Characterization of neurovascular relationships is critical to accurate interpretation of functional neuroimaging data. We have previously observed spatial uncoupling of optical intrinsic signal imaging (OIS) and evoked potential (EP) responses in rodent barrel cortex following simultaneous whisker and forelimb stimulation, leading to changes in OIS response magnitude. To further test the hypothesis that this uncoupling may have resulted from "passive" overspill of perfusion-related responses between functional regions, we conducted the present study using temporally staggered rather than simultaneous whisker and forelimb stimulation. This paradigm minimized overlap of neural responses in barrel cortex and forelimb primary somatosensory cortex (SI), while maintaining overlap of vascular response time courses between regions. When contrasted with responses to 1.5-s lone-whisker stimulation, staggered whisker and forelimb stimulation resulted in broadening of barrel cortex OIS response time course in the temporal direction of forelimb stimulation. OIS response peaks were also temporally shifted toward the forelimb stimulation period; time-to-peak was shorter (relative to whisker stimulus onset) when forelimb stimulation preceded whisker stimulation and longer when forelimb stimulation followed whisker stimulation. In contrast with OIS and EP magnitude decreases previously observed during simultaneous whisker/forelimb stimulation, barrel cortex OIS response magnitude increased during staggered stimulation and no detectable changes in underlying EP activity were observed. Spatial extent of barrel cortex OIS responses also increased during staggered stimulation. These findings provide further evidence for spatial uncoupling of OIS and EP responses, and emphasize the importance of temporal stimulus properties on the effects of this uncoupling. It is hypothesized that spatial uncoupling is a result of passive overspill of perfusion-related responses into regions distinct from those which are functionally active. It will be important to consider potential influences of this uncoupling when designing and interpreting functional imaging studies that use hemodynamic responses to infer underlying neural activity.
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