Aberrant Cortical Activity in Multiple GCaMP6-Expressing Transgenic Mouse Lines

Abstract

Transgenic mouse lines are invaluable tools for neuroscience but, as with any technique, care must be taken to ensure that the tool itself does not unduly affect the system under study. Here we report aberrant electrical activity, similar to interictal spikes, and accompanying fluorescence events in some genotypes of transgenic mice expressing GCaMP6 genetically encoded calcium sensors. These epileptiform events have been observed particularly, but not exclusively, in mice with Emx1-Cre and Ai93 transgenes, of either sex, across multiple laboratories. The events occur at >0.1 Hz, are very large in amplitude (>1.0 mV local field potentials, >10% df/f widefield imaging signals), and typically cover large regions of cortex. Many properties of neuronal responses and behavior seem normal despite these events, although rare subjects exhibit overt generalized seizures. The underlying mechanisms of this phenomenon remain unclear, but we speculate about possible causes on the basis of diverse observations. We encourage researchers to be aware of these activity patterns while interpreting neuronal recordings from affected mouse lines and when considering which lines to study.

Keywords: Cortex; GCaMP; epilepsy; transgenic.

Figure 1.

Epileptiform electrical activity in frontal cortex of some GCaMP6-expressing mice but not others. Left, each row contains an example segment of raw LFP data from each of six mice, with genotype identified in colored text. Middle, a plot of the prominence versus width of all peaks (see Methods) in the full LFP traces. Points highlighted in black were identified as a distinct cluster and included in the computation of event rate and the example events plotted at right. Right, 50 example events (gray) and the mean of all events (red). For the recording in row C, positive peaks rather than negative were analyzed.

Figure 2.

Comparison of events observed in LFP and widefield calcium imaging in the same mouse. Format as in Fig. 1. The two LFP recordings were made simultaneously with each other, but not simultaneously with the widefield imaging. In the frontal LFP recording, the two clusters in prominence versus width arise because of the double-peaked shape of the events in this mouse at this site: for some events, the width includes only the first peak; for some, it includes both.

Figure 3.

Incidence and spatial extent of epileptiform events observed in widefield calcium imaging. Format as in Fig. 1. The intensity trace and detected peaks come from the pixel indicated by the red circle on the brain image at right. Green coloration in the rightmost panel, overlaid on the mean image of the brain, represents the amplitude of the mean event at each point across the brain. See Videos 1, 2, and 3 for examples.

Figure 4.

Germline Cre recombination results in widespread GCaMP expression. A, B, Native GCaMP6 fluorescence obtained using two-photon serial tomography from two Rbp4-Cre/wt;Camk2a-tTA/wt;Ai93 STOP⁺/Ai93 STOP⁺ mice, showing expression of GCaMP only in restricted populations of L5 cortical neurons and moderate expression in hippocampus. C, D, Similar images with matched intensity scale but from a Rbp4-Cre/wt;Camk2a-tTA/wt;Ai93 STOP⁺/Ai93 STOPdel mouse that had germline Cre recombination, showing high, widespread expression across cortex and hippocampus.

Figure 5.

Epileptiform events observed in LFP, two-photon calcium imaging, and widefield calcium imaging in one individual mouse, but not simultaneously. Format as in Fig. 1. The genotype of the mouse was Emx1-Cre;Camk2a-tTA;Ai94 (expressing GCaMP6s). Two-photon trace was generated as the mean intensity of each frame across the entire field of view; widefield trace was generated as the mean within an ROI approximating the two-photon field of view.

Figure 6.

Epileptiform events fail to develop over time in mice treated with doxycycline until age 7 weeks. A, Measured rates of epileptiform events in untreated (red) and doxycycline treated (green) mice by age at time of measurement. Connected points indicate observations from the same mouse. All doxycycline-treated mice failed to develop events over the measured time period. Note that the mice represented in this figure are a small subset of all measured mice, and we do not intend to claim that the time courses followed here are representative of all mice developing events. B, C, and D refer to the measurements corresponding to the figure panels at right. B–D, Example traces and prominence versus width plots for an example untreated mouse showing development of events between 11 and 14 weeks.