Abstract
The interplay between hemodynamic-based markers of cortical activity (e.g. fMRI and optical intrinsic signal imaging), which are an indirect and relatively slow report of neural activity, and underlying synaptic electrical and metabolic activity through neurovascular coupling is a topic of ongoing research and debate. As application of resting state functional connectivity measures is extended further into topics such as brain development, aging and disease, the importance of understanding the fundamental physiological basis for functional connectivity will grow. Here we extend functional connectivity analysis from hemodynamic- to calcium-based imaging. Transgenic mice (n = 7) expressing a fluorescent calcium indicator (GCaMP6) driven by the Thy1 promoter in glutamatergic neurons were imaged transcranially in both anesthetized (using ketamine/xylazine) and awake states. Sequential LED illumination (λ = 454, 523, 595, 640nm) enabled concurrent imaging of both GCaMP6 fluorescence emission (corrected for hemoglobin absorption) and hemodynamics. Functional connectivity network maps were constructed for infraslow (0.009–0.08Hz), intermediate (0.08–0.4Hz), and high (0.4–4.0Hz) frequency bands. At infraslow and intermediate frequencies, commonly used in BOLD fMRI and fcOIS studies of functional connectivity and implicated in neurovascular coupling mechanisms, GCaMP6 and HbO2 functional connectivity structures were in high agreement, both qualitatively and also quantitatively through a measure of spatial similarity. The spontaneous dynamics of both contrasts had the highest correlation when the GCaMP6 signal was delayed with a ~0.6–1.5s temporal offset. Within the higher-frequency delta band, sensitive to slow wave sleep oscillations in non-REM sleep and anesthesia, we evaluate the speed with which the connectivity analysis stabilized and found that the functional connectivity maps captured putative network structure within time window lengths as short as 30 seconds. Homotopic GCaMP6 functional connectivity maps at 0.4–4.0Hz in the anesthetized states show a striking correlated and anti-correlated structure along the anterior to posterior axis. This structure is potentially explained in part by observed propagation of delta-band activity from frontal somatomotor regions to visuoparietal areas. During awake imaging, this spatio-temporal quality is altered, and a more complex and detailed functional connectivity structure is observed. The combined calcium/hemoglobin imaging technique described here will enable the dissociation of changes in ionic and hemodynamic functional structure and neurovascular coupling and provide a framework for subsequent studies of neurological disease such as stroke.
Highlights
Functional magnetic resonance imaging has been instrumental in unlocking the functional architecture of the brain and is used routinely to map patterns of resting-state functional connectivity brain networks in humans [1]
Because GCaMP6 is able to probe activity at frequencies higher than traditionally associated with hemodynamic functional connectivity (i.e.
There are a number of previously reported in Functional connectivity of calcium dynamics in anesthetized and awake mice vivo widefield optical neuroimaging studies in skull-intact mice [2, 15, 16] that have been successfully implemented
Summary
Functional magnetic resonance imaging (fMRI) has been instrumental in unlocking the functional architecture of the brain and is used routinely to map patterns of resting-state functional connectivity brain networks in humans [1]. Developed optical approaches for mapping resting-state functional connectivity in mice, including optical intrinsic signal imaging (fcOIS), image hemodynamics and have similar limitations [1, 2]. With the advent of genetic engineering techniques in mice, there are new opportunities for extending wide-field optical imaging to calcium dynamics and offer a more direct and faster measure of neural activity. Calcium concentration changes due to voltage-gated calcium channels can be imaged and visualized using fluorescent, genetically encoded calcium indicators (GECIs) [12]. These fluorophores, including GCaMP [13], are generated from a modified green fluorescent protein coupled to a calcium binding domain, enabling increased fluorescence in the presence of elevated calcium levels. GECI’s have been used extensively to study in vivo neural activity in C. elegans [13], mice [8, 13,14,15,16,17], rats [18], monkeys [19], and other animal models
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