Figures and data
Development of Ca2+ micro-waves travelling through hippocampus following GCaMP transduction.
a: Experimental protocol to examine CA1 neuronal activity using two-photon imaging following AAV transduction of genetically encoded Ca2+ indicators. b: Immunohistochemical sections following last imaging session. GCaMP6s (AAV1.syn.GCaMP6s.SV40, addgene #100843) expression throughout the ipsilateral hippocampus and projection pathways in the contralateral hippocampus. c: Two-photon Ca2+ imaging of FOV in CA1 at 4wks p.i. showing aberrant Ca2+ micro-waves (see also Supplementary video 1). Magnified inset shows 3 colored neuronal subgroups (blue, orange, magenta) based on their spatial vicinity from a total population of 100 identified neurons (green). right: time series of two-photon Ca2+ imaging FOVs showing two Ca2+ micro-waves, the first at 0 sec, the second appearing at 6 sec (asterisk). The second wave progresses through FOV over dozens of seconds. d: Raster-plot of individual neuronal Ca2+ activity (ΔF/F, 1min moving window, traces max-normalized per neuron) from neighboring subgroups (colors correspond to c). Asterisk (same as in c): a Ca2+ micro-wave advances through neighboring neuronal subgroups. e: Occurrence rate of aberrant Ca2+ micro-waves with increasing expression time, following viral transduction of AAV1.syn.GCaMP6s.SV40 in mature C57BL6 wild-type animals. n.d. = none detected. f: Two-photon Ca2+ imaging FOV in the visual cortex at 6wks p.i. (left) with normal sparse spontaneous Ca2+ activity and no detected Ca2+ micro-waves (right; raster plot of ΔF/F, 1min moving window, traces max-normalized per neuron).
Viruses used for expression of GECI.
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 to 1:2. Two-photon Ca2+ imaging was performed from 2 weeks after injection in the hippocampus (CA1, CA3 or DG) or neocortex (Ctx). Ca2+ micro-wave incidence was determined from the number of animals exhibiting Ca2+ micro-waves at the specified timepoint and region. * in heterozygous Scn2aA263V mice. # in PV-Cre::APPswe/PS1dE9 mice. $ Sourced from Viral Vector Facility University of Zurich (VVF/UZH). Inj. vol. = injection volume in µl; p.i. = post-injection; wks = weeks.
Aberrant Ca2+ micro-waves are consistent across laboratories and GECI variant.
a: Plot of the occurrence rate of aberrant Ca2+ micro-waves in CA1 at the different institutes at 6-8wks after injection of GCaMP6s or GCaMP6m. b: Ca2+ micro-wave diameters (left) and progression speed (right) in CA1 from each animal recorded across institutes. Inset: Histogram of fluorescent intensity taken across each Ca2+-wave within an animal. Green line is the average, areas outside dashed lines mark 10% lowest fluorescence values, which were excluded from analysis. c: Plot of the occurrence rate of aberrant Ca2+ micro-waves in CA1 following injection with commonly used GECIs (see table 1). d: Two-photon Ca2+ imaging FOV (left) in hippocampal CA1 following dual injection approach for conditional GCaMP6s expression (6 wks p.i.) with normal sparse spontaneous Ca2+ activity and no detection of Ca2+ micro-waves (right; raster plot of ΔF/F, 1min moving window, traces max-normalized per neuron).
