title: Nonlinearity of two-photon Ca2+ imaging yields distorted measurements of tuning for V1 neuronal populations.
abstract: We studied the relative accuracy of drifting gratings and noise stimuli for functionally characterizing neural populations using two-photon calcium imaging. Calcium imaging has the potential to distort measurements due to nonlinearity in the conversion from spikes to observed fluorescence. We demonstrate a dramatic impact of fluorescence saturation on functional measurements in ferret V1 by showing that responses to drifting gratings strongly violate contrast invariance of orientation tuning, a fundamental property of the spike rates. The observed relationship is consistent with saturation that clips the high-contrast tuning curve peaks by ∼40%. The nonlinearity was also apparent in mouse V1 responses to drifting gratings, but not as strong as in the ferret. Contrast invariance holds, however, for tuning curves measured with a randomized grating stimulus. This finding is consistent with prior work showing that the linear portion of a linear-nonlinear system can be recovered with reverse correlation. Furthermore, we demonstrate that a noise stimulus is more effective at keeping spike rates in the linear operating regime of a saturating nonlinearity, which both maximizes signal-to-noise ratios and simplifies the recovery of fast spike dynamics from slow calcium transients. Finally, we uncover spatiotemporal receptive fields by removing the nonlinearity and slow calcium transient from a model of fluorescence generation, which allowed us to observe dynamic sharpening of orientation tuning. We conclude that for two-photon recordings it is imperative that one considers the nonlinear distortion when designing stimuli and interpreting results, especially in sensory areas, species, or cell types with high firing rates.
authors: Systems Neurobiology Laboratories, Salk Institute for Biological Studies, La Jolla, CA 92037, USA. inauhaus@salk.edu
authors: Ian
authors: I
authors: Nauhaus
authors: Kristina J
authors: KJ
authors: Nielsen
authors: Edward M
authors: EM
authors: Callaway
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015350:Contrast Sensitivity / Q000502:physiology; D005289:Ferrets; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008959:Models, Neurological; D057054:Molecular Imaging / Q000379:methods*; D009474:Neurons / Q000502:physiology; D017711:Nonlinear Dynamics; D010775:Photic Stimulation / Q000379:methods; D059629:Signal-To-Noise Ratio; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014793:Visual Cortex / Q000502:physiology*
chemical_list: D002118:Calcium