Arousal state transitions occlude sensory-evoked neurovascular coupling in neonatal mice.

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.

Arousal state transitions occlude sensory-evoked neurovascular coupling in neonatal mice.

In the adult sensory cortex, increases in neural activity elicited by sensory stimulation usually drives 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. Significance Statement: In the adult brain, increases in neural activity are often followed by vasodilation, allowing activity to be monitored using optical or magnetic resonance imaging. However, in neonates, sensory stimulation can drive vasoconstriction, whose origin was not understood. We used optical and magnetic resonance imaging approaches to investigate hemodynamics in neonatal mice. We found that sensory-induced vasoconstriction occurred when the mice were asleep, as sleep is associated with dilation of the vasculature of the brain relative to the awake state. The stimulus awakens the mice, causing a constriction due to the arousal state change. Our study shows the importance of monitoring arousal state, particularly when investigating subjects that may sleep, and the dominance arousal effects on brain hemodynamics.

High-frequency neuronal signal better explains multi-phase BOLD response.

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.

Relating Pupil Diameter and Blinking to Cortical Activity and Hemodynamics across Arousal States.

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.

Photoacoustic imaging for microcirculation.

Microcirculation facilitates the blood-tissue exchange of nutrients and regulates blood perfusion. It is, therefore, essential in maintaining tissue health. Aberrations in microcirculation are potentially indicative of underlying cardiovascular and metabolic pathologies. Thus, quantitative information about it is of great clinical relevance. Photoacoustic imaging (PAI) is a capable technique that relies on the generation of imaging contrast via the absorption of light and can image at micron-scale resolution. PAI is especially desirable to map microvasculature as hemoglobin strongly absorbs light and can generate a photoacoustic signal. This paper reviews the current state of the art for imaging microvascular networks using photoacoustic imaging. We further describe how quantitative information about blood dynamics such as the total hemoglobin concentration, oxygen saturation, and blood flow rate is obtained using PAI. We also discuss its importance in understanding key pathophysiological processes in neurovascular, cardiovascular, ophthalmic, and cancer research fields. We then discuss the current challenges and limitations of PAI and the approaches that can help overcome these limitations. Finally, we provide the reader with an overview of future trends in the field of PAI for imaging microcirculation.

Behavioral and physiological monitoring for awake neurovascular coupling experiments: a how-to guide.

Significance: Functional brain imaging in awake animal models is a popular and powerful technique that allows the investigation of neurovascular coupling (NVC) under physiological conditions. However, ubiquitous facial and body motions (fidgeting) are prime drivers of spontaneous fluctuations in neural and hemodynamic signals. During periods without movement, animals can rapidly transition into sleep, and the hemodynamic signals tied to arousal state changes can be several times larger than sensory-evoked responses. Given the outsized influence of facial and body motions and arousal signals in neural and hemodynamic signals, it is imperative to detect and monitor these events in experiments with un-anesthetized animals. Aim: To cover the importance of monitoring behavioral state in imaging experiments using un-anesthetized rodents, and describe how to incorporate detailed behavioral and physiological measurements in imaging experiments. Approach: We review the effects of movements and sleep-related signals (heart rate, respiration rate, electromyography, intracranial pressure, whisking, and other body movements) on brain hemodynamics and electrophysiological signals, with a focus on head-fixed experimental setup. We summarize the measurement methods currently used in animal models for detection of those behaviors and arousal changes. We then provide a guide on how to incorporate this measurements with functional brain imaging and electrophysiology measurements. Results: We provide a how-to guide on monitoring and interpreting a variety of physiological signals and their applications to NVC experiments in awake behaving mice. Conclusion: This guide facilitates the application of neuroimaging in awake animal models and provides neuroscientists with a standard approach for monitoring behavior and other associated physiological parameters in head-fixed animals.

Awake mouse brain photoacoustic and optical imaging through a transparent ultrasound cranial window.

Optical resolution photoacoustic microscopy (OR-PAM) can map the cerebral vasculature at capillary-level resolution. However, the OR-PAM setup's bulky imaging head makes awake mouse brain imaging challenging and inhibits its integration with other optical neuroimaging modalities. Moreover, the glass cranial windows used for optical microscopy are unsuitable for OR-PAM due to the acoustic impedance mismatch between the glass plate and the tissue. To overcome these challenges, we propose a lithium niobate based transparent ultrasound transducer (TUT) as a cranial window on a thinned mouse skull. The TUT cranial window simplifies the imaging head considerably due to its dual functionality as an optical window and ultrasound transducer. The window remains stable for six weeks, with no noticeable inflammation and minimal bone regrowth. The TUT window's potential is demonstrated by imaging the awake mouse cerebral vasculature using OR-PAM, intrinsic optical signal imaging, and two-photon microscopy. The TUT cranial window can potentially also be used for ultrasound stimulation and simultaneous multimodal imaging of the awake mouse brain.

Brain-wide ongoing activity is responsible for significant cross-trial BOLD variability.

A notorious issue of task-based functional magnetic resonance imaging (fMRI) is its large cross-trial variability. To quantitatively characterize this variability, the blood oxygenation level-dependent (BOLD) signal can be modeled as a linear summation of a stimulation-relevant and an ongoing (i.e. stimulation-irrelevant) component. However, systematic investigation on the spatiotemporal features of the ongoing BOLD component and how these features affect the BOLD response is still lacking. Here we measured fMRI responses to light onsets and light offsets in awake rats. The neuronal response was simultaneously recorded with calcium-based fiber photometry. We established that between-region BOLD signals were highly correlated brain-wide at zero time lag, including regions that did not respond to visual stimulation, suggesting that the ongoing activity co-fluctuates across the brain. Removing this ongoing activity reduced cross-trial variability of the BOLD response by ~30% and increased its coherence with the Ca2+ signal. Additionally, the negative ongoing BOLD activity sometimes dominated over the stimulation-driven response and contributed to the post-stimulation BOLD undershoot. These results suggest that brain-wide ongoing activity is responsible for significant cross-trial BOLD variability, and this component can be reliably quantified and removed to improve the reliability of fMRI response. Importantly, this method can be generalized to virtually all fMRI experiments without changing stimulation paradigms.

Neurovascular coupling and bilateral connectivity during NREM and REM sleep.

To understand how arousal state impacts cerebral hemodynamics and neurovascular coupling, we monitored neural activity, behavior, and hemodynamic signals in un-anesthetized, head-fixed mice. Mice frequently fell asleep during imaging, and these sleep events were interspersed with periods of wake. During both NREM and REM sleep, mice showed large increases in cerebral blood volume ([HbT]) and arteriole diameter relative to the awake state, two to five times larger than those evoked by sensory stimulation. During NREM, the amplitude of bilateral low-frequency oscillations in [HbT] increased markedly, and coherency between neural activity and hemodynamic signals was higher than the awake resting and REM states. Bilateral correlations in neural activity and [HbT] were highest during NREM, and lowest in the awake state. Hemodynamic signals in the cortex are strongly modulated by arousal state, and changes during sleep are substantially larger than sensory-evoked responses.

Functional hyperemia drives fluid exchange in the paravascular space.

The brain lacks a conventional lymphatic system to remove metabolic waste. It has been proposed that directional fluid movement through the arteriolar paravascular space (PVS) promotes metabolite clearance. We performed simulations to examine if arteriolar pulsations and dilations can drive directional CSF flow in the PVS and found that arteriolar wall movements do not drive directional CSF flow. We propose an alternative method of metabolite clearance from the PVS, namely fluid exchange between the PVS and the subarachnoid space (SAS). In simulations with compliant brain tissue, arteriolar pulsations did not drive appreciable fluid exchange between the PVS and the SAS. However, when the arteriole dilated, as seen during functional hyperemia, there was a marked exchange of fluid. Simulations suggest that functional hyperemia may serve to increase metabolite clearance from the PVS. We measured blood vessels and brain tissue displacement simultaneously in awake, head-fixed mice using two-photon microscopy. These measurements showed that brain deforms in response to pressure changes in PVS, consistent with our simulations. Our results show that the deformability of the brain tissue needs to be accounted for when studying fluid flow and metabolite transport.

Ultra-slow Oscillations in fMRI and Resting-State Connectivity: Neuronal and Vascular Contributions and Technical Confounds.

Ultra-slow, ∼0.1-Hz variations in the oxygenation level of brain blood are widely used as an fMRI-based surrogate of "resting-state" neuronal activity. The temporal correlations among these fluctuations across the brain are interpreted as "functional connections" for maps and neurological diagnostics. Ultra-slow variations in oxygenation follow a cascade. First, they closely track changes in arteriole diameter. Second, interpretable functional connections arise when the ultra-slow changes in amplitude of γ-band neuronal oscillations, which are shared across even far-flung but synaptically connected brain regions, entrain the ∼0.1-Hz vasomotor oscillation in diameter of local arterioles. Significant confounds to estimates of functional connectivity arise from residual vasomotor activity as well as arteriole dynamics driven by self-generated movements and subcortical common modulatory inputs. Last, methodological limitations of fMRI can lead to spurious functional connections. The neuronal generator of ultra-slow variations in γ-band amplitude, including that associated with self-generated movements, remains an open issue.

Nucleolin mediates the binding of cancer cells to L-selectin under conditions of lymphodynamic shear stress.

Head and neck squamous cell carcinoma (HNSCC) cells bind to lymphocytes via L-selectin in a shear-dependent manner. This interaction takes place exclusively under low-shear stress conditions, such as those found within the lymph node parenchyma. This represents a novel functional role for L-selectin-selectin ligand interactions. Our previous work has characterized as-of-yet unidentified L-selectin ligands expressed by HNSCC cells that are specifically active under conditions of low shear stress consistent with lymph flow. Using an affinity purification approach, we now show that nucleolin expressed on the surface of HNSCC cells is an active ligand for L-selectin. Parallel plate chamber flow-based experiments and atomic force microscopy (AFM) experiments show that nucleolin is the main functional ligand under these low-force conditions. Furthermore, AFM shows a clear relationship between work of deadhesion and physiological loading rates. Our results reveal nucleolin as the first major ligand reported for L-selectin that operates under low-shear stress conditions.