@article{18603,
  abstract     = {It is widely believed that information storage in neuronal circuits involves nanoscopic structural changes at synapses, resulting in the formation of synaptic engrams. However, direct evidence for this hypothesis is lacking. To test this conjecture, we combined chemical potentiation, functional analysis by paired pre-postsynaptic recordings, and structural analysis by electron microscopy (EM) and freeze-fracture replica labeling (FRL) at the rodent hippocampal mossy fiber synapse, a key synapse in the trisynaptic circuit of the hippocampus. Biophysical analysis of synaptic transmission revealed that forskolin-induced chemical potentiation increased the readily releasable vesicle pool size and vesicular release probability by 146% and 49%, respectively. Structural analysis of mossy fiber synapses by EM and FRL demonstrated an increase in the number of vesicles close to the plasma membrane and the number of clusters of the priming protein Munc13-1, indicating an increase in the number of both docked and primed vesicles. Furthermore, FRL analysis revealed a significant reduction of the distance between Munc13-1 and CaV2.1 Ca2+ channels, suggesting reconfiguration of the channel-vesicle coupling nanotopography. Our results indicate that presynaptic plasticity is associated with structural reorganization of active zones. We propose that changes in potential nanoscopic organization at synaptic vesicle release sites may be correlates of learning and memory at a plastic central synapse.},
  author       = {Kim, Olena and Okamoto, Yuji and Kaufmann, Walter and Brose, Nils and Shigemoto, Ryuichi and Jonas, Peter M},
  issn         = {1545-7885},
  journal      = {PLoS Biology},
  number       = {11},
  publisher    = {Public Library of Science},
  title        = {{Presynaptic cAMP-PKA-mediated potentiation induces reconfiguration of synaptic vesicle pools and channel-vesicle coupling at hippocampal mossy fiber boutons}},
  doi          = {10.1371/journal.pbio.3002879},
  volume       = {22},
  year         = {2024},
}

@phdthesis{11196,
  abstract     = {One of the fundamental questions in Neuroscience is how the structure of synapses and their physiological properties are related. While synaptic transmission remains a dynamic process, electron microscopy provides images with comparably low temporal resolution (Studer et al., 2014). The current work overcomes this challenge and describes an improved “Flash and Freeze” technique (Watanabe et al., 2013a; Watanabe et al., 2013b) to study synaptic transmission at the hippocampal mossy fiber-CA3 pyramidal neuron synapses, using mouse acute brain slices and organotypic slices culture. The improved method allowed for selective stimulation of presynaptic mossy fiber boutons and the observation of synaptic vesicle pool dynamics at the active zones. Our results uncovered several intriguing morphological features of mossy fiber boutons. First, the docked vesicle pool was largely depleted (more than 70%) after stimulation, implying that the docked synaptic vesicles pool and readily releasable pool are vastly overlapping in mossy fiber boutons. Second, the synaptic vesicles are skewed towards larger diameters, displaying a wide range of sizes. An increase in the mean diameter of synaptic vesicles, after single and repetitive stimulation, suggests that smaller vesicles have a higher release probability. Third, we observed putative endocytotic structures after moderate light stimulation, matching the timing of previously described ultrafast endocytosis (Watanabe et al., 2013a; Delvendahl et al., 2016). 
	In addition, synaptic transmission depends on a sophisticated system of protein machinery and calcium channels (Südhof, 2013b), which amplifies the challenge in studying synaptic communication as these interactions can be potentially modified during synaptic plasticity. And although recent study elucidated the potential correlation between physiological and morphological properties of synapses during synaptic plasticity (Vandael et al., 2020), the molecular underpinning of it remains unknown. Thus, the presented work tries to overcome this challenge and aims to pinpoint changes in the molecular architecture at hippocampal mossy fiber bouton synapses during short- and long-term potentiation (STP and LTP), we combined chemical potentiation, with the application of a cyclic adenosine monophosphate agonist (i.e. forskolin) and freeze-fracture replica immunolabelling. This method allowed the localization of membrane-bound proteins with nanometer precision within the active zone, in particular, P/Q-type calcium channels and synaptic vesicle priming proteins Munc13-1/2. First, we found that the number of clusters of Munc13-1 in the mossy fiber bouton active zone increased significantly during STP, but decreased to lower than the control value during LTP. Secondly, although the distance between the calcium channels and Munc13-1s did not change after induction of STP, it shortened during the LTP phase. Additionally, forskolin did not affect Munc13-2 distribution during STP and LTP. These results indicate the existence of two distinct mechanisms that govern STP and LTP at mossy fiber bouton synapses: an increase in the readily realizable pool in the case of STP and a potential increase in release probability during LTP. “Flash and freeze” and functional electron microscopy, are versatile methods that can be successfully applied to intact brain circuits to study synaptic transmission even at the molecular level.
},
  author       = {Kim, Olena},
  issn         = {2663-337X},
  pages        = {132},
  publisher    = {Institute of Science and Technology Austria},
  title        = {{Nanoarchitecture of hippocampal mossy fiber-CA3 pyramidal neuron synapses}},
  doi          = {10.15479/at:ista:11196},
  year         = {2022},
}

@article{7473,
  abstract     = {How structural and functional properties of synapses relate to each other is a fundamental question in neuroscience. Electrophysiology has elucidated mechanisms of synaptic transmission, and electron microscopy (EM) has provided insight into morphological properties of synapses. Here we describe an enhanced method for functional EM (“flash and freeze”), combining optogenetic stimulation with high-pressure freezing. We demonstrate that the improved method can be applied to intact networks in acute brain slices and organotypic slice cultures from mice. As a proof of concept, we probed vesicle pool changes during synaptic transmission at the hippocampal mossy fiber-CA3 pyramidal neuron synapse. Our findings show overlap of the docked vesicle pool and the functionally defined readily releasable pool and provide evidence of fast endocytosis at this synapse. Functional EM with acute slices and slice cultures has the potential to reveal the structural and functional mechanisms of transmission in intact, genetically perturbed, and disease-affected synapses.},
  author       = {Borges Merjane, Carolina and Kim, Olena and Jonas, Peter M},
  issn         = {0896-6273},
  journal      = {Neuron},
  pages        = {992--1006},
  publisher    = {Elsevier},
  title        = {{Functional electron microscopy (“Flash and Freeze”) of identified cortical synapses in acute brain slices}},
  doi          = {10.1016/j.neuron.2019.12.022},
  volume       = {105},
  year         = {2020},
}

@inproceedings{11222,
  author       = {Kim, Olena and Borges Merjane, Carolina and Jonas, Peter M},
  booktitle    = {Intrinsic Activity},
  issn         = {2309-8503},
  keywords     = {hippocampus, mossy fibers, readily releasable pool, electron microscopy},
  location     = {Innsbruck, Austria},
  number       = {Suppl. 1},
  publisher    = {Austrian Pharmacological Society},
  title        = {{Functional analysis of the docked vesicle pool in hippocampal mossy fiber terminals by electron microscopy}},
  doi          = {10.25006/ia.7.s1-a3.27},
  volume       = {7},
  year         = {2019},
}