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 ...

Femtosecond phase-coherent two-dimensional (2D) spectroscopy has been experimentally demonstrated as the direct optical analog of 2D nuclear magnetic resonance. An acousto-optic pulse shaper created a collinear three-pulse sequence with well-controlled and variable interpulse delays and phases,which interacted with a model atomic system of rubidium vapor. The desired nonlinear polarization was selected by phase cycling (coadding experimental results obtained with different interpulse phases). This method may enhance our ability to probe the femtosecond structural dynamics of macromolecules.

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.

Identification of the cellular players and molecular messengers that communicate neuronal activity to the vasculature driving cerebral hemodynamics is important for (1) the basic understanding of cerebrovascular regulation and (2) interpretation of functional Magnetic Resonance Imaging (fMRI) signals. Using a combination of optogenetic stimulation and 2-photon imaging in mice, we demonstrate that selective activation of cortical excitation and inhibition elicits distinct vascular responses and identify the vasoconstrictive mechanism as Neuropeptide Y (NPY) acting on Y1 receptors. The latter implies that task-related negative Blood Oxygenation Level Dependent (BOLD) fMRI signals in the cerebral cortex under normal physiological conditions may be mainly driven by the NPY-positive inhibitory neurons. Further, the NPY-Y1 pathway may offer a potential therapeutic target in cerebrovascular disease.DOI: http://dx.doi.org/10.7554/eLife.14315.001

Calcium-dependent release of vasoactive gliotransmitters is widely assumed to trigger vasodilation associated with rapid increases in neuronal activity. Inconsistent with this hypothesis, intact stimulus-induced vasodilation was observed in inositol 1,4,5-triphosphate (IP3) type-2 receptor (R2) knockout (KO) mice, in which the primary mechanism of astrocytic calcium increase – the release of calcium from intracellular stores following activation of an IP3-dependent pathway – is lacking. Further, our results in wild type (WT) mice indicate that in vivo onset of astrocytic calcium increase in response to sensory stimulus could be considerably delayed relative to the simultaneously measured onset of arteriolar dilation. Delayed calcium increases in WT mice were observed in both astrocytic cell bodies and perivascular endfeet. Thus, astrocytes may not play a role in the initiation of blood flow response, at least not via calcium-dependent mechanisms. Moreover, an increase in astrocytic intracellular calcium was not required for normal vasodilation in the IP3R2-KO animals.

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.

In vivo optical imaging of cerebral blood flow (CBF) and metabolism did not exist 50 years ago. While point optical fluorescence and absorption measurements of cellular metabolism and hemoglobin concentrations had already been introduced by then, point blood flow measurements appeared only 40 years ago. The advent of digital cameras has significantly advanced two-dimensional optical imaging of neuronal, metabolic, vascular, and hemodynamic signals. More recently, advanced laser sources have enabled a variety of novel three-dimensional high-spatial-resolution imaging approaches. Combined, as we discuss here, these methods are permitting a multifaceted investigation of the local regulation of CBF and metabolism with unprecedented spatial and temporal resolution. Through multimodal combination of these optical techniques with genetic methods of encoding optical reporter and actuator proteins, the future is bright for solving the mysteries of neurometabolic and neurovascular coupling and translating them to clinical utility.

We demonstrate a vacuum-deposited organic light emitting device which emits from its top surface through a transparent indium-tin-oxide anode. This device employs a novel protective cap layer which prevents damage to the organic layers during sputter deposition of the anode, while also improving hole injection. Mechanisms of current transport and carrier injection from the contacts are investigated. This device configuration allows for integration of organic light emitting devices with n-channel field effect transistors used in display active matrix backplanes.