton counting. Boutons are bead-like structures at motor axon terminals that house synapses, and the number of boutons reflects the size of the NMJ. We also describe how to identify the different bouton types.
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- Award ID(s):
- 2048080
- PAR ID:
- 10564910
- Publisher / Repository:
- Cold Spring Harbor Laboratory Press
- Date Published:
- Journal Name:
- Cold Spring Harbor Protocols
- ISSN:
- 1940-3402
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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For decades, theDrosophilalarval neuromuscular junction (NMJ) has been a go-to model for synaptic development. This simple, accessible system is composed of a repeating pattern of 33 distinct neurons that stereotypically innervate 30 muscles. Fundamental mechanisms that underlie diverse aspects of axon pathfinding, synaptic form, and function have been uncovered at the NMJ, and new pathways continue to be uncovered. These discoveries are fueled by the ease of dissections and an extensive array of techniques. Chief among these techniques are various microscopy approaches, including super-resolution and electron microscopy. Functionally, theDrosophilaNMJ is glutamatergic, similar to the vertebrate central synapses, making it a great model to study normal development and neurological diseases. Here we provide a brief overview of the larval neuromuscular system, highlighting the connectivity patterns, development, and some of the mechanisms underlying these processes.more » « less
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Synapses maintain two forms of neurotransmitter release to support communication in the brain. First, evoked neurotransmitter release is triggered by the invasion of an action potential across en passant boutons that form along axons. The probability of evoked release (Pr) varies substantially across boutons, even within a single axon. Such heterogeneity is the result of differences in the probability of a single synaptic vesicle fusing (Pv) and in the number of vesicles available for immediate release, known as the readily-releasable pool (RRP). Spontaneous release (also known as a mini) is an important form of neurotransmission that occurs in the absence of action potentials. Because it cannot be triggered with electrical stimulation, much less is known about potential heterogeneity in the frequency of spontaneous release between boutons. We utilized a photostable and bright fluorescent indicator of glutamate release (iGluSnFR3) to quantify both spontaneous and evoked release at individual glutamatergic boutons. We found that the rate of spontaneous release is quite heterogenous at the level of individual boutons. Interestingly, when measuring both evoked and spontaneous release at single synapses, we found that boutons with the highest rates of spontaneous release also displayed the largest evoked responses.Using a new optical method to measure RRP at individual boutons, we found that this heterogeneity in spontaneous release was strongly correlated with the size of the RRP, but not related to Pv. We conclude that the RRP is a critical and dynamic aspect of synaptic strength that contributes to both evoked and spontaneous vesicle release. Significance StatementNeurotransmitter is released through two mechanisms: action potential-evoked and spontaneous vesicle fusion. It is unknown if some synapses specialize in either evoked or spontaneous release with an antagonistic relationship, or if the two forms of release coexist and have a cooperative relationship. We used a robust optical glutamate indicator to measure both forms of release at individual synapses. We found that the frequency of spontaneous release displays significant heterogeneity and is directly related to the size of the readily releasable pool of vesicles. This finding links both mechanisms of neurotransmitter release and suggests an important signaling mechanism to the postsynaptic neuron at individual synapses.more » « less
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In the nearly 50 years since the neuromuscular junction (NMJ) was first established as a model synapse, its molecular composition has been extensively characterized. Early work relied on fluorescent signals to determine whether proteins localized to the pre- and postsynaptic regions. As more synaptic molecules were identified, determining the localization of these proteins relative to each other became important. Conventional microscopy lacks the resolving power to assess whether two proteins are within an appropriate distance to bind directly or be part of a larger complex. Super-resolution and immunoelectron microscopies can improve spatial resolution, but these techniques can be difficult to execute and troubleshoot, and access to these instruments is limiting. However, another approach, proximity labeling, overcomes many of these limitations by using a DNA secondary label that can only be amplified if the two proteins of interest are within 40 nm of each other, which is ∼5× greater than the resolving power of conventional microscopy. In this protocol, we describe the use of the proximity ligation assay, which combines immunohistochemistry with DNA amplification, to reveal protein colocalization in theDrosophilaNMJ.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1101/pdb.prot108500
