Imaging Activity in Neurons and Glia with a Polr2a-Based and Cre-Dependent GCaMP5G-IRES-tdTomato Reporter Mouse

Abstract

SUMMARY New strategies for introducing genetically encoded activity indicators into animal models facilitate the investigation of nervous system function. We have developed the PC::G5-tdT mouse line that expresses the GCaMP5G calcium indicator in a Cre-dependent fashion. Instead of targeting the ROSA26 locus, we inserted the reporter cassette nearby the ubiquitously expressed Polr2a gene without disrupting locus integrity. The indicator was tagged with IRES-tdTomato to aid detection of positive cells. This reporter system is effective in a wide range of developmental and cellular contexts. We recorded spontaneous cortical calcium waves in intact awake newborns and evaluated concentration-dependent responses to odorants in the adult olfactory bulb. Moreover, PC::G5-tdT effectively reports intracellular calcium dynamics in somas and fine processes of astrocytes and microglial cells. Through electrophysiological and behavioral analyses, we determined that GCaMP5G expression had no major impact on nervous system performance. PC::G5-tdT will be instrumental for a variety of brain mapping experiments.

Long-term In Vivo Calcium Imaging of Astrocytes Reveals Distinct Cellular Compartment Responses to Sensory Stimulation

“…1E) and a greater number of signals ( Fig. 1D) than endfeet or somata, comparable to previous reports with other calcium indicators and in other brain regions (Shigetomi et al 2013;Gee et al 2014;Kanemaru et al 2014;Otsu et al 2015;Srinivasan et al 2015;Tang et al 2015). A previous study has also detected three different populations of GCaMP6s peaks in astrocytes: single peaks, multi peaks, and plateaus (Bonder and McCarthy 2014).…”

“…We also found that somata, processes and endfeet had a similar mean signal onset (~15 seconds), though peaks occurred earlier after the start of stimulation. Unlike Gee et al (2014), we did not observe similar somata and process signal amplitudes (Fig. 4D).…”

“…4B), similar to light-evoked astrocyte calcium signals in the visual cortex (Asada et al 2015). Processes also reportedly have an earlier calcium signal onset time after stimulation (Wang et al 2006;Gee et al 2014). When considering only the responding ROIs in each sub-cellular compartment, we found processes had the fastest mean time to reach peak maximum after 90 Hz whisker stimulation (14.41 ± 0.35 s), but there was no significant difference (P= 0.999 for all comparisons) between process, endfoot (14.73 ± 0.75 s), and soma (15.07 ± 0.51 s) peak times.…”

“…In terms of astrocyte compartments, a recent study compared calcium signals (from GCaMP5G) in astrocyte processes and somata during whisker stimulation and they found that somata and processes had similar mean amplitudes with a signal delay of ~25 s after the start of stimulation (Gee et al 2014). We also found that somata, processes and endfeet had a similar mean signal onset (~15 seconds), though peaks occurred earlier after the start of stimulation.…”

If I had a million neurons

“…This inspiration also explains the ambitious focus on a million neurons. Obviously, research can benefit tremendously from techniques for recording up to a thousand neurons (Dombeck et al, 2010;Gee et al, 2014;Ghosh et al, 2011;Heys et al, 2014;Sheffield and Dombeck, 2014), and further benefits will also arise from recording tens of thousands of neurons or hundreds of thousands of neurons.…”