Diversity of neurovascular coupling dynamics along vascular arbors in layer II/III somatosensory cortex.

The spatial-temporal sequence of cerebral blood flow (CBF), cerebral blood volume (CBV) and blood velocity changes triggered by neuronal activation is critical for understanding functional brain imaging. This sequence follows a stereotypic pattern of changes across different zones of the vasculature in the olfactory bulb, the first relay of olfaction. However, in the cerebral cortex, where most human brain mapping studies are performed, the timing of activity evoked vascular events remains controversial. Here we utilized a single whisker stimulation model to map out functional hyperemia along vascular arbours from layer II/III to the surface of primary somatosensory cortex, in anesthetized and awake Thy1-GCaMP6 mice. We demonstrate that sensory stimulation triggers an increase in blood velocity within the mid-capillary bed and a dilation of upstream large capillaries, and the penetrating and pial arterioles. We report that under physiological stimulation, response onset times are highly variable across compartments of different vascular arbours. Furthermore, generating transfer functions (TFs) between neuronal Ca2+ and vascular dynamics across different brain states demonstrates that anesthesia decelerates neurovascular coupling (NVC). This spatial-temporal pattern of vascular events demonstrates functional diversity not only between different brain regions but also at the level of different vascular arbours within supragranular layers of the cerebral cortex.

Obesity in Midlife Hampers Resting and Sensory-Evoked Cerebral Blood Flow in Mice.

OBJECTIVE: This study aimed to investigate the effects of a high-fat diet (HFD) and aging on resting and activity-dependent cerebral blood flow (CBF). METHODS: To run a comparison between obese and age-matched control animals, 6-week-old mice were fed either with regular chow or an HFD for 3 months or 8 months. Glucose tolerance and insulin sensitivity were assessed for metabolic phenotyping. Resting and odor-evoked CBF at the microvascular scale in the olfactory bulb (OB) was investigated by multiexposure speckle imaging. Immunolabeling-enabled imaging of solvent-cleared organs was used to analyze vascular density. The ejection fraction was studied by using cardioechography. Olfactory sensitivity was tested by using a buried-food test. RESULTS: Glucose intolerance and compromised odor-evoked CBF were observed in obese mice in the younger group. Prolonged HFD feeding triggered insulin resistance and stronger impairment in activity-dependent CBF. Aging had a specific negative impact on resting CBF. There was no decrease in vascular density in the OB of obese mice, although cardiac function was impaired at both ages. In addition, decreased olfactory sensitivity was observed only in the older, middle-aged obese mice. CONCLUSIONS: OB microvasculature in obese mice showed a specific functional feature characterized by impaired sensory-evoked CBF and a specific deleterious effect of aging on resting CBF.

Mild hypoxia triggers transient blood-brain barrier disruption: a fundamental protective role for microglia.

We recently demonstrated that when mice are exposed to chronic mild hypoxia (CMH, 8% O2), blood vessels in the spinal cord show transient vascular leak that is associated with clustering and activation of microglia around disrupted vessels. Importantly, microglial depletion profoundly increased hypoxia-induced vascular leak, implying that microglia play a critical role maintaining vascular integrity in the hypoxic spinal cord. The goal of the current study was to examine if microglia play a similar vasculo-protective function in the brain. Employing extravascular fibrinogen leak as an index of blood-brain barrier (BBB) disruption, we found that CMH provoked transient vascular leak in cerebral blood vessels that was associated with activation and aggregation of Mac-1-positive microglia around leaky vessels. Interestingly, CMH-induced vascular leak showed regional selectivity, being much more prevalent in the brainstem and olfactory bulb than the cerebral cortex and cerebellum. Pharmacological depletion of microglia with the colony stimulating factor-1 receptor inhibitor PLX5622, had no effect under normoxic conditions, but markedly increased hypoxia-induced cerebrovascular leak in all regions examined. As in the spinal cord, this was associated with endothelial induction of MECA-32, a marker of leaky CNS endothelium, and greater loss of endothelial tight junction proteins. Brain regions displaying the highest levels of hypoxic-induced vascular leak also showed the greatest levels of angiogenic remodeling, suggesting that transient BBB disruption may be an unwanted side-effect of hypoxic-induced angiogenic remodeling. As hypoxia is common to a multitude of human diseases including obstructive sleep apnea, lung disease, and age-related pulmonary, cardiac and cerebrovascular dysfunction, our findings have important translational implications. First, they point to a potential pathogenic role of chronic hypoxia in triggering BBB disruption and subsequent neurological dysfunction, and second, they demonstrate an important protective role for microglia in maintaining vascular integrity in the hypoxic brain.

Effects of nicotine on regional blood flow in the olfactory bulb in response to olfactory nerve stimulation.

This study examined the effect of olfactory nerve stimulation on regional cerebral blood flow and assessed the effect of intravenous nicotine administration on this response in anesthetized rats. Regional cerebral blood flow was measured with laser Doppler flowmetry or laser speckle contrast imaging. Unilateral olfactory nerve stimulation for 5 s produced current (≥ 100 µA) and frequency-dependent (≥ 5 Hz) increases in blood flow in the olfactory bulb ipsilateral to the stimulus. The increased olfactory bulb blood flow peaked at 30 ± 7% using stimulus parameters of 300 µA and 20 Hz. Nerve stimulation did not change frontal cortical blood flow or mean arterial pressure. The intravenous injection of nicotine (30 µg/kg) augmented the olfactory bulb blood flow response to nerve stimulation (20 Hz, 300 µA) by approximately 1.5-fold (60-s area after the stimulation). These results indicate that olfactory nerve stimulation increases olfactory bulb blood flow, and the response is potentiated by the activation of nicotinic cholinergic transmission.

Anosmin-1 activates vascular endothelial growth factor receptor and its related signaling pathway for olfactory bulb angiogenesis.

Anosmin-1 is a secreted glycoprotein encoded by the ANOS1 gene, and its loss of function causes Kallmann syndrome (KS), which is characterized by anosmia and hypogonadism due to olfactory bulb (OB) dysfunction. However, the physiological function of anosmin-1 remains to be elucidated. In KS, disordered angiogenesis is observed in OB, resulting in its hypoplasia. In this study, we examined the involvement of anosmin-1 in angiogenic processes. Anosmin-1 was detected on the vessel-like structure in OB of chick embryos, and promoted the outgrowth of vascular sprouts as shown by assays of OB tissue culture. Cell migration, proliferation, and tube formation of endothelial cells were induced by treatment with anosmin-1 as well as vascular endothelial growth factor-A (VEGF-A), and further enhanced by treatment with both of them. We newly identified that anosmin-1 activated VEGF receptor-2 (VEGFR2) by binding directly to it, and its downstream signaling molecules, phospholipase Cγ1 (PLCγ1) and protein kinase C (PKC). These results suggest that anosmin-1 plays a key role in the angiogenesis of developing OB through the VEGFR2-PLCγ1-PKC axis by enhancing the VEGF function.

Mesoscopic and microscopic imaging of sensory responses in the same animal.

Imaging based on blood flow dynamics is widely used to study sensory processing. Here we investigated the extent to which local neuronal and capillary responses (two-photon microscopy) are correlated to mesoscopic responses detected with fast ultrasound (fUS) and BOLD-fMRI. Using a specialized chronic olfactory bulb preparation, we report that sequential imaging of the same mouse allows quantitative comparison of odour responses, imaged at both microscopic and mesoscopic scales. Under these conditions, functional hyperaemia occurred at the threshold of neuronal activation and fUS-CBV signals could be detected at the level of single voxels with activation maps varying according to blood velocity. Both neuronal and vascular responses increase non-linearly as a function of odour concentration, whereas both microscopic and mesoscopic vascular responses are linearly correlated to local neuronal calcium. These data establish strengths and limits of mesoscopic imaging techniques to report neural activity.

Dominance of layer-specific microvessel dilation in contrast-enhanced high-resolution fMRI: Comparison between hemodynamic spread and vascular architecture with CLARITY.

Contrast-enhanced cerebral blood volume-weighted (CBVw) fMRI response peaks are specific to the layer of evoked synaptic activity (Poplawsky et al., 2015), but the spatial resolution limit of CBVw fMRI is unknown. In this study, we measured the laminar spread of the CBVw fMRI evoked response in the external plexiform layer (EPL, 265 ± 65 µm anatomical thickness, mean ± SD, n = 30 locations from 5 rats) of the rat olfactory bulb during electrical stimulation of the lateral olfactory tract and examined its potential vascular source. First, we obtained the evoked CBVw fMRI responses with a 55 × 55 µm2 in-plane resolution and a 500-µm thickness at 9.4 T, and found that the fMRI signal peaked predominantly in the inner half of EPL (136 ± 54 µm anatomical thickness). The mean full-width at half-maximum of these fMRI peaks was 347 ± 102 µm and the functional spread was approximately 100 or 200 µm when the effects of the laminar thicknesses of EPL or inner EPL were removed, respectively. Second, we visualized the vascular architecture of EPL from a different rat using a Clear Lipid-exchanged Anatomically Rigid Imaging/immunostaining-compatible Tissue hYdrogel (CLARITY)-based tissue preparation method and confocal microscopy. Microvascular segments with an outer diameter of <11 µm accounted for 64.3% of the total vascular volume within EPL and had a mean segment length of 55 ± 40 µm (n = 472). Additionally, vessels that crossed the EPL border had a mean segment length outside of EPL equal to 73 ± 61 µm (n = 28), which is comparable to half of the functional spread (50-100 µm). Therefore, we conclude that dilation of these microvessels, including capillaries, likely dominate the CBVw fMRI response and that the biological limit of the fMRI spatial resolution is approximately the average length of 1-2 microvessel segments, which may be sufficient for examining sublaminar circuits.

Effect of basal forebrain stimulation on extracellular acetylcholine release and blood flow in the olfactory bulb.

The olfactory bulb receives cholinergic basal forebrain input, as does the neocortex; however, the in vivo physiological functions regarding the release of extracellular acetylcholine and regulation of regional blood flow in the olfactory bulb are unclear. We used in vivo microdialysis to measure the extracellular acetylcholine levels in the olfactory bulb of urethane-anesthetized rats. Focal chemical stimulation by microinjection of L-glutamate into the horizontal limb of the diagonal band of Broca (HDB) in the basal forebrain, which is the main source of cholinergic input to the olfactory bulb, increased extracellular acetylcholine release in the ipsilateral olfactory bulb. When the regional cerebral blood flow was measured using laser speckle contrast imaging, the focal chemical stimulation of the HDB did not significantly alter the blood flow in the olfactory bulb, while increases were observed in the neocortex. Our results suggest a functional difference between the olfactory bulb and neocortex regarding cerebral blood flow regulation through the release of acetylcholine by cholinergic basal forebrain input.

Layer-Specific fMRI Responses to Excitatory and Inhibitory Neuronal Activities in the Olfactory Bulb.

High-resolution functional magnetic resonance imaging (fMRI) detects localized neuronal activity via the hemodynamic response, but it is unclear whether it accurately identifies neuronal activity specific to individual layers. To address this issue, we preferentially evoked neuronal activity in superficial, middle, and deep layers of the rat olfactory bulb: the glomerular layer by odor (5% amyl acetate), the external plexiform layer by electrical stimulation of the lateral olfactory tract (LOT), and the granule cell layer by electrical stimulation of the anterior commissure (AC), respectively. Electrophysiology, laser-Doppler flowmetry of cerebral blood flow (CBF), and blood oxygenation level-dependent (BOLD) and cerebral blood volume-weighted (CBV) fMRI at 9.4 T were performed independently. We found that excitation of inhibitory granule cells by stimulating LOT and AC decreased the spontaneous multi-unit activities of excitatory mitral cells and subsequently increased CBF, CBV, and BOLD signals. Odor stimulation also increased the hemodynamic responses. Furthermore, the greatest CBV fMRI responses were discretely separated into the same layers as the evoked neuronal activities for all three stimuli, whereas BOLD was poorly localized with some exception to the poststimulus undershoot. In addition, the temporal dynamics of the fMRI responses varied depending on the stimulation pathway, even within the same layer. These results indicate that the vasculature is regulated within individual layers and CBV fMRI has a higher fidelity to the evoked neuronal activity compared with BOLD. Our findings are significant for understanding the neuronal origin and spatial specificity of hemodynamic responses, especially for the interpretation of laminar-resolution fMRI. SIGNIFICANCE STATEMENT: Functional magnetic resonance imaging (fMRI) is a noninvasive, in vivo technique widely used to map function of the entire brain, including deep structures, in animals and humans. However, it measures neuronal activity indirectly by way of the vascular response. It is currently unclear how finely the hemodynamic response is regulated within single cortical layers and whether increased inhibitory neuronal activities affect fMRI signal changes. Both laminar specificity and the neural origins of fMRI are important to interpret functional maps properly, which we investigated by activating discrete rat olfactory bulb circuits.

Aneurysm of the A1 Segment of the Anterior Cerebral Artery Associated with the Persistent Primitive Olfactory Artery.

BACKGROUND: The persistent primitive artery constitutes the anterior cerebral artery proper. When the persistent primitive artery keeps its embryologic course along the olfactory bulb, it is called the persistent primitive olfactory artery (PPOA). CASE DESCRIPTION: A 69-year-old man presented with an incidentally discovered unruptured aneurysm at the origin of the PPOA. The PPOA originated at the A1 segment of the anterior cerebral artery, coursed anteromedially along the olfactory tract, made a hairpin turn posterosuperior to the midline, and formed the callosomarginal branch of the anterior cerebral artery. The anomalous artery was interpreted as a PPOA (type 3). Type 3 PPOA associated with an unruptured aneurysm is rare. CONCLUSIONS: There is a high incidence of aneurysms associated with a PPOA. Follow-up studies are necessary in the present case to monitor for the development of another aneurysm at the hairpin bend.

Artery-on-a-chip platform for automated, multimodal assessment of cerebral blood vessel structure and function.

We present a compact microfluidic platform for the automated, multimodal assessment of intact small blood vessels. Mouse olfactory artery segments were reversibly loaded onto a microfluidic device and kept under physiological (i.e., close to in vivo) environmental conditions. For immunohistochemical endpoint protein analysis, automated on chip fixation and staining of the artery eliminated the need for any subsequent tissue sectioning or processing outside the chip. In a first case study, we demonstrate the blood vessel abluminal structure based on the positions of smooth muscle cell nuclei, actin filaments and voltage gated calcium channels. In a second case study we incubated smooth muscle cells (SMCs) with a calcium-sensitive dye to simultaneously assess time-dependent, agonist-induced calcium and diameter changes of pressurized resistance arteries. We expect the presented microfluidic platform to promote routine on-chip staining and quantitative fluorescence imaging of intact blood vessels from different vascular beds, tissue engineered vascular constructs and vascularized microtissues. The at least tenfold reduction in required aliquot volumes for functional assessment and staining was achieved by on-board fluid manipulation of the syringe-pump free platform and may promote its applications for screening of newly synthesized compounds.

Capillaries in the olfactory bulb but not the cortex are highly susceptible to virus-induced vascular leak and promote viral neuroinvasion.

Viral neuroinvasion is a critical step in the pathogenesis of viral encephalitis. Multiple mechanisms of neuroinvasion have been identified, but their relative contribution to central nervous system (CNS) infection remains unclear for many viruses. In this study, we examined neuroinvasion of the mosquito-borne bunyavirus La Crosse (LACV), the leading cause of pediatric viral encephalitis in the USA. We found that the olfactory bulb (OB) and tract were the initial areas of CNS virus infection in mice. Removal of the OB reduced the incidence of LACV-induced disease demonstrating the importance of this area to neuroinvasion. However, we determined that infection of the OB was not due to axonal transport of virus from olfactory sensory neurons as ablation of these cells did not affect viral pathogenesis. Instead, we found that OB capillaries were compromised allowing leakage of virus-sized particles into the brain. Analysis of OB capillaries demonstrated specific alterations in cytoskeletal and Rho GTPase protein expression not observed in capillaries from other brain areas such as the cortex where leakage did not occur. Collectively, these findings indicate that LACV neuroinvasion occurs through hematogenous spread in specific brain regions where capillaries are prone to virus-induced activation such as the OB. Capillaries in these areas may be "hot spots" that are more susceptible to neuroinvasion not only for LACV, but other neurovirulent viruses as well.

Functional imaging of olfaction by CBV fMRI in monkeys: insight into the role of olfactory bulb in habituation.

Cerebral blood volume (CBV) fMRI with superparamagnetic iron oxide nanoparticles (USPIO) as contrast agent was used to investigate the odorant-induced olfaction in anesthetized rhesus monkeys. fMRI data were acquired in 24 axial slices covering the entire brain, with isoamyl-acetate as the odor stimulant. For each experiment, multiple fMRI measurements were made during a 1- or 2-h period, with each measurement consisting of a baseline period, a stimulation period, and a recovery period. Three different stimulation paradigms with a stimulation period of 1 min, 2 min, or 8 min, respectively, were used to study the olfactory responses in the olfactory bulb (OB). Odorant-induced CBV increases were observed in the OB of each individual monkey. The spatial and temporal activation patterns were reproducible within and between animals. The sensitivity of CBV fMRI in OB was comparable with the sensitivities reported in previous animal fMRI studies. The CBV responses during the 1-min, 2-min, or 8-min odor stimulation period were relatively stable, and did not show attenuation. The amplitudes of CBV response to the repeated stimuli during the 1- or 2-h period were also stable. The stable CBV response in the OB to both continuous and repeated odor stimuli suggests that the OB may not play a major role in olfactory habituation. The technical approach described in this report can enable more extensive fMRI studies of olfactory processing in OB of both humans and non-human primates.

Functional ultrasound imaging reveals different odor-evoked patterns of vascular activity in the main olfactory bulb and the anterior piriform cortex.

Topographic representation of the outside world is a key feature of sensory systems, but so far it has been difficult to define how the activity pattern of the olfactory information is distributed at successive stages in the olfactory system. We studied odor-evoked activation patterns in the main olfactory bulb and the anterior piriform cortex of rats using functional ultrasound (fUS) imaging. fUS imaging is based on the use of ultrafast ultrasound scanners and detects variations in the local blood volume during brain activation. It makes deep brain imaging of ventral structures, such as the piriform cortex, possible. Stimulation with two different odors (hexanal and pentylacetate) induced the activation of odor-specific zones that were spatially segregated in the main olfactory bulb. Interestingly, the same odorants triggered the activation of the entire anterior piriform cortex, in all layers, with no distinguishable odor-specific areas detected in the power Doppler images. These fUS imaging results confirm the spatial distribution of odor-evoked activity in the main olfactory bulb, and furthermore, they reveal the absence of such a distribution in the anterior piriform cortex at the macroscopic scale in vivo.

Odor processing by adult-born neurons.

The adult mammalian brain is continuously supplied with adult-born neurons in the olfactory bulb (OB) and hippocampus, where they are thought to be important for circuit coding and plasticity. However, direct evidence for the actual involvement of these neurons in neural processing is still lacking. We recorded the spiking activity of adult-born periglomerular neurons in the mouse OB in vivo using two-photon-targeted patch recordings. We show that odor responsiveness reaches a peak during neuronal development and then recedes at maturity. Sensory enrichment during development enhances the selectivity of adult-born neurons after maturation, without affecting neighboring resident neurons. Thus, in the OB circuit, adult-born neurons functionally integrate into the circuit, where they acquire distinct response profiles in an experience-dependent manner. The constant flow of these sensitive neurons into the circuit provides it with a mechanism of long-term plasticity, wherein new neurons mature to process odor information based on past demands.

Layer-dependent BOLD and CBV-weighted fMRI responses in the rat olfactory bulb.

The olfactory bulb is a laminarized brain structure involved in odor sensation that has important implications to basic neuroscience research, like mechanisms for neurovascular coupling and early disease diagnosis. To investigate laminar-dependent responses to odor exposure, blood oxygenation level-dependent (BOLD) and cerebral blood volume weighted (CBVw) fMRI with iron oxide nanoparticle contrast agent were obtained with 110×110×500µm(3) resolution in urethane-anesthetized rats at 9.4T. The baseline total CBV is the largest at the olfactory bulb surface and midline, and decreases in the deeper layers, while a band of increased microvasculature density is observed at the glomerular, external plexiform and mitral cell layers. With odor exposure, CBVw fMRI is more sensitive and reproducible than BOLD. BOLD fMRI had the greatest activation on the bulb surface, midline, olfactory nerve and glomerular layers, while CBVw activation peaked in glomerular and external plexiform layers, but was still significant in mitral cell layer. Negative BOLD responses were observed in the bulb midline and near large blood vessels. CBVw laminar profiles are similar to the layer-dependent metabolic changes to the same odor exposure reported by previous glucose metabolism studies. Unique activation patterns for two different odor conditions were also differentiated with CBVw fMRI. Our study suggests that CBVw activation better represents the spatial location of metabolic activity in the olfactory bulb than BOLD.

Persistent primitive olfactory artery: CT angiographic diagnosis and literature review for classification and clinical significance.

OBJECTIVES: The aim of this study was to investigate the incidence of a persistent primitive olfactory artery (POA) and to review the literatures focusing on the classification and clinical significance of this variant. DESIGN: To identify cases with persistent POA, we reviewed the records of computed tomography (CT) angiography performed on 3,067 patients in our institution from January 1, 2011 to August 31, 2013. Literatures on the incidence and classification of a persistent POA were reviewed. RESULTS: Among these patients, eight were diagnosed with a persistent POA (five men, three women, aged 44-82 years), an incidence of 0.26 %. Six persistent POAs terminated as a distal anterior cerebral artery (ACA) and two as a distal middle cerebral artery. Previous studies applied similar definitions for the classification of persistent POA; however, there has been confusion on the definition of variant 2. CONCLUSION: In our institution, the incidence of persistent POA seen on CT angiography was 0.26 %. An artery with its embryological course along the olfactory bulb should be classified as a persistent POA and differentiated from dural artery from ACA.

Multispectral reflectance imaging of brain activation in rodents: methodological study of the differential path length estimations and first in vivo recordings in the rat olfactory bulb.

Dynamic maps of relative changes in blood volume and oxygenation following brain activation are obtained using multispectral reflectance imaging. The technique relies on optical absorption modifications linked to hemodynamic changes. The relative variation of hemodynamic parameters can be quantified using the modified Beer-Lambert Law if changes in reflected light intensities are recorded at two wavelengths or more and the differential path length (DP) is known. The DP is the mean path length in tissues of backscattered photons and varies with wavelength. It is usually estimated using Monte Carlo simulations in simplified semi-infinite homogeneous geometries. Here we consider the use of multilayered models of the somatosensory cortex (SsC) and olfactory bulb (OB), which are common physiological models of brain activation. Simulations demonstrate that specific DP estimation is required for SsC and OB, specifically for wavelengths above 600 nm. They validate the hypothesis of a constant path length during activation and show the need for specific DP if imaging is performed in a thinned-skull preparation. The first multispectral reflectance imaging data recorded in vivo during OB activation are presented, and the influence of DP on the hemodynamic parameters and the pattern of oxymetric changes in the activated OB are discussed.

Outer skull landmark-based coordinates for measurement of cerebral blood flow and intracranial pressure in rabbits.

Despite the increased use of intracranial neuromonitoring during experimental subarachnoid hemorrhage (SAH), coordinates for probe placement in rabbits are lacking. This study evaluates the safety and reliability of using outer skull landmarks to identify locations for placement of cerebral blood flow (CBF) and intraparenchymal intracranial pressure (ICP) probes. Experimental SAH was performed in 17 rabbits using an extracranial-intracranial shunt model. ICP probes were placed in the frontal lobe and compared to measurements recorded from the olfactory bulb. CBF probes were placed in various locations in the frontal cortex anterior to the coronary suture. Insertion depth, relation to the ventricular system, and ideal placement location were determined by post-mortem examination. ICP recordings at the time of SAH from the frontal lobe did not differ significantly from those obtained from the right olfactory bulb. Ideal coordinates for intraparenchymal CBF probes in the left and right frontal lobe were found to be located 4.6±0.9 and 4.5±1.2 anterior to the bregma, 4.7±0.7mm and 4.7±0.5mm parasagittal, and at depths of 4±0.5mm and 3.9±0.5mm, respectively. The results demonstrate that the presented coordinates based on skull landmarks allow reliable placement of intraparenchymal ICP and CBF probes in rabbit brains without the use of a stereotactic frame.

Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels.

Uncovering principles that regulate energy metabolism in the brain requires mapping of partial pressure of oxygen (PO(2)) and blood flow with high spatial and temporal resolution. Using two-photon phosphorescence lifetime microscopy (2PLM) and the oxygen probe PtP-C343, we show that PO(2) can be accurately measured in the brain at depths up to 300 µm with micron-scale resolution. In addition, 2PLM allowed simultaneous measurements of blood flow and of PO(2) in capillaries with less than one-second temporal resolution. Using this approach, we detected erythrocyte-associated transients (EATs) in oxygen in the rat olfactory bulb and showed the existence of diffusion-based arterio-venous shunts. Sensory stimulation evoked functional hyperemia, accompanied by an increase in PO(2) in capillaries and by a biphasic PO(2) response in the neuropil, consisting of an 'initial dip' and a rebound. 2PLM of PO(2) opens new avenues for studies of brain metabolism and blood flow regulation.