AbstractFiber photometry recordings are commonly used as a proxy for neuronal activity, based on the assumption that increases in bulk calcium fluorescence reflect increases in spiking of the underlying neural population. However, this assumption has not been adequately tested. Here, using endoscopic calcium imaging in the striatum we report that the bulk fluorescence signal correlates weakly with somatic calcium signals, suggesting that this signal does not reflect spiking activity, but may instead reflect subthreshold changes in neuropil calcium. Consistent with this suggestion, the bulk fluorescence photometry signal correlated strongly with neuropil calcium signals extracted from these same endoscopic recordings. We further confirmed that photometry did not reflect striatal spiking activity with simultaneous in vivo extracellular electrophysiology and fiber photometry recordings in awake behaving mice. We conclude that the fiber photometry signal should not be considered a proxy for spiking activity in neural populations in the striatum.Significance statementFiber photometry is a technique for recording brain activity that has gained popularity in recent years due to it being an efficient and robust way to record the activity of genetically defined populations of neurons. However, it remains unclear what cellular events are reflected in the photometry signal. While it is often assumed that the photometry signal reflects changes in spiking of the underlying cell population, this has not been adequately tested. Here, we processed calcium imaging recordings to extract both somatic and non-somatic components of the imaging field, as well as a photometry signal from the whole field. Surprisingly, we found that the photometry signal correlated much more strongly with the non-somatic than the somatic signals. This suggests that the photometry signal most strongly reflects subthreshold changes in calcium, and not spiking. We confirmed this point with simultaneous fiber photometry and extracellular spiking recordings, again finding that photometry signals relate poorly to spiking in the striatum. Our results may change interpretations of studies that use fiber photometry as an index of spiking output of neural populations.
In vivo direct imaging of neuronal activity at high temporo-spatial resolution
