Abstract
Fluorescent calcium (Ca2+) indicator proteins (FCIPs) are promising tools for functional imaging of cellular activity in living animals. However, they have still not reached their full potential for in vivo imaging of neuronal activity due to limitations in expression levels, dynamic range, and sensitivity for reporting action potentials. Here, we report that viral expression of the ratiometric Ca2+ sensor yellow cameleon 3.60 (YC3.60) in pyramidal neurons of mouse barrel cortex enables in vivo measurement of neuronal activity with high dynamic range and sensitivity across multiple spatial scales. By combining juxtacellular recordings and two-photon imaging in vitro and in vivo, we demonstrate that YC3.60 can resolve single action potential (AP)-evoked Ca2+ transients and reliably reports bursts of APs with negligible saturation. Spontaneous and whisker-evoked Ca2+ transients were detected in individual apical dendrites and somata as well as in local neuronal populations. Moreover, bulk measurements using wide-field imaging or fiber-optics revealed sensory-evoked YC3.60 signals in large areas of the barrel field. Fiber-optic recordings in particular enabled measurements in awake, freely moving mice and revealed complex Ca2+ dynamics, possibly reflecting different behavior-related brain states. Viral expression of YC3.60 – in combination with various optical techniques – thus opens a multitude of opportunities for functional studies of the neural basis of animal behavior, from dendrites to the levels of local and large-scale neuronal populations.
Highlights
Neuronal circuits are organized at diverse spatial scales, from subcellular compartments such as dendrites to local neuronal populations to whole brain areas
AAV-INDUCED EXPRESSION OF THE CA2+ SENSOR YC3.60 IN MOUSE BARREL CORTEX YC3.60 is a genetic Ca2+ sensor based on a cyano fluorescent protein (CFP), a Ca2+-sensitive linker, and a bright circularly permuted yellow fluorescent protein (YFP) (Nagai et al, 2004)
We have shown that upon neuron-specific expression in the mouse neocortex, the Fluorescent calcium (Ca2+) indicator proteins (FCIPs) YC3.60 can be used to read out activation of neocortical areas at different spatial scales both in anesthetized and awake, freely moving animals
Summary
Neuronal circuits are organized at diverse spatial scales, from subcellular compartments such as dendrites to local neuronal populations to whole brain areas. Advanced in vivo fluorescence staining and imaging techniques, in particular using fluorescent Ca2+ indicators, permit functional studies of neuronal activity in the living brain across all spatial scales (Helmchen and Denk, 2005; Kerr and Denk, 2008; Grewe and Helmchen, 2009; Wilt et al, 2009). Probing of neuronal network activity in vivo became possible by a multi-cell bolus loading technique with membrane-permeant Ca2+ indicators (Stosiek et al, 2003; Kerr et al, 2005), enabling for example the study of the functional organization of sensory-evoked neuronal population responses in the rodent barrel cortex (Kerr et al, 2007; Sato et al, 2007). A great advantage of the latter approach is that it permits measurements in awake and freely moving animals
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