Parvalbumin interneuron activity induces slow cerebrovascular fluctuations in awake mice.

Neuronal regulation of cerebrovasculature underlies brain imaging techniques reliant on cerebral blood flow (CBF) changes. However, interpreting these signals requires understanding their neural correlates. Parvalbumin (PV) interneurons are crucial in network activity, but their impact on CBF is not fully understood. Optogenetic studies show that stimulating cortical PV interneurons induces diverse CBF responses, including rapid increases, decreases, and slower delayed increases. To clarify this relationship, we measured hemodynamic and neural responses to optogenetic stimulation of PV interneurons expressing Channelrhodopsin-2 during evoked and ongoing resting-state activity in the somatosensory cortex of awake mice. Two-photon microscopy (2P) Ca2+ imaging showed robust activation of PV-positive (PV+) cells and inhibition of PV-negative (PV-) cells. Prolonged PV+ cell stimulation led to a delayed, slow CBF increase, resembling a secondary peak in the CBF response to whisker stimulation. 2P vessel diameter measurements revealed that PV+ cell stimulation induced rapid arterial vasodilation in superficial layers and delayed vasodilation in deeper layers. Ongoing activity recordings indicated that both PV+ and PV- cell populations modulate arterial fluctuations at rest, with PV+ cells having a greater impact. These findings show that PV interneurons generate a complex depth-dependent vascular response, dominated by slow vascular changes in deeper layers.

Long-range inhibitory neurons mediate cortical neurovascular coupling.

To meet the high energy demands of brain function, cerebral blood flow (CBF) parallels changes in neuronal activity by a mechanism known as neurovascular coupling (NVC). However, which neurons play a role in mediating NVC is not well understood. Here, we identify in mice and humans a specific population of cortical GABAergic neurons that co-express neuronal nitric oxide synthase and tachykinin receptor 1 (Tacr1). Through whole-tissue clearing, we demonstrate that Tacr1 neurons extend local and long-range projections across functionally connected cortical areas. We show that whisker stimulation elicited Tacr1 neuron activity in the barrel cortex through feedforward excitatory pathways. Additionally, through optogenetic experiments, we demonstrate that Tacr1 neurons are instrumental in mediating CBF through the relaxation of mural cells in a similar fashion to whisker stimulation. Finally, by electron microscopy, we observe that Tacr1 processes contact astrocytic endfeet. These findings suggest that Tacr1 neurons integrate cortical activity to mediate NVC.

Differences in cerebral blood vasculature and flow in awake and anesthetized mouse cortex revealed by quantitative optical coherence tomography angiography.

BACKGROUND: Most of the in vivo neurovascular imaging studies are performed in anesthetized animals. However, anesthesia significantly affects cerebral hemodynamics. NEW METHOD: We applied optical coherence tomography (OCT) methods such as optical microangiography (OMAG) and Doppler optical microangiography (DOMAG) to quantitatively evaluate the effect of anesthesia in cerebral vasculature and blood flow in mouse brain. RESULTS: The OMAG results indicated the increase of large vessel diameter and capillary density induced by ketamine-xylazine and isoflurane, meaning that both anesthetics caused vasodilation. In addition, the preliminary results from DOMAG showed that isoflurane increased the baseline cerebral blood flow. COMPARISON WITH EXISTING METHODS: In comparison with other in vivo imaging modalities, OCT can provide label-free assessment of cortical tissue including tissue morphology, cerebral blood vessel network and flow information down to capillary level, with a large field of view and high imaging speed. CONCLUSIONS: OCT angiography methods demonstrated the ability to measure the differences in the baseline morphological and flow parameters of both large and capillary cerebrovascular networks between awake and anesthetized mice.

Optical microangiography reveals temporal and depth-resolved hemodynamic change in mouse barrel cortex during whisker stimulation.

SIGNIFICANCE: Cerebral blood flow (CBF) regulation at neurovascular coupling (NVC) plays an important role in normal brain functioning to support oxygen delivery to activating neurons. Therefore, studying the mechanisms of CBF adjustment is crucial for the improved understanding of brain activity. AIM: We investigated the temporal profile of hemodynamic signal change in mouse cortex caused by neural activation and its variation over cortical depth. APPROACH: Following the cranial window surgery, intrinsic optical signal imaging (IOSI) was used to spatially locate the activated region in mouse cortex during whisker stimulation. Optical microangiography (OMAG), the functional extension of optical coherence tomography, was applied to image the activated and control regions identified by IOSI. Temporal profiles of hemodynamic response signals obtained by IOSI and OMAG were compared, and OMAG signal was analyzed over cortical layers. RESULTS: Our results showed that the hemodynamic response to neural activity revealed by blood flow change signal signal through IOSI is slower than that observed by OMAG signal. OMAG also indicated the laminar variation of the response over cortical depth, showing the largest response in cortical layer IV. CONCLUSIONS: Overall, we demonstrated the development and application of dual-modality imaging system composed of IOSI and OMAG, which may have potential to enable the future investigations of depth-resolved CBF and to provide the insights of hemodynamic events associated with the NVC.

Procedure and protocols for optical imaging of cerebral blood flow and hemodynamics in awake mice.

We describe a method and procedure that allows for the optical coherence tomography angiography (OCTA) and intrinsic optical signal imaging (IOSI) of cerebral blood flow and hemodynamics in fully awake mice. We detail the procedure of chronic cranial window preparation, the use of an air-lift mobile homecage to achieve stable optical recording in the head-restrained awake mouse, and the imaging methods to achieve multiparametric hemodynamic measurements. The results show that by using a collection of OCTA algorithms, the high-resolution cerebral vasculature can be reliably mapped at a fully awake state, including flow velocity measurements in penetrating arterioles and capillary bed. Lastly, we demonstrate how the awake imaging paradigm is used to study cortical hemodynamics in the mouse barrel cortex during whisker stimulation. The method presented here will facilitate optical recording in the awake, active mice and open the door to many projects that can bridge the hemodynamics in neurovascular units to naturalistic behavior.

Measurement and visualization of stimulus-evoked tissue dynamics in mouse barrel cortex using phase-sensitive optical coherence tomography.

We describe a method to measure tissue dynamics in mouse barrel cortex during functional activation via phase-sensitive optical coherence tomography (PhS-OCT). The method measures the phase changes in OCT signals, which are induced by the tissue volume change, upon which to localize the activated tissue region. Phase unwrapping, compensation and normalization are applied to increase the dynamic range of the OCT phase detection. To guide the OCT scanning, intrinsic optical signal imaging (IOSI) system equipped with a green light laser source (532 nm) is integrated with the PhS-OCT system to provide a full field time-lapsed images of the reflectance that is used to identify the transversal 2D localized tissue response in the mouse brain. The OCT results show a localized decrease in the OCT phase signal in the activated region of the mouse brain tissue. The decrease in the phase signal may be originated from the brain tissue compression caused by the vasodilatation in the activated region. The activated region revealed in the cross-sectional OCT image is consistent with that identified by the IOSI imaging, indicating the phase change in the OCT signals may associate with the changes in the corresponding hemodynamics. In vivo localized tissue dynamics in the barrel cortex at depth during whisker stimulation is observed and monitored in this study.

Electrically tunable lens integrated with optical coherence tomography angiography for cerebral blood flow imaging in deep cortical layers in mice.

We report the use of an electrically tunable lens (ETL) in a 1.3 µm spectral-domain optical coherence tomography (SD-OCT) system to overcome the depth of focus (DOF) limitation in conventional OCT systems for OCT angiography (OCTA) in a mouse cerebral cortex. The ETL provides fast and dynamic control of the axial focus of the probe beam along the entire range of the mouse cortex, upon which we performed cerebral blood flow imaging of all cortical layers by stitching the OCTA images automatically captured at six focal depths. Capillary vasculature and axial blood flow velocity were revealed in distinctive cortical layers and, for the first time, to the best of our knowledge, in white matter. The results have shown the system capability to conveniently investigate the hemodynamics in deep cortical layers in the mouse brain. More importantly, the compact integration of an ETL will benefit the future design of handheld or intra-cavity OCT probes for a wide range of applications in research and clinical fields.