Two-Photon Microscopy as a Tool to Study Blood Flow and Neurovascular Coupling in the Rodent Brain

Functional magnetic resonance imaging (fMRI) is a unique window to the brain, enabling scientists to follow changes in brain activity in response to hormones, ageing, environment, drugs of abuse and other stimuli. There are two features that make fMRI unique when compared with other imaging modalities used in behavioural neuroscience. First, it can be entirely noninvasive: each animal can serve as its own control over the natural course of its life, vital for following neuroadaptation and other developmental processes critical to understanding behaviour. Second, fMRI has the spatial and temporal resolution to observe patterns of neuronal activity across the entire brain in less than a minute. Although fMRI does not have the cellular spatial resolution of immunostaining, nor the millisecond temporal resolution of electrophysiology, synchronised changes in neuronal activity across multiple brain areas seen with functional MRI can be viewed as functional neuroanatomical circuits coordinating the thoughts, memories and emotions for particular behaviours. Thus, fMRI affords a systems approach to the study of the brain, complementing and building from other neurobiological techniques to understand how behaviour is organised across multiple brain regions. In this review, we present a general background to fMRI and the different imaging modalities that can be used in fMRI studies. Included are examples of the application of fMRI in behavioural neuroscience research, along with discussion of the advantages and disadvantages of this technology.

Functional hyperemia is a well-established hallmark of healthy brain function, whereby local brain blood flow adjusts in response to a change in the activity of the surrounding neurons. Although functional hyperemia has been extensively studied at the level of both tissue and individual vessels, vascular network-level coordination remains largely unknown. To bridge this gap, we developed a deep learning-based pipeline that uses two-photon fluorescence microscopy images of cerebral microcirculation to enable automated reconstruction and quantification of the geometric changes across the microvascular network, comprising hundreds of interconnected blood vessels, pre and post-activation of the neighboring neurons. The pipeline's utility was demonstrated in the Thy1-ChR2 optogenetic mouse model, where we observed network-wide vessel radius changes to depend on the photostimulation intensity, with both dilations and constrictions occurring across the cortical depth, at an average of 16.1±14.3 μm (mean ± SD) away from the most proximal neuron for dilations; and at 21.9±14.6 μm away for constrictions. We observed a significant heterogeneity of the vascular radius changes within vessels, with radius adjustment varying by an average of 24 ± 28% of the resting diameter, likely reflecting the heterogeneity of the distribution of contractile cells on the vessel walls. A graph theory-based network analysis revealed that the assortativity of adjacent blood vessel responses rose by 152 ± 65% at 4.3 mW/mm2 of blue photostimulation vs. the control, with a 4% median increase in the efficiency of the capillary networks during this level of blue photostimulation in relation to the baseline. Interrogating individual vessels is thus not sufficient to predict how the blood flow is modulated in the network. Our pipeline, enables tracking of the microvascular network geometry over time, relating caliber adjustments to vessel wall-associated cells' state, and mapping network-level flow distribution impairments in experimental models of disease.

In the adult brain, increases in local neural activity are accompanied by increases in regional blood flow. This relationship between neural activity and hemodynamics is termed neurovascular coupling and provides the blood flow-dependent contrast detected in functional magnetic resonance imaging (fMRI). Neurovascular coupling is commonly assumed to be consistent and reliable from birth; however, numerous studies have demonstrated markedly different hemodynamics in the early postnatal brain. Our recent study in J. Neuroscience examined whether different hemodynamics in the immature brain are driven by differences in the underlying spatiotemporal properties of neural activity during this period of robust neural circuit expansion. Using a novel wide-field optical imaging technique to visualize both neural activity and hemodynamics in the mouse brain, we observed longer duration and increasingly complex patterns of neural responses to stimulus as cortical connectivity developed over time. However, imaging of brain blood flow, oxygenation, and metabolism in the same mice demonstrated an absence of coupled blood flow responses in the newborn brain. This lack of blood flow coupling was shown to lead to oxygen depletions following neural activations - depletions that may affect the duration of sustained neural responses and could be important to the vascular patterning of the rapidly developing brain. These results are a step toward understanding the unique neurovascular and neurometabolic environment of the newborn brain, and provide new insights for interpretation of fMRI BOLD studies of early brain development.

Reduced blood flow and impaired neurovascular coupling are recognized features of glaucoma, the leading cause of irreversible blindness worldwide, but the mechanisms underlying these defects are unknown. Retinal pericytes regulate microcirculatory blood flow and coordinate neurovascular coupling through interpericyte tunneling nanotubes (IP-TNTs). Using two-photon microscope live imaging of the mouse retina, we found reduced capillary diameter and impaired blood flow at pericyte locations in eyes with high intraocular pressure, the most important risk factor to develop glaucoma. We show that IP-TNTs are structurally and functionally damaged by ocular hypertension, a response that disrupted light-evoked neurovascular coupling. Pericyte-specific inhibition of excessive Ca2+ influx rescued hemodynamic responses, protected IP-TNTs and neurovascular coupling, and enhanced retinal neuronal function as well as survival in glaucomatous retinas. Our study identifies pericytes and IP-TNTs as potential therapeutic targets to counter ocular pressure-related microvascular deficits, and provides preclinical proof of concept that strategies aimed to restore intrapericyte calcium homeostasis rescue autoregulatory blood flow and prevent neuronal dysfunction.

P.4.023 The social cognition network and its impairment in schizophrenia: medial prefrontal cortex functional deficit in an emotional task

.We explore cortical microvasculature changes during the progression of atherosclerosis using young and old transgenic atherosclerotic (ATX) mice with thinned-skull cranial window. In awake animals, exploiting intrinsic signal optical imaging, Doppler optical coherence tomography, and two-photon microscopy, we investigate how the progression of atherosclerotic disease affects the morphology and function of cortical microvasculature as well as baseline cerebral tissue oxygenation. Results show that aged ATX mice exhibited weaker hemodynamic response in the somatosensory cortex to whisker stimulation and that the diameter of their descending arterioles and associated mean blood flow decreased significantly compared with the young ATX group. Data from two-photon phosphorescence lifetime microscopy indicate that old ATX mice had lower and more heterogeneous partial pressure of oxygen () in cortical tissue than young ATX mice. In addition, hypoxic micropockets in cortical tissue were found in old, but not young, ATX mice. Capillary red blood cell (RBC) flux, RBC velocity, RBC velocity heterogeneity, hematocrit, and diameter were also measured using line scans with two-photon fluorescence microscopy. When compared with the young group, RBC flux, velocity, and hematocrit decreased and RBC velocity heterogeneity increased in old ATX mice, presumably due to disturbed blood supply from arterioles that were affected by atherosclerosis. Finally, dilation of capillaries in old ATX mice was observed, which suggests that capillaries play an active role in compensating for an oxygen deficit in brain tissue.

Neurovascular coupling (NVC) is the functional mechanism that links brain neural activity with the dynamic regulation of local blood flow and oxygenation. In recent years, there has been an increasing academic attention to the role of NVC in its pathophysiology and the application of new technologies. This study aims to map the research landscape related to NVC through scientometric analysis. Publications from the past 30 years were retrieved from the Web of Science Core Collection (WoSCC) database. Data were analyzed using CiteSpace, VOSviewer, and the bibliometrix R package, including co-citation and keyword co-occurrence network analyses. Key metrics such as publication counts and citation frequencies were assessed to identify trends and collaboration patterns among countries, institutions, and authors. Among the 2047 articles included in the study, United States has maintained a clear leading position. Meanwhile, the number of Chinese research participants has grown rapidly over the past decade. The most prolific authors were Professors Iadecola Costantino and Tarantini Stefano. The research findings of Professor Tarantini Stefano have been widely recognized by researchers in the field. Keyword analysis identified "cerebral blood flow," "neuronal activity," and "neurovascular coupling" as dominant terms, emphasizing the central role of brain function and imaging techniques such as fMRI, TCD, and optical imaging. The emergence of "fNIRS," "resting-state fMRI," and "autoregulation" highlights the growing impact of noninvasive neuroimaging in studying brain-blood flow interactions. Cluster analysis revealed key research themes including functional connectivity, nitric oxide-mediated vascular regulation, cerebral autoregulation, Alzheimer's disease metabolism, and CO2-induced hemodynamic modulation. Over the past three decades, NVC has emerged as a key research focus, driven by interdisciplinary collaboration and advances in brain connectivity, dysfunction, and technology. In the future, integrating artificial intelligence, multi-omics analysis, and high-resolution imaging will further elucidate NVC mechanisms in health and disease, promoting interdisciplinary translation and breakthroughs in neuroscience and brain health.

Prostaglandin E2 (PGE2) has been widely proposed to mediate neurovascular coupling by dilating brain parenchymal arterioles through activation of prostanoid EP4 receptors. However, our previous report that direct application of PGE2 induces an EP1-mediated constriction strongly argues against its direct action on arterioles during neurovascular coupling, the mechanisms sustaining functional hyperemia. Recent advances have highlighted the role of capillaries in sensing neuronal activity and propagating vasodilatory signals to the upstream penetrating parenchymal arteriole. Here, we examined the effect of capillary stimulation with PGE2 on upstream arteriolar diameter using an ex vivo capillary-parenchymal arteriole preparation and in vivo cerebral blood flow measurements with two-photon laser-scanning microscopy. We found that PGE2 caused upstream arteriolar dilation when applied onto capillaries with an EC50 of 70 nM. The response was inhibited by EP1 receptor antagonist and was greatly reduced, but not abolished, by blocking the strong inward-rectifier K+ channel. We further observed a blunted dilatory response to capillary stimulation with PGE2 in a genetic mouse model of cerebral small vessel disease with impaired functional hyperemia. This evidence casts previous findings in a different light, indicating that capillaries are the locus of PGE2 action to induce upstream arteriolar dilation in the control of brain blood flow, thereby providing a paradigm-shifting view that nonetheless remains coherent with the broad contours of a substantial body of existing literature.

Neurovascular coupling is a process through which neuronal activity leads to local increases in blood flow in the central nervous system. In brain slices, 100% O(2) has been shown to alter neurovascular coupling, suppressing activity-dependent vasodilation. However, in vivo, hyperoxia reportedly has no effect on blood flow. Resolving these conflicting findings is important, given that hyperoxia is often used in the clinic in the treatment of both adults and neonates, and a reduction in neurovascular coupling could deprive active neurons of adequate nutrients. Here we address this issue by examining neurovascular coupling in both ex vivo and in vivo rat retina preparations. In the ex vivo retina, 100% O(2) reduced light-evoked arteriole vasodilations by 3.9-fold and increased vasoconstrictions by 2.6-fold. In vivo, however, hyperoxia had no effect on light-evoked arteriole dilations or blood velocity. Oxygen electrode measurements showed that 100% O(2) raised pO(2) in the ex vivo retina from 34 to 548 mm Hg, whereas hyperoxia has been reported to increase retinal pO(2) in vivo to only ~53 mm Hg [Yu DY, Cringle SJ, Alder VA, Su EN (1994) Am J Physiol 267:H2498-H2507]. Replicating the hyperoxic in vivo pO(2) of 53 mm Hg in the ex vivo retina did not alter vasomotor responses, indicating that although O(2) can modulate neurovascular coupling when raised sufficiently high, the hyperoxia-induced rise in retinal pO(2) in vivo is not sufficient to produce a modulatory effect. Our findings demonstrate that hyperoxia does not alter neurovascular coupling in vivo, ensuring that active neurons receive an adequate supply of nutrients.

Neurovascular coupling (NVC) describes the effective matching of cerebral blood flow (CBF) to regions of neuro‐metabolic demand. There is increasing interest to assess human NVC for both basic research and its potential role in vascular‐cognitive impairment. The clinical utility of NVC relies on a standardized protocol for which the driving metabolic demands are highly‐selective. Various research groups deploy divergent strategies to elicit visual NVC responses, including inactive processes (visual grating), passive visual tracking (target with predictable motion) and active visual tracking (target with unpredictable motion). These strategies differ in degree of cognitive and metabolic demand and may elicit different NVC responses, thus precluding study comparison.The present NVC assessment evaluated temporal and regional responsiveness of blood flow (transcranial Doppler) to the visual cortex [via the posterior cerebral artery (PCA)] and blood pressure (Finapres NOVA) during visual stimulation in 19 healthy subjects while also measuring middle cerebral artery (MCA) blood flow. Visual stimulation included 10 cycles of 30‐seconds with eyes‐closed, followed by 30‐seconds with eyes‐open tracking a moving computerized target. Each subject completed three trials of passive tracking and three trials of active tracking (114 NVC protocols, 1140 individual hyperemias). A custom eye‐scanning apparatus followed eye motion to quantify visual target‐tracking vigilance. Additional custom software was used to quantify NVC.The data demonstrated that active tracking elicited greater NVC responses compared to passive tracking. Specifically, there was 26% greater change in the mean elevation of PCA blood velocity (p=<0.000) and 13% greater peak NVC response (p<0.01). The MCA response was also greater during active tracking (mean response 111% greater, peak response 41% greater; both p<0.001). Visual target‐tracking vigilance was linearly correlated to the degree of hyperemia in the MCA and PCA, as well blood pressure during NVC.It was observed that active and passive visual tracking elicit different NVC responses and cannot be reliably compared. That PCA and MCA responses were greater with active tracking suggests an elevated global CBF (i.e. not selective to regions perfused by the PCA) that may result from recruitment of brain centres responsible for sustained attention and executive function. In other words, active tracking leads to non‐selective elevations in global CBF and greater target‐tracking vigilance impacts the NVC response. These findings are a critical step to better understand and standardize the evaluation of NVC in humans and for potential clinical deployment of NVC assessments.Support or Funding InformationNatural Sciences and Engineering Research Council; Alberta Innovates Research Studentship; NSERC; CIHR; Libin Cardiovascular Institute; Hotchkiss Brain Institute; Compute CanadaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neuronal activity evokes localized changes in blood flow, a response termed neurovascular coupling. One widely recognized hypothesis of neurovascular coupling holds that glial cell depolarization evoked by neuronal activity leads to the release of K+ onto blood vessels (K+ siphoning) and to vessel relaxation. We now present two direct tests of this glial cell-K+ siphoning hypothesis of neurovascular coupling. Potassium efflux was evoked from glial cells in the rat retina by applying depolarizing current pulses to individual cells. Glial depolarizations as large as 100 mV produced no change in the diameter of adjacent arterioles. We also monitored light-evoked vascular responses in Kir4.1 knock-out mice, where functional Kir K+ channels are absent from retinal glial cells. The magnitude of light-evoked vasodilations was identical in Kir4.1 knock-out and wild-type animals. Contrary to the hypothesis, the results demonstrate that glial K+ siphoning in the retina does not contribute significantly to neurovascular coupling.

The cerebral vasculature provides blood flow throughout the brain, and local changes in blood flow are regulated to match the metabolic demands of the active brain regions. This neurovascular coupling is mediated by real-time changes in vessel diameter and depends on the underlying vascular network structure. Neurovascular structure is configured during development by genetic and activity-dependent factors. In adulthood, it can be altered by experiences such as prolonged hypoxia, sensory deprivation and seizure. Here, we have sought to determine whether exercise could alter cerebral vascular structure in the adult mouse. We performed repeated in vivo two-photon imaging in the motor cortex of adult transgenic mice expressing membrane-anchored green fluorescent protein in endothelial cells (tyrosine endothelial kinase 2 receptor (Tie2)-Cre:mTmG). This strategy allows for high-resolution imaging of the vessel walls throughout the lifespan. Vascular structure, as measured by capillary branch point number and position, segment diameter and length remained stable over a time scale of months as did pericyte number and position. Furthermore, we compared the vascular structure before, during, and after periods of voluntary wheel running and found no alterations in these same parameters. In both running and control mice, we observed a low rate of capillary segment subtraction. Interestingly, these rare subtraction events preferentially remove short vascular loops.

Retinal vessel responses to flickering light are different in various systemic and ocular diseases and can be improved after successful therapy. We investigated retinal vessel response to flickering light in age-related macular degeneration (AMD) patients before and after treatment with a single intravitreal bevacizumab (Avastin(®) ) injection. In 10 patients with exudative AMD [age: median (1.quartile; 3.quartile) 76.0 (73.5; 80.0) years], retinal vessel reactions were examined by Dynamic Vessel Analyser (DVA) before and 3 months after a single intravitreal application of bevacizumab (1.25 mg). A baseline measurement was followed by three consecutive monochromatic flicker stimulations (530-600 nm, 12.5 Hz, 20 seconds). Temporal retinal vessel reaction was analysed and compared with the reaction in healthy controls. Mean arterial dilation at the end of flicker was not different in all groups. For veins this parameter amounted to: pre-treatment, 2.6 (1.7; 3.9)%; post-treatment, 2.9 (2.4; 4.0)%; control, 4.3 (3.2; 5.7)%; significant: pre-treatment - control (Dunnett's procedure, p < 0.05). Maximal dilation occurred in arteries at: pre-treatment, 17.5 (14.8; 32.5) seconds; post-treatment, 18.0 (16.6; 30.6) seconds; control, 14.5 (10.8; 17.3) seconds. Both AMD groups were slower (p < 0.05): in veins at 17.0 (14.5; 20.0) seconds, 12.8 (8.6; 14.8) seconds and 18.5 (17.1; 19.9) seconds, respectively; significant post-treatment - control (p < 0.05). In the post-treatment AMD group arterial constriction after stimulation occurred more slowly compared with the control group (p < 0.05). Dynamic retinal arterial and venous reactions to flickering light are altered in AMD compared with controls. Three months after a single injection of a vascular endothelial growth factor inhibitor, the investigated retinal dynamic vascular parameters were not altered in our study.

Purpose The retina and optic nerve, both optically accessible components of the central nervous system, are ideally suited for the investigation of the intrinsic physiological process by which blood flow, metabolism and neural activity are tightly coupled (Roy and Sherrington. 1890). Methods Using various techniques in the cat and human eye, the changes in blood flow in the retina and optic nerve in response to increased neural activity by flicker stimulation have been determined. The effect of varying the stimulus parameters, such as flicker modulation depth, frequency, luminance and red‐green color ratio, on the blood flow response was investigated. Putative mediators of the activity‐induced flow changes and the relationship between activity, blood flow and metabolic changes were assessed. Results Visual stimulation with flicker increases rapidly and markedly both retinal and optic nerve blood flows (functional hyperemia). Moreover, the data reveal unequivocally the presence, under specific conditions of flicker, of a neurovascular/neurometabolic coupling in these tissues, partly mediated by local potassium ions and nitric oxide production. Furthermore, the activity‐induced hyperemia is altered during a number of physiological and pharmacological interventions and in some pathologic conditions. Conclusion Flicker stimulation of the retina offers a new and powerful means to modulate blood flow and investigate the neurovascular coupling in the neural eye tissues. Exploration of this activity‐induced hyperemia and the mechanism(s) underlying the neurovascular coupling will lead to an increased understanding of the pathophysiology of various ocular diseases.

In the past the microkinetics of blood flow in the infrared pit organs of pit vipers has been studied with Doppler flowmetry using various infrared stimuli such as a human hand or soldering iron at various distances, lasers of various wavelengths, etc. Quick-acting variations in blood flow were recorded, and interpreted as a cooling mechanism for avoiding afterimage in the infrared receptors. However, the Doppler measurements provided only the summation of blood flow in a number of vessels covered by the sensing probe, but did not give data on flow in individual vessels. In the present work the authors introduced into the bloodstream of Gloydius and Trimeresurus pit vipers fluorescent microspheres labeled with fluorescein isothiocyanate (FITC) contained in a solution of FITC-dextran in physiological saline. They observed the passage of the microspheres through individual pit organ vessels with a fluorescent microscope to which was attached a high-speed video camera and image intensifier. Output of the camera was recorded before, during, and after stimulus with a 810-nm diode laser. Recording was done at 250 frames/s on high-speed video apparatus and downloaded to a hard disk. Disk files were loaded into proprietary software and particles were tracked and average velocities calculated. The data were then tested for significance by ANOVA with post hoc tests. A significant (p<.05) increase in blood velocity was found at the focal point of the stimulus laser, but not anywhere removed from this point. Proximal severing of the pit sensory nerves caused degeneration of the pit receptor terminals and abolished stimulus-induced blood flow changes, but did not affect normal blood flow. The authors conclude that the receptors themselves are directly and locally controlling the smooth muscle elements of the blood vessels, in response to heating of the receptors by infrared radiation. They speculate that the heavy vascularization constitutes a cooling system for the radiation-encoding receptors, and further that the agent of control may be a volatile neuromediator such as nitric oxide.