@article{12759,
  abstract     = {Stereological methods for estimating the 3D particle size and density from 2D projections are essential to many research fields. These methods are, however, prone to errors arising from undetected particle profiles due to sectioning and limited resolution, known as ‘lost caps’. A potential solution developed by Keiding, Jensen, and Ranek in 1972, which we refer to as the Keiding model, accounts for lost caps by quantifying the smallest detectable profile in terms of its limiting ‘cap angle’ (ϕ), a size-independent measure of a particle’s distance from the section surface. However, this simple solution has not been widely adopted nor tested. Rather, model-independent design-based stereological methods, which do not explicitly account for lost caps, have come to the fore. Here, we provide the first experimental validation of the Keiding model by comparing the size and density of particles estimated from 2D projections with direct measurement from 3D EM reconstructions of the same tissue. We applied the Keiding model to estimate the size and density of somata, nuclei and vesicles in the cerebellum of mice and rats, where high packing density can be problematic for design-based methods. Our analysis reveals a Gaussian distribution for ϕ rather than a single value. Nevertheless, curve fits of the Keiding model to the 2D diameter distribution accurately estimate the mean ϕ and 3D diameter distribution. While systematic testing using simulations revealed an upper limit to determining ϕ, our analysis shows that estimated ϕ can be used to determine the 3D particle density from the 2D density under a wide range of conditions, and this method is potentially more accurate than minimum-size-based lost-cap corrections and disector methods. Our results show the Keiding model provides an efficient means of accurately estimating the size and density of particles from 2D projections even under conditions of a high density.},
  author       = {Rothman, Jason Seth and Borges Merjane, Carolina and Holderith, Noemi and Jonas, Peter M and Angus Silver, R.},
  issn         = {1932-6203},
  journal      = {PLoS ONE},
  number       = {3 March},
  publisher    = {Public Library of Science},
  title        = {{Validation of a stereological method for estimating particle size and density from 2D projections with high accuracy}},
  doi          = {10.1371/journal.pone.0277148},
  volume       = {18},
  year         = {2023},
}

@article{9438,
  abstract     = {Rigorous investigation of synaptic transmission requires analysis of unitary synaptic events by simultaneous recording from presynaptic terminals and postsynaptic target neurons. However, this has been achieved at only a limited number of model synapses, including the squid giant synapse and the mammalian calyx of Held. Cortical presynaptic terminals have been largely inaccessible to direct presynaptic recording, due to their small size. Here, we describe a protocol for improved subcellular patch-clamp recording in rat and mouse brain slices, with the synapse in a largely intact environment. Slice preparation takes ~2 h, recording ~3 h and post hoc morphological analysis 2 d. Single presynaptic hippocampal mossy fiber terminals are stimulated minimally invasively in the bouton-attached configuration, in which the cytoplasmic content remains unperturbed, or in the whole-bouton configuration, in which the cytoplasmic composition can be precisely controlled. Paired pre–postsynaptic recordings can be integrated with biocytin labeling and morphological analysis, allowing correlative investigation of synapse structure and function. Paired recordings can be obtained from mossy fiber terminals in slices from both rats and mice, implying applicability to genetically modified synapses. Paired recordings can also be performed together with axon tract stimulation or optogenetic activation, allowing comparison of unitary and compound synaptic events in the same target cell. Finally, paired recordings can be combined with spontaneous event analysis, permitting collection of miniature events generated at a single identified synapse. In conclusion, the subcellular patch-clamp techniques detailed here should facilitate analysis of biophysics, plasticity and circuit function of cortical synapses in the mammalian central nervous system.},
  author       = {Vandael, David H and Okamoto, Yuji and Borges Merjane, Carolina and Vargas Barroso, Victor M and Suter, Benjamin and Jonas, Peter M},
  issn         = {17502799},
  journal      = {Nature Protocols},
  number       = {6},
  pages        = {2947–2967},
  publisher    = {Springer Nature},
  title        = {{Subcellular patch-clamp techniques for single-bouton stimulation and simultaneous pre- and postsynaptic recording at cortical synapses}},
  doi          = {10.1038/s41596-021-00526-0},
  volume       = {16},
  year         = {2021},
}

@article{8001,
  abstract     = {Post-tetanic potentiation (PTP) is an attractive candidate mechanism for hippocampus-dependent short-term memory. Although PTP has a uniquely large magnitude at hippocampal mossy fiber-CA3 pyramidal neuron synapses, it is unclear whether it can be induced by natural activity and whether its lifetime is sufficient to support short-term memory. We combined in vivo recordings from granule cells (GCs), in vitro paired recordings from mossy fiber terminals and postsynaptic CA3 neurons, and “flash and freeze” electron microscopy. PTP was induced at single synapses and showed a low induction threshold adapted to sparse GC activity in vivo. PTP was mainly generated by enlargement of the readily releasable pool of synaptic vesicles, allowing multiplicative interaction with other plasticity forms. PTP was associated with an increase in the docked vesicle pool, suggesting formation of structural “pool engrams.” Absence of presynaptic activity extended the lifetime of the potentiation, enabling prolonged information storage in the hippocampal network.},
  author       = {Vandael, David H and Borges Merjane, Carolina and Zhang, Xiaomin and Jonas, Peter M},
  issn         = {10974199},
  journal      = {Neuron},
  number       = {3},
  pages        = {509--521},
  publisher    = {Elsevier},
  title        = {{Short-term plasticity at hippocampal mossy fiber synapses is induced by natural activity patterns and associated with vesicle pool engram formation}},
  doi          = {10.1016/j.neuron.2020.05.013},
  volume       = {107},
  year         = {2020},
}

@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},
}

@article{1323,
  abstract     = {Mossy fiber synapses on CA3 pyramidal cells are 'conditional detonators' that reliably discharge postsynaptic targets. The 'conditional' nature implies that burst activity in dentate gyrus granule cells is required for detonation. Whether single unitary excitatory postsynaptic potentials (EPSPs) trigger spikes in CA3 neurons remains unknown. Mossy fiber synapses exhibit both pronounced short-term facilitation and uniquely large post-tetanic potentiation (PTP). We tested whether PTP could convert mossy fiber synapses from subdetonator into detonator mode, using a recently developed method to selectively and noninvasively stimulate individual presynaptic terminals in rat brain slices. Unitary EPSPs failed to initiate a spike in CA3 neurons under control conditions, but reliably discharged them after induction of presynaptic short-term plasticity. Remarkably, PTP switched mossy fiber synapses into full detonators for tens of seconds. Plasticity-dependent detonation may be critical for efficient coding, storage, and recall of information in the granule cell–CA3 cell network.},
  author       = {Vyleta, Nicholas and Borges Merjane, Carolina and Jonas, Peter M},
  journal      = {eLife},
  publisher    = {eLife Sciences Publications},
  title        = {{Plasticity-dependent, full detonation at hippocampal mossy fiber–CA3 pyramidal neuron synapses}},
  doi          = {10.7554/eLife.17977},
  volume       = {5},
  year         = {2016},
}