Changes in neuronal activity are accompanied by the release of vasoactive mediators that cause microscopic dilation and constriction of the cerebral microvasculature and are manifested in macroscopic blood oxygenation level-dependent (BOLD) functional MRI (fMRI) signals. We used two-photon microscopy to measure the diameters of single arterioles and capillaries at different depths within the rat primary somatosensory cortex. These measurements were compared with cortical depth-resolved fMRI signal changes. Our microscopic results demonstrate a spatial gradient of dilation onset and peak times consistent with "upstream" propagation of vasodilation toward the cortical surface along the diving arterioles and "downstream" propagation into local capillary beds. The observed BOLD response exhibited the fastest onset in deep layers, and the "initial dip" was most pronounced in layer I. The present results indicate that both the onset of the BOLD response and the initial dip depend on cortical depth and can be explained, at least in part, by the spatial gradient of delays in microvascular dilation, the fastest response being in the deep layers and the most delayed response in the capillary bed of layer I.blood flow | cortical layer | hemodynamic | imaging | somatosensory N euroglial activation is accompanied by release of vasoactive mediators that dilate and constrict the surrounding arterioles (1, 2) and capillaries (3, 4). These changes in diameter in turn lead to changes in blood flow throughout the vascular matrix and can be detected on the macroscopic level as a positive blood oxygenation level-dependent (BOLD) functional MRI (fMRI) signal when blood flow response exceeds oxygen consumption (5-7). Under the assumption of local neurovascular coupling, the onset of the changes in diameter is determined by the following three factors, any of which may differ as a function of the cortical depth and branching order within the vascular tree: (i) the onset and peak time of the neuronal activity evoking the response; (ii) the time needed to release a vascular messenger [e.g., prostaglandin or NO (8)]; and (iii) the time needed for the target vessel to respond. However, in addition to local neurovascular coupling, vascular responses can propagate within the arteriolar/capillary networks (3, 9, 10). Indeed, propagation of dilation and constriction has been observed on the cortical surface (11-15), in excised cerebral vessels, and in noncerebral preparations (16,17).Previous studies with single-vessel resolution in vivo have been limited to the cortical surface, but recent improvements in twophoton microscopy technology allow direct imaging of singlevessel diameters and flow velocities within a 3D geometry of vascular trees (1,2,18,19). In the present study, we used this technology to examine microvascular responses to sensory stimulation down to 550 μm below the cortical surface in the rat primary somatosensory cortex (SI). We then compared the results with highresolution BOLD fMRI to investigate the extent to which laminar ...

Synaptic transmission initiates a cascade of signal transduction events that couple neuronal activity to local changes in blood flow and oxygenation. Although a number of vasoactive molecules and specific cell types have been implicated, the transformation of stimulusinduced activation of neuronal circuits to hemodynamic changes is still unclear. We use somatosensory stimulation and a suite of in vivo imaging tools to study neurovascular coupling in rat primary somatosensory cortex. Our stimulus evoked a central region of net neuronal depolarization surrounded by net hyperpolarization. Hemodynamic measurements revealed that predominant depolarization corresponded to an increase in oxygenation, whereas predominant hyperpolarization corresponded to a decrease in oxygenation. On the microscopic level of single surface arterioles, the response was composed of a combination of dilatory and constrictive phases. Critically, the relative strength of vasoconstriction covaried with the relative strength of oxygenation decrease and neuronal hyperpolarization. These results suggest that a neuronal inhibition and concurrent arteriolar vasoconstriction correspond to a decrease in blood oxygenation, which would be consistent with a negative blood oxygenation level-dependent functional magnetic resonance imaging signal.

The present study addresses the relationship between blood flow and glucose consumption in rat primary somatosensory cortex (SI) in vivo. We examined bilateral neuronal and hemodynamic changes and 2-deoxyglucose (2DG) uptake, as measured by autoradiography, in response to unilateral forepaw stimulation. In contrast to the contralateral forepaw area, where neuronal activity, blood oxygenation/ flow and 2DG uptake increased in unison, we observed, in the ipsilateral SI, a blood oxygenation/flow decrease and arteriolar vasoconstriction in the presence of increased 2DG uptake. Laminar electrophysiological recordings revealed an increase in ipsilateral spiking consistent with the observed increase in 2DG uptake. The vasoconstriction and the decrease in blood flow in the presence of an increase in 2DG uptake in the ipsilateral SI contradict the prominent metabolic hypothesis regarding the regulation of cerebral blood flow, which postulates that the state of neuroglial energy consumption determines the regional blood flow through the production of vasoactive metabolites. We propose that other factors, such as neuronal (and glial) release of messenger molecules, might play a dominant role in the regulation of blood flow in vivo in response to a physiological stimulus.

The relationship between measurements of cerebral blood oxygenation and neuronal activity is highly complex and depends on both neurovascular and neurometabolic biological coupling. While measurements of blood oxygenation changes via optical and MRI techniques have been developed to map functional brain activity, there is evidence that the specific characteristics of these signals are sensitive to the underlying vascular physiology and structure of the brain. Since baseline blood flow and oxygen saturation may vary between sessions and across subjects, functional blood oxygenation changes may be a less reliable indicator of brain activity in comparison to blood flow and metabolic changes. In this work, we use a biomechanical model to examine the relationships between neural, vascular, metabolic, and hemodynamic responses to parametric whisker stimulation under both NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript normal and hypercapnic conditions in a rat model. We find that the relationship between neural activity and oxy-and deoxyhemoglobin changes is sensitive to hypercapnia-induced changes in baseline cerebral blood flow. In contrast, the underlying relationships between evoked neural activity, blood flow, and model-estimated oxygen metabolism changes are unchanged by the hypercapnic challenge. We conclude that evoked changes in blood flow and cerebral oxygen metabolism are more closely associated with underlying evoked neuronal responses.

Microscopic in vivo measurements of cerebral oxygenation are of key importance for understanding normal cerebral energy metabolism and its dysregulation in a wide range of clinical conditions. Relevant cerebral pathologies include compromised blood perfusion following stroke and a decrease in efficiency of single-cell respiratory processes that occurs in neurodegenerative diseases such as Alzheimer's and Parkinson's disease. In this chapter we review a number of quantitative optical approaches to measuring oxygenation of blood and cerebral tissue. These methods can be applied to map the hemodynamic response and study neurovascular and neurometabolic coupling, and can provide microscopic imaging of biomarkers in animal models of human disease, which would be useful for screening potential therapeutic approaches.Key words O 2 sensing, Phosphorescence quenching, Intrinsic optical signals, Energy metabolism, In vivo imaging, Hemoglobin, Two-photon microscopy, CCD loading of extrinsic O 2 -sensitive probes. Following the original demonstration [1], the intrinsic imaging method was widely used for investigation of neuro-hemodynamic coupling [2][3][4][5][6][7][8][9][10][11] and mapping of cortical neuronal responses [12][13][14][15] (Box 1).The hemoglobin molecule is composed of four monomers, each containing an O 2 -binding heme group. Hemoglobin tetramer Box 1 Optical Imaging of Intrinsic Signals-A Historical PerspectiveOne hundred and twenty years ago, Roy and Sherrington [16] argued that neuronal activation causes the local vasculature to respond. While unequivocal confirmation of this claim had to wait nearly a century, until radioactive methods became available [17][18][19][20], large reflectance changes of brain tissue during localized seizure activity could be visualized already in the late 1930s [21]. A few decades later advances made it possible to detect and analyze the much smaller optical signals during activity of the normal cortex. These were accounted for by activity-associated changes in cerebral blood flow (CBF) and volume (CBV) [18, 22]; in addition, Chance [23] and Jöbsis [22] observed that neuronal activity is often accompanied by oximetric signals that can be detected optically by monitoring the absorption (and/or fluorescence) of hemoglobin and other intrinsic chromophores.In the late 1980s, Grinvald et al. [1] showed that the small light absorption changes induced by these activity-evoked hemodynamic responses can be used to explore cortical functional architecture in vivo, by using a CCD camera to image the cortex upon illumination at specific wavelengths during the presentation of sensory stimuli. The resulting cortical images can then be used to produce functional maps at the spatial resolution of a few tens of microns, more than enough to image the columnar structure of the mammalian neocortex.Since then, the interpretation of intrinsic signals in terms of neuronal activity-and thus their utility for functional brain mapping-has been tightly linked to our understanding of the mechanisms unde...