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Resting-state functional magnetic resonance imaging has been increasingly used for examining connectivity across brain regions. The spatial scale by which hemodynamic imaging can resolve functional connections at rest remains unknown. To examine this issue, deoxyhemoglobin-weighted intrinsic optical imaging data were acquired from the visual cortex of lightly anesthetized ferrets. The neural activity of orientation domains, which span a distance of 0.7-0.8 mm, has been shown to be correlated during evoked activity and at rest. We performed separate analyses to assess the degree to which the spatial and temporal characteristics of spontaneous hemodynamic signals depend on the known functional organization of orientation columns. As a control, artificial orientation column maps were generated. Spatially, resting hemodynamic patterns showed a higher spatial resemblance to iso-orientation maps than artificially generated maps. Temporally, a correlation analysis was used to establish whether iso-orientation domains are more correlated than orthogonal orientation domains. After accounting for a significant decrease in correlation as a function of distance, a small but significant temporal correlation between iso-orientation domains was found, which decreased with increasing difference in orientation preference. This dependence was abolished when using artificially synthetized orientation maps. Finally, the temporal correlation coefficient as a function of orientation difference at rest showed a correspondence with that calculated during visual stimulation suggesting that the strength of resting connectivity is related to the strength of the visual stimulation response. Our results suggest that temporal coherence of hemodynamic signals measured by optical imaging of intrinsic signals exists at a submillimeter columnar scale in resting state.

Functional Connectivity of Resting Hemodynamic Signals in Submillimeter Orientation Columns of the Visual Cortex