Understanding stimulus-evoked cerebral hemodynamics is critical for elucidating brain function and neurological disease mechanisms. However, existing neuroimaging approaches are often limited by spatial or temporal resolution, imaging depth, sensitivity, or the ability to simultaneously capture multiple hemodynamic parameters. To address these challenges, we developed a hybrid functional ultrasound–photoacoustic (fUSPA) imaging platform integrating ultrafast ultrasound with multispectral photoacoustic techniques. This system enables real-time quantitative mapping of cerebral blood volume (CBV), cerebral blood flow (CBF), and blood oxygen saturation (SO2) at high spatiotemporal resolution using a compact head-mounted probe. In addition to intrinsic hemodynamic imaging, the platform supports microbubble-enhanced super-resolution ultrasound for microvascular flow measurements and photoacoustic contrast agent-based imaging, such as indocyanine green, for vascular perfusion assessment. Using fUSPA, we investigated brain-wide cerebrovascular reactivity (CVR) at single-vessel resolution by quantifying relative changes in CBV, CBF, and SO2 during hypercapnic stimulation. Functional analyses reveal distinct CVR responses between cortical arteries and veins, as well as anti-correlated CBV oscillations during resting conditions. We demonstrate the potential of multiparametric fUSPA imaging to interrogate complex cerebrovascular dynamics and advance studies of brain function and neurodiseases, such as brain cancer.

Background: Cortical arteries exhibit larger dilations and constrictions during sleep than wakefulness. These arterial dynamics are thought to drive periarterial pumping of cerebrospinal fluid (CSF) that helps clear waste from the brain. Although norepinephrine (NE) mediated vascular dynamics during sleep have been linked to CSF flow, the mechanism by which NE interacts with local cortical activity to control cerebrovascular dynamics remain unclear. Objectives: We tested the hypothesis that NE regulates cerebrovascular dynamics in a state-dependent manner by interacting with local neuron activity and other neuromodulators. Methods: Using fiber photometry in head-fixed mice (n = 6 mice), we simultaneously measured cerebral blood volume (CBV), NE (GRAB-NE), acetylcholine (GRAB-ACh) in barrel cortex during wakefulness, NREM, and REM sleep. We have also recorded pyramidal calcium activity (CaMKIIa-GCaMP7s) and CBV in a separate cohort (n = 6 mice). To test causality, we performed optogenetic activation of locus coeruleus (LC) NE neurons and pharmacological blockade of adrenergic receptors (n = 8 mice). Results: CBV increased significantly during sleep, exceeding sensory-evoked vascular responses. Pyramidal calcium activity preceded CBV changes and was strongly positively correlated. Both acetylcholine (ACh) and NE levels were low during sleep with ACh rebounding to slightly higher-level during REM sleep. Awakening from either sleep stages produced large increases in NE, ACh, while causing a drop in CBV and Pyramidal calcium activity. Mathematical modeling showed NE and Pyramidal activity can accurately predict the CBV changes during sleep. Pharmacological blockade of adrenergic receptors weakened the neurovascular coupling between NE and vascular response. Optogenetic stimulation of LC-NE neurons increased cortical NE levels and pupil diameter and causing vasoconstriction. LC activation resulted in inhibition of pyramidal calcium activity preceding vasoconstriction in the cortex. Conclusions: Cerebrovascular dynamics are strongly state dependent. During sleep, low NE tone permits coupling between pyramidal neuron activity and vasculature allowing large CBV increases. Optogenetic activation of LC-NE elevates cortical NE, suppresses pyramidal calcium activity potentially, and causes vasoconstriction. These findings identify norepinephrine as a state-dependent regulator that overrides local control of cerebrovascular tone.

The brain moves within the skull, but the drivers and function of this motion are not understood. We visualized brain motion relative to the skull in awake head-fixed mice using high-speed, multi-plane two-photon microscopy. Brain motion was primarily rostrally and laterally directed, and was tightly correlated with locomotion, but not with respiration or the cardiac cycle. Electromyography recordings in abdominal muscles and microCT reconstructions of the trunk and spinal vasculature showed that brain motion was driven by abdominal muscle contractions that activate a hydraulic-like vascular connection between the nervous system and the abdominal cavity. Externally-applied abdominal pressure generated brain motion similar to those seen during abdominal muscle contractions. Simulations showed that brain motion drives substantial volumes of interstitial fluid through and out of the brain (at volumetric rates several times higher than production) into the subarachnoid space, in the opposite direction of fluid flow seen during sleep. The brain is hydraulically linked to the abdominal compartment, and fluid flow in the brain is coupled to body movements, providing a mechanism by which the mechanics of the viscera directly impact brain health.

Cortical arteries undergo coordinated dilations and constrictions during non-rapid eye movement (NREM) sleep that can drive the circulation of cerebrospinal fluid (CSF). CSF flow is thought to stimulate glymphatic clearance and remove metabolic waste, but the drivers of arterial dynamics during sleep remain poorly understood. We explored how norepinephrine (NE), a vasoconstrictor and arousal promoting neuromodulator, could control arterial dynamics during NREM sleep in mice. We used two-photon microscopy and fiber photometry to simultaneously measure cortical NE levels and vascular dynamics in head-fixed mice during sleep. NE was measured using a virally expressed GPCR activation-based NE sensor (GRABNE-2m). Blood plasma was visualized with fluorescent albumin (Alb.mScarlet) and intravascular injections of tetramethylrhodamine-isothiocyanate (TRITC). Sleep state was monitored and classified using electrocorticography, electromyography, pupillometry, and behavioral tracking. We found norepinephrine (NE) levels in the somatosensory cortex were highest during wakefulness, decreased during NREM, and were lowest during rapid eye movement (REM) sleep. Arteriole diameter and blood volume increased during NREM with pulsations consistent with periarterial pumping and was highest during REM sleep. Immediately prior to arousal from sleep, local NE levels rapidly increased back to waking baseline levels, followed by vasoconstriction. To determine if NE release is sufficient to produce these hemodynamic changes at arousal events, we optogenetically stimulated the locus coeruleus (LC) of transgenic mice (TH-cre+ and DBH-cre+) expressing a cre-dependent channelrhodopsin (ChR2) in noradrenergic neurons. Fiber photometry recordings showed that optogenetic stimulation of the LC triggered NE increases and causes vasoconstrictions analogous to those in natural awakening events. Pharmacologic inhibition of NE receptors attenuated vasoconstriction seen during optogenetic stimulation. Together these experiments suggest that NE signaling is a key driver of the hemodynamic changes seen throughout NREM sleep and at arousal events. Knowledge of the underlying mechanism governing CSF movement and glymphatic clearance during sleep can inform the development of NE-modulating therapies to improve waste clearance and slow neurodegeneration in patients with neurological disorders. This research was supported by NIH/NINDS (U19NS128613 PI: Drew). MW is supported by T32NS115667 fellowship. MH is supported by T32NS115667 trainee fellowship and AHA predoctoral fellowship (24PRE1201066).

The prefrontal cortex (PFC) is one of the last brain regions to fully mature, making it particularly sensitive to stress and drug use early in life. Both human and rodent studies find long-lasting behavioral changes after adolescent alcohol exposure that implicate underlying disruptions in PFC development, including structural abnormalities and altered brain functional connectivity. Few rodent studies have been conducted to understand the network-level implications of these disruptions. We assessed how adolescent binge-like alcohol consumption in a drinking in the dark (DID) model affected adult aversion-resistant alcohol consumption, exploration, and brain-wide functional connectivity in mice. Approximately one month after the conclusion of DID, only female mice exposed to alcohol during adolescence exhibited aversion-resistant alcohol preference in adulthood. Adult females exhibited additional sex-specific changes in exploratory behavior in the elevated plus maze after adolescent alcohol consumption. Resting state neuroimaging revealed changes in prefrontal cortical connections with sensory motor, hippocampal, striatal, and other networks, providing insights into the putative systems underlying deficits caused by adolescent alcohol exposure. Critically, our data corroborate a growing body of literature in human and rodent studies demonstrating that adolescent alcohol use may increase risk for adult alcohol use more strongly in females. Finally, we identify neural correlates of this effect that include both known and novel networks and tie these back to human datasets, allowing biological and mechanistic targets to be further explored for future study and interventions.

The prefrontal cortex (PFC), which is thought to be disrupted early in the cycle of substance use and addiction [1], is comprised of a complex microcircuit of long-range glutamatergic pyramidal neurons controlled by GABAergic-expressing local inhibitory neurons [2, 3]. Somatostatin (SST)-expressing neurons are a subpopulation of these local GABAergic inhibitory cells and provide both peptidergic and GABAergic control over these PFC circuits [3, 4], and are disturbed following alcohol consumption in humans [5] and in rodent models [6, 7]. However, little is known about how endogenous SST peptide signaling is affected by alcohol. Using ex vivo electrophysiology, immunohistochemistry, in situ hybridization, and behavior, we demonstrate robust down-regulation of SST control over pyramidal output activity in the prelimbic (PL), but not infralimbic (IL), PFC after alcohol exposure. We also show this is likely mediated by changes in SST receptor expression levels and not disrupted expression or capacity for release of SST peptide, suggesting postsynaptic homeostatic changes to SST signaling following binge alcohol consumption in mice that may underlie post-alcohol dysregulation in mood. This provides insight into how voluntary alcohol consumption disrupts PFC peptide signaling and suggests a potential therapeutic target for the treatment of alcohol use disorder (AUD).

It is unknown how the brain orchestrates coordination of global neural and vascular dynamics. We sought to uncover the role of a sparse but unusual population of genetically distinct interneurons known as type-I nNOS neurons, using a novel pharmacological strategy to unilaterally ablate these neurons from the somatosensory cortex of mice. Region-specific ablation produced changes in both neural activity and vascular dynamics, decreased power in the delta-band of the local field potential, reduced sustained vascular responses to prolonged sensory stimulation, and abolished the post-stimulus undershoot in cerebral blood volume. Coherence between the left and right somatosensory cortex gamma-band power envelope and blood volume at ultra-low frequencies was decreased, suggesting type-1 nNOS neurons integrate long-range coordination of brain signals. Lastly, we observed decreases in the amplitude of resting-state blood volume oscillations and decreased vasomotion following the ablation of type-I nNOS neurons. This demonstrates that a small population of nNOS-positive neurons is indispensable for regulating both neural and vascular dynamics in the whole brain, raising the possibility that loss of these neurons could contribute to the development of neurodegenerative diseases and sleep disturbances.

Ultrasound neuromodulation is a rapidly advancing, non-invasive technique with significant therapeutic potential for treating various neurological disorders. Although extensive in vitro and in vivo studies have provided valuable insights into its modulatory effects, the underlying mechanisms remain poorly understood, limiting its clinical translation. Optical neuroimaging techniques can help investigate these mechanisms; however, the opacity and bulkiness of conventional ultrasound transducers pose significant challenges for their integration with in vivo ultrasound neuromodulation studies, particularly in awake rodents. To address these limitations, we propose a straightforward solution: a miniaturized lithium niobate-based transparent ultrasound transducer (TUT) integrated as a thinned-skull cranial window for ultrasound stimulation while facilitating multimodal optical neuroimaging in awake mice brain. Using laser speckle contrast imaging and intrinsic optical signal imaging, we studied changes in brain hemodynamics in response to various ultrasound stimulation sequences. Our experiments demonstrated that TUT cranial window can robustly induce neuromodulatory effects with observed increase in both cerebral blood flow and total hemoglobin, with peak and cumulative hemodynamic changes directionally correlated with ultrasound stimulation duration and intensity. Overall, these findings highlight that TUT cranial window can seamlessly integrate ultrasound stimulation and optical neuroimaging in awake mouse brain models, offering promising prospects for uncovering the underlying mechanisms of ultrasound neuromodulation.

Studying brain-wide hemodynamic responses to different stimuli at high spatiotemporal resolutions can help gain new insights into the mechanisms of neuro- diseases and -disorders. Nonetheless, this task is challenging, primarily due to the complexity of neurovascular coupling, which encompasses interdependent hemodynamic parameters including cerebral blood volume (CBV), cerebral blood flow (CBF), and cerebral oxygen saturation (SO2). The current brain imaging technologies exhibit inherent limitations in resolution, sensitivity, and imaging depth, restricting their capacity to comprehensively capture the intricacies of cerebral functions. To address this, a multimodal functional ultrasound and photoacoustic (fUSPA) imaging platform is reported, which integrates ultrafast ultrasound and multispectral photoacoustic imaging methods in a compact head-mountable device, to quantitatively map individual dynamics of CBV, CBF, and SO2 as well as contrast agent enhanced brain imaging at high spatiotemporal resolutions. Following systematic characterization, the fUSPA system is applied to study brain-wide cerebrovascular reactivity (CVR) at single-vessel resolution via relative changes in CBV, CBF, and SO2 in response to hypercapnia stimulation. These results show that cortical veins and arteries exhibit differences in CVR in the stimulated state and consistent anti-correlation in CBV oscillations during the resting state, demonstrating the multiparametric fUSPA system's unique capabilities in investigating complex mechanisms of brain functions.

Introduction: Cerebrovascular dysfunction has been implicated in age-related cognitive decline and dementia, but the underlying vascular mechanisms are not well understood. An improved understanding of the nature of normal cerebrovascular aging is needed to help to establish the role that vascular dysfunction might play in cognitive decline and dementia. Methods: Here, we asked how normal aging differentially impacts the vascular structure and function in different brain areas in mice. We investigated structural changes in aged cerebrovascular networks and pericytes utilizing serial two-photon tomography (STPT). To further investigate potential remodeling of different vascular compartments and pericyte subtypes, we utilized tissue clearing, 3D immunolabeling, and light sheet fluorescence microscopy (LSFM) imaging. We also assessed how healthy aging impacts brain hemodynamics in response to voluntary locomotion and whisker stimulation in awake, head-fixed mice using wide field optical signal imaging and two-photon imaging. We tested mice of both sexes at 2-month, 18-month (early aging), and 24-month (late aging) of age. Results: Whole-brain vascular tracing using STPT showed an overall ~10% decrease in vascular length and branching density, and LSFM imaging with 3D immunolabeling further revealed increased arteriole tortuosity in aged brains. We also uncovered a selective vascular and pericyte loss in deep cortical layers, basal forebrain regions, and the hippocampal network. This may contribute to their regional vulnerabilities in neurodegenerative disorders. Moreover, our in vivo imaging in awake, head-fixed mice identified delayed neurovascular coupling response and inefficient oxygen delivery in aged brains. Conclusions: Our study reveals aging-related brain-wide changes in vascular and mural cell types that can explain vulnerability and resilience of different brain areas in normal aging. Moreover, we identified an age-related decrease in brain oxygenation and delayed neurovascular coupling responses which can be linked with cognitive impairment in aged brains. These aging-related changes will serve as a common factor to understand many neurodegenerative disorders and cognition decline in the elderly population.

Arousal state affects neural activity and vascular dynamics in the cortex, with sleep associated with large changes in the local field potential and increases in cortical blood flow. We investigated the relationship between pupil diameter and blink rate with neural activity and blood volume in the somatosensory cortex in male and female unanesthetized, head-fixed mice. We monitored these variables while the mice were awake, during periods of rapid eye movement (REM), and non-rapid eye movement (NREM) sleep. Pupil diameter was smaller during sleep than in the awake state. Changes in pupil diameter were coherent with both gamma-band power and blood volume in the somatosensory cortex, but the strength and sign of this relationship varied with arousal state. We observed a strong negative correlation between pupil diameter and both gamma-band power and blood volume during periods of awake rest and NREM sleep, although the correlations between pupil diameter and these signals became positive during periods of alertness, active whisking, and REM. Blinking was associated with increases in arousal and decreases in blood volume when the mouse was asleep. Bilateral coherence in gamma-band power and in blood volume dropped following awake blinking, indicating a reset of neural and vascular activity. Using only eye metrics (pupil diameter and eye motion), we could determine the arousal state of the mouse ('Awake,' 'NREM,' 'REM') with >90% accuracy with a 5 s resolution. There is a strong relationship between pupil diameter and hemodynamics signals in mice, reflecting the pronounced effects of arousal on cerebrovascular dynamics. SIGNIFICANCE STATEMENT Determining arousal state is a critical component of any neuroscience experiment. Pupil diameter and blinking are influenced by arousal state, as are hemodynamics signals in the cortex. We investigated the relationship between cortical hemodynamics and pupil diameter and found that pupil diameter was strongly related to the blood volume in the cortex. Mice were more likely to be awake after blinking than before, and blinking resets neural activity. Pupil diameter and eye motion can be used as a reliable, noninvasive indicator of arousal state. As mice transition from wake to sleep and back again over a timescale of seconds, monitoring pupil diameter and eye motion permits the noninvasive detection of sleep events during behavioral or resting-state experiments.

The role of parvalbumin (PV) interneurons in vascular control is poorly understood. Here, we investigated the hemodynamic responses elicited by optogenetic stimulation of PV interneurons using electrophysiology, functional magnetic resonance imaging (fMRI), wide-field optical imaging (OIS), and pharmacological applications. As a control, forepaw stimulation was used. Stimulation of PV interneurons in the somatosensory cortex evoked a biphasic fMRI response in the photostimulation site and negative fMRI signals in projection regions. Activation of PV neurons engaged two separable neurovascular mechanisms in the stimulation site. First, an early vasoconstrictive response caused by the PV-driven inhibition is sensitive to the brain state affected by anesthesia or wakefulness. Second, a later ultraslow vasodilation lasting a minute is closely dependent on the sum of interneuron multiunit activities, but is not due to increased metabolism, neural or vascular rebound, or increased glial activity. The ultraslow response is mediated by neuropeptide substance P (SP) released from PV neurons under anesthesia, but disappears during wakefulness, suggesting that SP signaling is important for vascular regulation during sleep. Our findings provide a comprehensive perspective about the role of PV neurons in controlling the vascular response.

Aging is frequently associated with compromised cerebrovasculature and pericytes. However, we do not know how normal aging differentially impacts vascular structure and function in different brain areas. Here we utilize mesoscale microscopy methods and in vivo imaging to determine detailed changes in aged murine cerebrovascular networks. Whole-brain vascular tracing shows an overall ~10% decrease in vascular length and branching density with ~7% increase in vascular radii in aged brains. Light sheet imaging with 3D immunolabeling reveals increased arteriole tortuosity of aged brains. Notably, vasculature and pericyte densities show selective and significant reductions in the deep cortical layers, hippocampal network, and basal forebrain areas. We find increased blood extravasation, implying compromised blood-brain barrier function in aged brains. Moreover, in vivo imaging in awake mice demonstrates reduced baseline and on-demand blood oxygenation despite relatively intact neurovascular coupling. Collectively, we uncover regional vulnerabilities of cerebrovascular network and physiological changes that can mediate cognitive decline in normal aging.

Somatostatin (SST) neurons in the prelimbic (PL) cortex mediate a variety of behavioral states, ranging from alcohol consumption to fear learning and avoidance-related behaviors. However, little is known about the role of somatostatin peptide signaling in cortical functioning or behavior.

Understanding brain-wide hemodynamic responses to different stimuli at high spatiotemporal resolutions can help study neuro-disorders and brain functions. However, the existing brain imaging technologies have limited resolution, sensitivity, imaging depth and provide information about only one or two hemodynamic parameters. To address this, we propose a multimodal functional ultrasound and photoacoustic (fUSPA) imaging platform, which integrates ultrafast ultrasound and multispectral photoacoustic imaging methods in a compact head-mountable device, to quantitatively map cerebral blood volume (CBV), cerebral blood flow (CBF), oxygen saturation (SO2) dynamics as well as contrast agent enhanced brain imaging with high spatiotemporal resolutions. After systematic characterization, the fUSPA system was applied to quantitatively study the changes in brain hemodynamics and vascular reactivity at single vessel resolution in response to hypercapnia stimulation. Our results show an overall increase in brain-wide CBV, CBF, and SO2, but regional differences in singular cortical veins and arteries and a reproducible anti-correlation pattern between venous and cortical hemodynamics, demonstrating the capabilities of the fUSPA system for providing multiparametric cerebrovascular information at high-resolution and sensitivity, that can bring insights into the complex mechanisms of neurodiseases.

Visual stimulation-evoked blood-oxygen-level dependent (BOLD) responses can exhibit more complex temporal dynamics than a simple monophasic response. For instance, BOLD responses sometimes include a phase of positive response followed by a phase of post-stimulus undershoot. Whether the BOLD response during these phases reflects the underlying neuronal signal fluctuations or is contributed by non-neuronal physiological factors remains elusive. When presenting blocks of sustained (i.e. DC) light ON-OFF stimulations to unanesthetized rats, we observed that the response following a decrease in illumination (i.e. OFF stimulation-evoked BOLD response) in the visual cortices displayed reproducible multiple phases, including an initial positive BOLD response, followed by an undershoot and then an overshoot before the next ON trial. This multi-phase BOLD response did not result from the entrainment of the periodic stimulation structure. When we measured the neural correlates of these responses, we found that the high-frequency band from the LFP power (300 – 3000 Hz, multi-unit activity (MUA)), but not the power in the gamma band (30 – 100 Hz) exhibited the same multiphasic dynamics as the BOLD signal. This study suggests that the post-stimulus phases of the BOLD response can be better explained by the high-frequency neuronal signal.

In the adult sensory cortex, increases in neural activity elicited by sensory stimulation usually drive vasodilation mediated by neurovascular coupling. However, whether neurovascular coupling is the same in neonatal animals as adults is controversial, as both canonical and inverted responses have been observed. We investigated the nature of neurovascular coupling in unanesthetized neonatal mice using optical imaging, electrophysiology, and BOLD fMRI. We find in neonatal (postnatal day 15, P15) mice, sensory stimulation induces a small increase in blood volume/BOLD signal, often followed by a large decrease in blood volume. An examination of arousal state of the mice revealed that neonatal mice were asleep a substantial fraction of the time, and that stimulation caused the animal to awaken. As cortical blood volume is much higher during REM and NREM sleep than the awake state, awakening occludes any sensory-evoked neurovascular coupling. When neonatal mice are stimulated during an awake period, they showed relatively normal (but slowed) neurovascular coupling, showing that that the typically observed constriction is due to arousal state changes. These result show that sleep-related vascular changes dominate over any sensory-evoked changes, and hemodynamic measures need to be considered in the context of arousal state changes.

The movement of fluid into, through, and out of the brain plays an important role in clearing metabolic waste. However, there is controversy regarding the mechanisms driving fluid movement in the fluid-filled paravascular spaces (PVS), and whether the movement of metabolic waste in the brain extracellular space (ECS) is primarily driven by diffusion or convection. The dilation of penetrating arterioles in the brain in response to increases in neural activity (neurovascular coupling) is an attractive candidate for driving fluid circulation, as it drives deformation of the brain tissue and of the PVS around arteries, resulting in fluid movement. We simulated the effects of vasodilation on fluid movement into and out of the brain ECS using a novel poroelastic model of brain tissue. We found that arteriolar dilations could drive convective flow through the ECS radially outward from the arteriole, and that this flow is sensitive to the dynamics of the dilation. Simulations of sleep-like conditions, with larger vasodilations and increased extracellular volume in the brain showed enhanced movement of fluid from the PVS into the ECS. Our simulations suggest that both sensory-evoked and sleep-related arteriolar dilations can drive convective flow of cerebrospinal fluid not just in the PVS, but also into the ECS through the PVS around arterioles.