ROUGH ENDOPLASMIC RETICULUM TETHERING IMPROVES SOMATIC CALCIUM IMAGING

Calcium imaging using genetically encoded calcium indicators such as the GCaMP family is one of the most widely used approaches for monitoring neural activity. However, fluorescence from the surrounding neuropil can contaminate somatic calcium signals, thereby complicating signal extraction and interpretation. A direct strategy to address this issue is to restrict indicator expression to neuronal cell bodies. Inspired by two previously reported soma-targeted GCaMP variants—soma-GCaMP and riboL1-GCaMP—which exhibit limited soma specificity or reduced brightness, we sought to develop a soma-targeted GCaMP that preserves signal strength while minimizing neuropil contamination by tethering GCaMP to the rough endoplasmic reticulum (RER). To this end, we systematically screened a panel of RER membrane proteins. Through in vitro screening in U87 cells and in vivo wide-field imaging in mice, we identified a construct based on the protein KTN1 that reliably reports neural activity with somatic localization, which we termed ER-GCaMP. Using two-photon imaging in mice, we compared ER-GCaMP8s with the previously reported soma-GCaMP8s and riboL1-GCaMP8s. ER-GCaMP8s exhibited soma specificity comparable to riboL1-GCaMP8s while displaying ~3-fold higher fluorescence brightness. Consistent with this advantage, wide-field imaging revealed that ER-GCaMP8s produced higher baseline fluorescence and larger visually evoked ΔF/F responses than riboL1-GCaMP8s. Finally, we validated ER-GCaMP8s in the macaque primary visual cortex (V1), confirming spatially restricted expression and functional viability for wide-field imaging. We further show that this ER-targeting sequence is compatible with other calcium indicators, highlighting the broad applicability of this strategy for high-performance somatic calcium imaging.