Abstract
BackgroundBrain visual circuits are often studied in vivo by imaging Ca2+ indicators with green-shifted emission spectra. Polychromatic white visual stimuli have a spectrum that partially overlaps indicators´ emission spectra, resulting in significant contamination of calcium signals. New methodTo overcome light contamination problems we choose blue visual stimuli, having a spectral composition not overlapping with Ca2+ indicator´s emission spectrum. To compare visual responsiveness to blue and white stimuli we used electrophysiology (visual evoked potentials –VEPs) and 3D acousto-optic two-photon (2P) population Ca2+ imaging in mouse primary visual cortex (V1). ResultsVEPs in response to blue and white stimuli had comparable peak amplitudes and latencies. Ca2+ imaging in a Thy1 GP4.3 line revealed that the populations of neurons responding to blue and white stimuli were largely overlapping, that their responses had similar amplitudes, and that functional response properties such as orientation and direction selectivities were also comparable. Comparison with existing methodsMasking or shielding the microscope are often used to minimize the contamination of Ca2+ signal by white light, but they are time consuming, bulky and thus can limit experimental design, particularly in the more and more frequently used awake set-up. Blue stimuli not interfering with imaging allow to omit shielding. ConclusionsTogether, our results show that the selected blue light stimuli evoke responses comparable to those evoked by white stimuli in mouse V1. This will make complex designs of imaging experiments in behavioral set-ups easier, and facilitate the combination of Ca2+ imaging with electrophysiology and optogenetics.
Highlights
Ca2+ imaging is widely used to monitor neuronal responses to different stimuli and conditions at population, axonal, dendritic and dendritic spine levels both in vitro and in vivo (Dana et al, 2019; Grienberger and Konnerth, 2012)
We first selected a “blue filter” transmitting a light spectrum (392–497 nm) from the CRT screen that would not overlap with the acquired GCaMP6s emission spectrum, band-passed by the “green filter” (503–539 nm, FBH520–40, Thorlabs) before the microscope’s photo multiplier tube (PMT)
To estimate the effectiveness of the blue light in driving mouse V1 activation compared to the white light, we first recorded VEPs in layer 2/3 in response to blue/black or white/black stationary gratings alter nating in counter-phase presented to the contralateral eye of anes thetized mice, adapted to the ambient light level of the set-up used for physiology experiments (Fig. 1B; for details see the Section 2.2.3 - “Vi sual stimulation during LFP recording”)
Summary
Ca2+ imaging is widely used to monitor neuronal responses to different stimuli and conditions at population, axonal, dendritic and dendritic spine levels both in vitro and in vivo (Dana et al, 2019; Grienberger and Konnerth, 2012). Brain visual circuits are often studied in vivo by imaging Ca2+ indicators with green-shifted emission spectra. Polychromatic white visual stimuli have a spectrum that partially overlaps indicatorsemission spectra, resulting in significant contamination of calcium signals. New method: To overcome light contamination problems we choose blue visual stimuli, having a spectral composition not overlapping with Ca2+ indicators emission spectrum. Conclusions: Together, our results show that the selected blue light stimuli evoke responses comparable to those evoked by white stimuli in mouse V1. This will make complex designs of imaging experiments in behavioral setups easier, and facilitate the combination of Ca2+ imaging with electrophysiology and optogenetics
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