PMID- 31057389
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20200930
IS  - 1663-3563 (Print)
IS  - 1663-3563 (Electronic)
IS  - 1663-3563 (Linking)
VI  - 11
DP  - 2019
TI  - Imaging and Analysis of Presynaptic Calcium Influx in Cultured Neurons Using 
      synGCaMP6f.
PG  - 12
LID - 10.3389/fnsyn.2019.00012 [doi]
LID - 12
AB  - Presynaptic Ca(2+) 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(2+) 
      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(2+) transients in individual 
      presynaptic terminals. The indicator GCaMP6f was developed for fast speed and 
      high sensitivity in detecting Ca(2+) 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(2+) 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(2+) 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(2+) 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(2+) transients. In addition, synGCaMP6f could be used to dissect 
      the contribution of different subtypes of VGCCs to presynaptic Ca(2+) 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.
FAU - Brockhaus, Johannes
AU  - Brockhaus J
AD  - Institute of Anatomy and Molecular Neurobiology, Westfalische 
      Wilhelms-University, Munster, Germany.
FAU - Bruggen, Bianca
AU  - Bruggen B
AD  - Institute of Anatomy and Molecular Neurobiology, Westfalische 
      Wilhelms-University, Munster, Germany.
FAU - Missler, Markus
AU  - Missler M
AD  - Institute of Anatomy and Molecular Neurobiology, Westfalische 
      Wilhelms-University, Munster, Germany.
LA  - eng
PT  - Journal Article
DEP - 20190416
PL  - Switzerland
TA  - Front Synaptic Neurosci
JT  - Frontiers in synaptic neuroscience
JID - 101548972
PMC - PMC6477507
OTO - NOTNLM
OT  - genetically encoded calcium indicator
OT  - high voltage activated calcium channels
OT  - live imaging
OT  - paired pulse facilitation
OT  - synapse
OT  - synaptic transmission
EDAT- 2019/05/07 06:00
MHDA- 2019/05/07 06:01
PMCR- 2019/01/01
CRDT- 2019/05/07 06:00
PHST- 2018/12/18 00:00 [received]
PHST- 2019/03/26 00:00 [accepted]
PHST- 2019/05/07 06:00 [entrez]
PHST- 2019/05/07 06:00 [pubmed]
PHST- 2019/05/07 06:01 [medline]
PHST- 2019/01/01 00:00 [pmc-release]
AID - 10.3389/fnsyn.2019.00012 [doi]
PST - epublish
SO  - Front Synaptic Neurosci. 2019 Apr 16;11:12. doi: 10.3389/fnsyn.2019.00012. 
      eCollection 2019.