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

Abstract

Genetically encoded calcium indicators (GECIs) such as GCaMP are invaluable tools in neuroscience to monitor neuronal activity using optical imaging. The viral transduction of GECIs is commonly used to target expression to specific brain regions, can be conveniently used with any mouse strain of interest without the need for prior crossing with a GECI mouse line, and avoids potential hazards due to the chronic expression of GECIs during development. A key requirement for monitoring neuronal activity with an indicator is that the indicator itself minimally affects activity. Here, using common adeno-associated viral (AAV) transduction procedures, we describe spatially confined aberrant Ca²⁺ microwaves slowly travelling through the hippocampus following expression of GCaMP6, GCaMP7, or R-CaMP1.07 driven by the synapsin promoter with AAV-dependent gene transfer in a titre-dependent fashion. Ca²⁺ microwaves developed in hippocampal CA1 and CA3, but not dentate gyrus nor neocortex, were typically first observed at 4 wk after viral transduction, and persisted up to at least 8 wk. The phenomenon was robust and observed across laboratories with various experimenters and setups. Our results indicate that aberrant hippocampal Ca²⁺ microwaves depend on the promoter and viral titre of the GECI, density of expression, as well as the targeted brain region. We used an alternative viral transduction method of GCaMP which avoids this artefact. The results show that commonly used Ca²⁺-indicator AAV transduction procedures can produce artefactual Ca²⁺ responses. Our aim is to raise awareness in the field of these artefactual transduction-induced Ca²⁺ microwaves, and we provide a potential solution.

Keywords: AAV; GCaMP; GECI; in vivo; mouse; neuroscience.

Figure 1.. 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) Immunohistochemical sections following the 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 field of view (FOV) in CA1 at 4 wk post-injection (p.i.) showing aberrant Ca²⁺ microwaves (see also Video 1). Magnified inset shows three coloured 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⁺ microwaves, the first at 0 s, the second appearing at 6 s (asterisk). The second wave progresses through FOV over dozens of seconds. (d) Raster plot of individual neuronal Ca²⁺ activity (ΔF/F, 1 min moving window, traces max-normalized per neuron) from neighbouring subgroups (colours correspond to c). Asterisk (same as in c): a Ca²⁺ microwave advances through neighbouring neuronal subgroups. (e) Occurrence rate (mean ± 95% CI) of aberrant Ca²⁺ microwaves with increasing expression time, following viral transduction of AAV1.syn.GCaMP6s.SV40 in mature C57BL/6 wildtype animals (n=4). n.d. = none detected. (f) Two-photon Ca²⁺ imaging FOV in the visual cortex at 6 wk p.i. (left) with normal sparse spontaneous Ca²⁺ activity and no detected Ca²⁺ microwaves (right; raster plot of ΔF/F, 1 min moving window, traces max-normalized per neuron).

Figure 2.. 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 parenthesis). (b) Boxplots (median ± quartiles and range) of Ca²⁺ microwave 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) Boxlot (median ± quartiles and range) of the occurrence rate of aberrant Ca²⁺ microwaves in CA1 following injection with commonly used GECIs (number of animals in parenthesis; see Table 1). (d) Two-photon Ca²⁺ imaging field of view (FOV) (left) in hippocampal CA1 following dual injection approach for conditional GCaMP6s expression (6 wk post-injection [p.i.]) with normal sparse spontaneous Ca²⁺ activity and no detection of Ca²⁺ microwaves (right; raster plot of ΔF/F, 1 min moving window, traces max-normalized per neuron).

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.