Transgenic mice expressing genetically encoded activity indicators are an attractive means of mapping mesoscopic regional functional cortical connectivity given widespread stable and cell-specific expression compatible with chronic recordings. Cortical functional connectivity was evaluated using wide-field imaging in lightly anesthetized Emx1-creXRosa26-GCaMP3 mice expressing calcium sensor in cortical neurons. Challenges exist because green fluorescence signals overlap with endogenous activity-dependent autofluorescence and are affected by changes in blood volume and oxygenation. Under the conditions used for imaging and analysis (0.1-1 Hz frequency band), autofluorescence and hemodynamic effects contributed 3% and 8% of the SD of spontaneous activity-dependent GCaMP3 fluorescence when signals were recorded through intact bone. To evaluate the accuracy and sensitivity of this approach, the topology of functional connections between somatomotor cortex (primary S1 and secondary S2 somatosensory, and primary motor cortex M1) was estimated. During sequences of spontaneous activity, calcium signals recorded at each location of area S1 were correlated with activity in contralateral area S1, ipsilateral area S2, and bilateral areas M1. Reciprocal results were observed when "seed pixels" were placed in S2 and M1. Coactivation of areas implies functional connections but could also be attributed to both regions receiving common upstream drive. These apparent connections revealed during spontaneous activity coactivation by GCaMP3 were confirmed by intracortical microstimulation but were more difficult to detect using intrinsic signals from reflected red light. We anticipate GCAMP wide-field imaging will enable longitudinal studies during plasticity paradigms or after models of CNS disease, such as stroke, where the weighting within these connectivity maps may be altered.

Connectivity mapping based on resting-state activity in mice has revealed functional motifs of correlated activity. However, the rules by which motifs organize into larger functional modules that lead to hemisphere wide spatial-temporal activity sequences is not clear. We explore cortical activity parcellation in head-fixed, quiet awake GCaMP6 mice from both sexes by using mesoscopic calcium imaging. Spectral decomposition of spontaneous cortical activity revealed the presence of two dominant frequency modes (Ͻ1 and ϳ3 Hz), each of them associated with a unique spatial signature of cortical macro-parcellation not predicted by classical cytoarchitectonic definitions of cortical areas. Based on assessment of 0.1-1 Hz activity, we define two macro-organizing principles: the first being a rotating polymodal-association pinwheel structure around which activity flows sequentially from visual to barrel then to hindlimb somatosensory; the second principle is correlated activity symmetry planes that exist on many levels within a single domain such as intrahemispheric reflections of sensory and motor cortices. In contrast, higher frequency activity Ͼ1 Hz yielded two larger clusters of coactivated areas with an enlarged default mode network-like posterior region. We suggest that the apparent constrained structure for intra-areal cortical activity flow could be exploited in future efforts to normalize activity in diseases of the nervous system.

Understanding the basis of brain function requires knowledge of cortical operations over wide-spatial scales, but also within the context of single neurons. In vivo, wide-field GCaMP imaging and sub-cortical/cortical cellular electrophysiology were used in mice to investigate relationships between spontaneous single neuron spiking and mesoscopic cortical activity. We make use of a rich set of cortical activity motifs that are present in spontaneous activity in anesthetized and awake animals. A mesoscale spike-triggered averaging procedure allowed the identification of motifs that are preferentially linked to individual spiking neurons by employing genetically targeted indicators of neuronal activity. Thalamic neurons predicted and reported specific cycles of wide-scale cortical inhibition/excitation. In contrast, spike-triggered maps derived from single cortical neurons yielded spatio-temporal maps expected for regional cortical consensus function. This approach can define network relationships between any point source of neuronal spiking and mesoscale cortical maps.DOI: http://dx.doi.org/10.7554/eLife.19976.001

Background-Craniotomy-based window implants are commonly used for microscopic imaging, in head-fixed rodents, however their field of view is typically small and incompatible with mesoscopic functional mapping of cortex.New Method-We describe a reproducible and simple procedure for chronic through-bone widefield imaging in awake head-fixed mice providing stable optical access for chronic imaging over large areas of the cortex for months.Results-The preparation is produced by applying clear-drying dental cement to the intact mouse skull, followed by a glass coverslip to create a partially transparent imaging surface. Surgery time takes about 30 minutes. A single set-screw provides a stable means of attachment for mesoscale assessment without obscuring the cortical field of view.Comparison with Existing Methods-We demonstrate the utility of this method by showing seed-pixel functional connectivity maps generated from spontaneous cortical activity of GCAMP6 signals in both awake and anesthetized mice.Conclusions-We propose that the intact skull preparation described here may be used for most longitudinal studies that do not require micron scale resolution and where cortical neural or vascular signals are recorded with intrinsic sensors.