Aberrant hippocampal Ca2+ microwaves following synapsin-dependent adeno-associated viral expression of Ca2+ indicators

  1. Nicola Masala
  2. Manuel Mittag
  3. Eleonora Ambrad Giovannetti
  4. Darik A O'Neil
  5. Fabian J Distler
  6. Peter Rupprecht
  7. Fritjof Helmchen
  8. Rafael Yuste
  9. Martin Fuhrmann
  10. Heinz Beck
  11. Michael Wenzel  Is a corresponding author
  12. Tony Kelly  Is a corresponding author
  1. University of Bonn, Faculty of Medicine, Institute for Experimental Epileptology and Cognition Research (IEECR), Germany
  2. University Hospital Bonn, Germany
  3. Department of Epileptology, University Hospital Bonn, Germany
  4. Neuroimmunology and Imaging Group, German Center for Neurodegenerative Diseases (DZNE), Germany
  5. NeuroTechnology Center, Columbia University, United States
  6. Brain Research Institute, University of Zurich, Switzerland
  7. Neuroscience Center Zurich, University of Zurich, Switzerland
  8. German Center for Neurodegenerative Diseases (DZNE), Germany
3 figures, 6 videos, 2 tables and 1 additional file

Figures

Development of Ca2+ microwaves travelling through hippocampus following GCaMP transduction.

(a) Experimental protocol to examine CA1 neuronal activity using two-photon imaging following adeno-associated viral (AAV) transduction of genetically encoded Ca2+ indicators. (b) …

Aberrant Ca2+ microwaves are consistent across laboratories and genetically encoded calcium indicator (GECI) variant.

(a) Boxplot (median ± quartiles and range) of the occurrence rate of aberrant Ca2+ microwaves in CA1 at the different institutes at 6–8 wk after injection of GCaMP6s or GCaMP6m (number of animals in …

Author response image 1
Plot of Ca2+ micro-wave frequency (left: number of Ca2+ waves/min) or occurrence (right: yes/no) against the animal age at the time of viral injection.

Blue line is linear (left) or logistic (right) fit to the data with 95% confidence level.

Videos

Video 1
GCaMP6s two-photon calcium imaging in the hippocampal CA1 region, around 100 µm beneath the hippocampal surface (stratum pyramidale), FOV ~600x600 µm, ~4 wk after transduction of AAV1 particles containing pAAV.Syn.GCaMP6s.WPRE.SV40 (Addgene plasmid #100843) in a mature bl6 wildtype mouse.

Imaging wavelength = 940 nm, acquisition speed = 15 frames/s. Movie played at ×5 acquisition speed. Imaging was performed at the IEECR/University of Bonn.

Video 2
GCaMP6s two-photon calcium imaging in the hippocampal CA1 region, around 100 µm beneath the hippocampal surface (stratum pyramidale), FOV ~450x450 µm, ~7 wk after transduction of AAV1 particles containing pAAV.Syn.GCaMP6s.WPRE.SV40 (Addgene plasmid #100843) in an ~3-month-old transgenic mouse (same as in Video 2; Scn2aA263V model of genetic epilepsy).

Imaging wave length = 940 nm, acquisition speed = 15 frames/s. Movie played at ×5 acquisition speed. Imaging was performed at the IEECR/University of Bonn.

Video 3
Same animal (Scn2aA263V model of genetic epilepsy) and time point of imaging as in Video 4.

GCaMP6s two-photon calcium imaging in the hippocampal CA1 region, around 25 µm beneath the hippocampal surface (stratum oriens), FOV ~350x350 µm, ~7 wk after transduction of AAV1 particles …

Video 4
GCaMP6s two-photon calcium imaging in the hippocampal CA1 region, around 100 µm beneath the hippocampal surface (stratum pyramidale), FOV ~450x450 µm, ~2 wk after transduction of AAV1 particles containing pAAV.Syn.GCaMP6s.WPRE.SV40 (Addgene plasmid #100843) in an ~2-month-old transgenic mouse model of genetic epilepsy (heterozygous Scn2aA263V mouse).

Imaging wavelength = 940 nm, acquisition speed = 15 frames/s. Movie played at ×5 acquisition speed. Imaging was performed at the IEECR/University of Bonn.

Video 5
R-CaMP1.07 two-photon calcium imaging in the hippocampal CA1 region, around 100 µm beneath the hippocampal surface (stratum pyramidale), FOV ~200x200 µm, ~10 wk after transduction of AAV1 particles containing ssAAV-9/2-hSyn1-chI-RCaMP1.07-WPRE-SV40p(A) (Viral Vector Core UZH #V224-9) in a mature (~5 mo) bl6 wildtype mouse.

Imaging wavelength = 960 nm, acquisition speed = 30.88 frames/s. Movie played at ×5 acquisition speed. Imaging was performed at the Neuroscience Center Zurich (UZH).

Video 6
GCaMP6s two-photon calcium imaging in the hippocampal CA3 region, stratum pyramidale, FOV ~600x600 µm, ~7 wk after transduction of AAV1 particles containing pAAV.Syn.GCaMP6s.WPRE.SV40 (Addgene plasmid #100843) in a mature bl6 wildtype mouse.

Imaging wavelength = 940 nm, acquisition speed = 30.206 frames/s. Movie played at ×5 acquisition speed. Imaging was performed at Columbia University.

Tables

Table 1
Viruses used for the expression of genetically encoded calcium indicators (GECIs).

Viral titre is from Addgene documentation and was used at original concentration (dilution of 1:1) or at a dilution of 1:2. Syn.Flex.GCaMP6s and CamKII0.4.Cre were co-injected and therefore diluted …

AAVConstructSource(Addgene id)Original titre(vg/ml)DilutionInjection volumePost-injection(wk)RegionCa2+-wave incidence (%)nMouse modelInstitute
AAV1Syn.GCaMP6s1008431 × 10131:10.52CA100/4wtUoB
AAV1Syn.GCaMP6s1008431 × 10131:10.54–6CA11004/4wtUoB
AAV1Syn.GCaMP6s1008431 × 10131:10.52CA1201/5Scn2a*UoB
AAV1Syn.GCaMP6s1008431 × 10131:10.54–8CA11005/5Scn2a*UoB
AAV1Syn.GCaMP6m1008411 × 10131:118CA1804/5wtDZNE
AAV1Syn.GCaMP6m1008411 × 10131:216CA1433/7wtDZNE
AAV1Syn.GCaMP6s1008431 × 10131:20.54–10DG00/3wtUoB
AAV9Syn.jGCaMP7f1044882.5 × 10131:2110–14CA1503/6APPsweDZNE
AAV1Syn.GCaMP6s1008431 × 10131:10.253–12CA3100 (1 exp.)1/1wtCU
AAV1Syn.GCaMP6s1008431 × 10131:10.253–5CA1100 (1 exp.)1/1wtCU
AAV1Syn.GCaMP6s1008431 × 10131:20.84–5Ctx00/>20
0/>20
wtCU
AAV1Syn.GCaMP6f1008377 × 10121:20.753–6Ctx0wtCU
AAV9hSyn1.R-CaMP1.07V224-9 4.3 × 10121:10.28CA11002/2wtUZH
AAV1syn.Flex.GCaMPm1008381 × 10131:20.534–38CA100/3wtDZNE
AAV9CamKII0.4.Cre.SV401055581 × 10131:2
AAV1syn.Flex.GCaMP6s1008451 × 10131:20.56CA100/2wtUoB
AAV1CamKII0.4.Cre.SV401055581 × 10131:2
  1. *

    In heterozygous Scn2aA263V mice.

  2. In PV-Cre::APPswe/PS1dE9 mice.

  3. Sourced from the Viral Vector Facility University of Zurich (VVF/UZH).

Table 2
Viral constructs used.
Resources table
Genetic reagent (Mus musculus)Recombinant DNA reagentAAVSourceID
Syn.GCaMP6sSyn.GCaMP6s.WPRE.SV40AAV1Addgene100843
Syn.GCaMP6mSyn.GCaMP6m.WPRE.SV40AAV1Addgene100841
Syn.jGCaMP7fSyn-jGCaMP7f-WPREAAV9Addgene104488
Syn.GCaMP6fSyn.GCaMP6f.WPRE.SV40AAV1Addgene100837
hSyn1.R-CaMP1.07hSyn1-chI-RCaMP1.07-WPRE-SV40p(A)AAV9VVF/UZHV224-9
syn.Flex.GCaMP6sSyn.Flex.GCaMP6s.WPRE.SV40AAV1Addgene100845
syn.Flex.GCaMP6mSyn.Flex.GCaMP6m.WPRE.SV40AAV1Addgene100838
CamKII0.4.Cre.SV40CamKII 0.4.Cre.SV40AAV1 or 9Addgene105558

Additional files

Download links