Visualizing presynaptic function

Kavalali, Ege T.; Jørgensen, Erik M.

doi:10.1038/nn.3578

Cited by 111 publications

(105 citation statements)

References 99 publications

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“…22,27 Changes in fluorescence (Δ F ) during electrical stimulation of AP firing reflect alkalization of pHlourin due to exocytosis, while changes during the post-stimulus period reflect re-acidification following endocytosis. 22 The transfection method yielded only several transfected cells per dish due to the low transfection efficiency such that boutons from single cells could be identified and imaged without interference from other neurons.…”

Section: Methodsmentioning

confidence: 99%

α2-Adrenergic Receptor and Isoflurane Modulation of Presynaptic Ca2+ Influx and Exocytosis in Hippocampal Neurons

2016

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Background Evidence indicates that the anesthetic-sparing effects of α2-adrenergic receptor (AR) agonists involve α2A-AR heteroreceptors on nonadrenergic neurons. Since volatile anesthetics inhibit neurotransmitter release by reducing synaptic vesicle (SV) exocytosis, the authors hypothesized that α2-AR agonists inhibit nonadrenergic SV exocytosis and thereby potentiate presynaptic inhibition of exocytosis by isoflurane. Methods Quantitative imaging of fluorescent biosensors of action potential–evoked SV exocytosis (synaptophysin-pHluorin) and Ca2+ influx (GCaMP6) were used to characterize presynaptic actions of the clinically used α2-AR agonists dexmedetomidine and clonidine, and their interaction with isoflurane, in cultured rat hippocampal neurons. Results Dexmedetomidine (0.1 μM, n = 10) or clonidine (0.5 μM, n = 8) inhibited action potential–evoked exocytosis (54 ± 5% and 59 ± 8% of control, respectively; P < 0.001). Effects on exocytosis were blocked by the subtype-nonselective α2-AR antagonist atipamezole or the α2A-AR–selective antagonist BRL 44408 but not by the α2C-AR–selective antagonist JP 1302. Dexmedetomidine inhibited exocytosis and presynaptic Ca2+ influx without affecting Ca2+ coupling to exocytosis, consistent with an effect upstream of Ca2+–exocytosis coupling. Exocytosis coupled to both N-type and P/Q-type Ca2+ channels was inhibited by dexmedetomidine or clonidine. Dexmedetomidine potentiated inhibition of exocytosis by 0.7 mM isoflurane (to 42 ± 5%, compared to 63 ± 8% for isoflurane alone; P < 0.05). Conclusions Hippocampal SV exocytosis is inhibited by α2A-AR activation in proportion to reduced Ca2+ entry. These effects are additive with those of isoflurane, consistent with a role for α2A-AR presynaptic heteroreceptor inhibition of nonadrenergic synaptic transmission in the anesthetic-sparing effects of α2A-AR agonists.

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Section: Methodsmentioning

confidence: 99%

α2-Adrenergic Receptor and Isoflurane Modulation of Presynaptic Ca2+ Influx and Exocytosis in Hippocampal Neurons

2016

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“…The abundance of proteins localised, for example, at the pre- and post-synapse, and reflected in the content of sub-cellular fractions like the post-synaptic density (PSD) is dependent on cell-type and brain region 116–118 . Due to the various confounds introduced by the trafficking of proteins and RNAs and/or local translation at synapses (or potentially in axons) 115 , proteomic profiling of enriched synaptic fractions can provide greater sensitivity and specificity than transcriptomics to assessing molecular regulation of synaptic proteins.…”

Section: Challenges In Assessing the Proteome(s) Of The Cnsmentioning

confidence: 99%

Decoding neuroproteomics: integrating the genome, translatome and functional anatomy

et al. 2014

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The immense inter- and intra-cellular heterogeneity of the central nervous system (CNS) presents major challenges for high-throughput *omic analyses. Transcriptional, translational, and post-translational regulatory events are localised to specific neuronal cell-types, or sub-cellular compartments, resulting in discrete patterns of protein expression and activity. A spatial and quantitative knowledge of the “neuroproteome” is therefore critical to understanding normal and pathological aspects of functional genomics and anatomy of the CNS. Improvements in mass-spectrometry allow profiling of proteins at sufficient depth to complement results from high-throughput genomic and transcriptomic assays. However, there are challenges in integrating proteomic data with other data modalities and even greater challenges obtaining comprehensive neuroproteomic data with cell-type specificity. Here we discuss how proteomics should be exploited to enhance high-throughput functional genomic analysis by tighter integration of data analyses. We also discuss experimental strategies to achieve finer cellular and sub-cellular resolution in transcriptomic and proteomic studies of neural tissues.

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“…These units are either called "release sites" or "docking sites," and their number per AZ represents the maximum SV output that this structure can provide per action potential (AP). In recent years, optical methods have been developed to record single-SV release (4). Notably, studies using superresolution and total internal reflection fluorescence imaging in functioning central synapses have provided information on the kinetics (5) and subsynaptic localization (6) of single-SV docking and release.…”

mentioning

confidence: 99%

Numbers of presynaptic Ca ²⁺ channel clusters match those of functionally defined vesicular docking sites in single central synapses

Miki

Kaufmann

Malagón

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

107

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Many central synapses contain a single presynaptic active zone and a single postsynaptic density. Vesicular release statistics at such "simple synapses" indicate that they contain a small complement of docking sites where vesicles repetitively dock and fuse. In this work, we investigate functional and morphological aspects of docking sites at simple synapses made between cerebellar parallel fibers and molecular layer interneurons. Using immunogold labeling of SDS-treated freeze-fracture replicas, we find that Ca v 2.1 channels form several clusters per active zone with about nine channels per cluster. The mean value and range of intersynaptic variation are similar for Ca v 2.1 cluster numbers and for functional estimates of docking-site numbers obtained from the maximum numbers of released vesicles per action potential. Both numbers grow in relation with synaptic size and decrease by a similar extent with age between 2 wk and 4 wk postnatal. Thus, the mean docking-site numbers were 3.15 at 2 wk (range: 1-10) and 2.03 at 4 wk (range: 1-4), whereas the mean numbers of Ca v 2.1 clusters were 2.84 at 2 wk (range: 1-8) and 2.37 at 4 wk (range: 1-5). These changes were accompanied by decreases of miniature current amplitude (from 93 pA to 56 pA), active-zone surface area (from 0.0427 μm 2 to 0.0234 μm 2 ), and initial success rate (from 0.609 to 0.353), indicating a tightening of synaptic transmission with development. Altogether, these results suggest a close correspondence between the number of functionally defined vesicular docking sites and that of clusters of voltage-gated calcium channels.neurotransmitter release | active zone | calcium channel | release site | parallel fiber I n presynaptic terminals, the active zone (AZ) represents a highly specialized structure allowing the binding and Ca 2+ -dependent release of synaptic vesicles (SVs) (1). Fluctuation analysis of synaptic signals suggests the presence of one or several functional units per AZ (2, 3). These units are either called "release sites" or "docking sites," and their number per AZ represents the maximum SV output that this structure can provide per action potential (AP). In recent years, optical methods have been developed to record single-SV release (4). Notably, studies using superresolution and total internal reflection fluorescence imaging in functioning central synapses have provided information on the kinetics (5) and subsynaptic localization (6) of single-SV docking and release. Despite these advances, electrophysiological methods remain the most rapid and sensitive method to assess SV exocytosis at central synapses such as the calyx of Held (7) or cerebellar mossy fiber terminals (8). In recent years, electrophysiological recordings performed at special synapses containing a single AZ and a single postsynaptic density (PSD), called "simple synapses," revealed a fixed maximum in the number of SVs that can be released per AZ by a short calcium stimulus (9-11), providing a direct evaluation of the number of docking sites per AZ. This number ran...

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Visualizing presynaptic function

Cited by 111 publications

References 99 publications

α2-Adrenergic Receptor and Isoflurane Modulation of Presynaptic Ca2+ Influx and Exocytosis in Hippocampal Neurons

α2-Adrenergic Receptor and Isoflurane Modulation of Presynaptic Ca2+ Influx and Exocytosis in Hippocampal Neurons

Decoding neuroproteomics: integrating the genome, translatome and functional anatomy

Numbers of presynaptic Ca ²⁺ channel clusters match those of functionally defined vesicular docking sites in single central synapses

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Visualizing presynaptic function

Cited by 111 publications

References 99 publications

α2-Adrenergic Receptor and Isoflurane Modulation of Presynaptic Ca2+ Influx and Exocytosis in Hippocampal Neurons

α2-Adrenergic Receptor and Isoflurane Modulation of Presynaptic Ca2+ Influx and Exocytosis in Hippocampal Neurons

Decoding neuroproteomics: integrating the genome, translatome and functional anatomy

Numbers of presynaptic Ca 2+ channel clusters match those of functionally defined vesicular docking sites in single central synapses

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Numbers of presynaptic Ca ²⁺ channel clusters match those of functionally defined vesicular docking sites in single central synapses