Presynaptic Ca influx through voltage-gated calcium channels (VGCCs) is a key step in synaptic transmission that links action potential (AP)-derived depolarization to vesicle release. However, investigation of presynaptic Ca influx by patch clamp recordings is difficult due to the small size of the majority of synaptic boutons along thin axons that hamper clamp control. Genetically encoded calcium indicators (GECIs) in combination with live cell imaging provide an alternative method to study Ca transients in individual presynaptic terminals. The indicator GCaMP6f was developed for fast speed and high sensitivity in detecting Ca transients even in subcellular compartments. We fused GCaMP6f to synaptophysin (synGCaMP6f) to enrich the calcium indicator in presynaptic boutons of transfected primary hippocampal neurons to study presynaptic Ca changes in response to individual APs or short bursts. Changes in fluorescence intensity were evaluated by normalization to control level or, alternatively, by normalization to maximal fluorescence using the calcium ionophore ionomycin. Measurements revealed robust Ca transients with amplitudes that depend on parameters like the number of APs, stimulation frequency or external calcium concentration. Our findings indicate an appropriate sensitivity of synGCaMP6f for studying total presynaptic Ca transients induced by single APs or short bursts that showed little rundown of the response after repeated bursts. Moreover, these recordings are fast enough to even study short-term plasticity like paired pulse facilitation (PPF) and frequency dependence of Ca transients. In addition, synGCaMP6f could be used to dissect the contribution of different subtypes of VGCCs to presynaptic Ca influx. Our results demonstrate that synGCaMP6f allows the reliable analysis of changes in presynaptic calcium concentration at many individual synaptic boutons in parallel and provides the possibility to study the regulation of this important step in synaptic transmission.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6477507 | PMC |
| http://dx.doi.org/10.3389/fnsyn.2019.00012 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Minimal presynaptic protein machinery governing diverse kinetics of calcium-evoked neurotransmitter release.
Nat Commun
December 2024
Nanobiology Institute, Yale University, West Haven, CT, USA.
Neurotransmitters are released from synaptic vesicles with remarkable precision in response to presynaptic calcium influx but exhibit significant heterogeneity in exocytosis timing and efficacy based on the recent history of activity. This heterogeneity is critical for information transfer in the brain, yet its molecular basis remains poorly understood. Here, we employ a biochemically-defined fusion assay under physiologically relevant conditions to delineate the minimal protein machinery sufficient to account for various modes of calcium-triggered vesicle fusion dynamics.
View Article and Find Full Text PDFDendritic, delayed, stochastic CaMKII activation in behavioural time scale plasticity.
Nature
November 2024
Neuronal Signal Transduction Group, Max Planck Florida Institute for Neuroscience, Jupiter, FL, USA.
Behavioural time scale plasticity (BTSP) is non-Hebbian plasticity induced by integrating presynaptic and postsynaptic components separated by a behaviourally relevant time scale (seconds). BTSP in hippocampal CA1 neurons underlies place cell formation. However, the molecular mechanisms that enable synapse-specific plasticity on a behavioural time scale are unknown.
View Article and Find Full Text PDFDistinct input-specific mechanisms enable presynaptic homeostatic plasticity.
bioRxiv
September 2024
University of Southern California, Department of Neurobiology, Los Angeles, CA USA.
Synapses are endowed with the flexibility to change through experience, but must be sufficiently stable to last a lifetime. This tension is illustrated at the neuromuscular junction (NMJ), where two motor inputs that differ in structural and functional properties co-innervate most muscles to coordinate locomotion. To stabilize NMJ activity, motor neurons augment neurotransmitter release following diminished postsynaptic glutamate receptor functionality, termed presynaptic homeostatic potentiation (PHP).
View Article and Find Full Text PDFSimultaneous recordings from vestibular Type I hair cells and their calyceal afferents in mice.
Front Neurol
August 2024
Anatomy and Cell Biology, College of Medicine, University of Illinois Chicago, Chicago, IL, United States.
The vestibular hair cell receptors of anamniotes, designated Type II, are presynaptic to bouton endings of vestibular nerve distal neurites. An additional flask-shaped hair cell receptor, Type I, is present in amniotes, and communicates with a chalice-shaped afferent neuritic ending that surrounds the entire hair cell except its apical neck. Since the full repertoire of afferent fiber dynamics and sensitivities observed throughout the vertebrate phyla can be accomplished through Type II hair cell-bouton synapses, the functional contribution(s) of Type I hair cells and their calyces to vestibular performance remains a topic of great interest.
View Article and Find Full Text PDFGraded control of Purkinje cell outputs by cAMP through opposing actions on axonal action potential and transmitter release.
J Physiol
July 2024
Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan.
All-or-none signalling by action potentials (APs) in neuronal axons is pivotal for the precisely timed and identical size of outputs to multiple distant targets. However, technical limitations with respect to measuring the signalling in small intact axons have hindered the evaluation of high-fidelity signal propagation. Here, using direct recordings from axonal trunks and/or terminals of cerebellar Purkinje cells in slice and culture, we demonstrate that the timing and amplitude of axonal outputs are gradually modulated by cAMP depending on the length of axon.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!