Baseline oxygen consumption decreases with cortical depth.

The cerebral cortex is organized in cortical layers that differ in their cellular density, composition, and wiring. Cortical laminar architecture is also readily revealed by staining for cytochrome oxidase-the last enzyme in the respiratory electron transport chain located in the inner mitochondrial membrane. It has been hypothesized that a high-density band of cytochrome oxidase in cortical layer IV reflects higher oxygen consumption under baseline (unstimulated) conditions. Here, we tested the above hypothesis using direct measurements of the partial pressure of O2 (pO2) in cortical tissue by means of 2-photon phosphorescence lifetime microscopy (2PLM). We revisited our previously developed method for extraction of the cerebral metabolic rate of O2 (CMRO2) based on 2-photon pO2 measurements around diving arterioles and applied this method to estimate baseline CMRO2 in awake mice across cortical layers. To our surprise, our results revealed a decrease in baseline CMRO2 from layer I to layer IV. This decrease of CMRO2 with cortical depth was paralleled by an increase in tissue oxygenation. Higher baseline oxygenation and cytochrome density in layer IV may serve as an O2 reserve during surges of neuronal activity or certain metabolically active brain states rather than reflecting baseline energy needs. Our study provides to our knowledge the first quantification of microscopically resolved CMRO2 across cortical layers as a step towards better understanding of brain energy metabolism.

Awake Mouse Imaging: From Two-Photon Microscopy to Blood Oxygen Level-Dependent Functional Magnetic Resonance Imaging.

BACKGROUND: Functional magnetic resonance imaging (fMRI) in awake behaving mice is well positioned to bridge the detailed cellular-level view of brain activity, which has become available owing to recent advances in microscopic optical imaging and genetics, to the macroscopic scale of human noninvasive observables. However, though microscopic (e.g., two-photon imaging) studies in behaving mice have become a reality in many laboratories, awake mouse fMRI remains a challenge. Owing to variability in behavior among animals, performing all types of measurements within the same subject is highly desirable and can lead to higher scientific rigor. METHODS: We demonstrated blood oxygenation level-dependent fMRI in awake mice implanted with long-term cranial windows that allowed optical access for microscopic imaging modalities and optogenetic stimulation. We started with two-photon imaging of single-vessel diameter changes (n = 1). Next, we implemented intrinsic optical imaging of blood oxygenation and flow combined with laser speckle imaging of blood flow obtaining a mesoscopic picture of the hemodynamic response (n = 16). Then we obtained corresponding blood oxygenation level-dependent fMRI data (n = 5). All measurements could be performed in the same mice in response to identical sensory and optogenetic stimuli. RESULTS: The cranial window did not deteriorate the quality of fMRI and allowed alternation between imaging modalities in each subject. CONCLUSIONS: This report provides a proof of feasibility for multiscale imaging approaches in awake mice. In the future, this protocol could be extended to include complex cognitive behaviors translatable to humans, such as sensory discrimination or attention.

Deep 2-photon imaging and artifact-free optogenetics through transparent graphene microelectrode arrays.

Recent advances in optical technologies such as multi-photon microscopy and optogenetics have revolutionized our ability to record and manipulate neuronal activity. Combining optical techniques with electrical recordings is of critical importance to connect the large body of neuroscience knowledge obtained from animal models to human studies mainly relying on electrophysiological recordings of brain-scale activity. However, integration of optical modalities with electrical recordings is challenging due to generation of light-induced artifacts. Here we report a transparent graphene microelectrode technology that eliminates light-induced artifacts to enable crosstalk-free integration of 2-photon microscopy, optogenetic stimulation, and cortical recordings in the same in vivo experiment. We achieve fabrication of crack- and residue-free graphene electrode surfaces yielding high optical transmittance for 2-photon imaging down to ~ 1 mm below the cortical surface. Transparent graphene microelectrode technology offers a practical pathway to investigate neuronal activity over multiple spatial scales extending from single neurons to large neuronal populations.

Cell type specificity of neurovascular coupling in cerebral cortex.

Identification of the cellular players and molecular messengers that communicate neuronal activity to the vasculature driving cerebral hemodynamics is important for (1) the basic understanding of cerebrovascular regulation and (2) interpretation of functional Magnetic Resonance Imaging (fMRI) signals. Using a combination of optogenetic stimulation and 2-photon imaging in mice, we demonstrate that selective activation of cortical excitation and inhibition elicits distinct vascular responses and identify the vasoconstrictive mechanism as Neuropeptide Y (NPY) acting on Y1 receptors. The latter implies that task-related negative Blood Oxygenation Level Dependent (BOLD) fMRI signals in the cerebral cortex under normal physiological conditions may be mainly driven by the NPY-positive inhibitory neurons. Further, the NPY-Y1 pathway may offer a potential therapeutic target in cerebrovascular disease.

In vivo stimulus-induced vasodilation occurs without IP3 receptor activation and may precede astrocytic calcium increase.

Calcium-dependent release of vasoactive gliotransmitters is widely assumed to trigger vasodilation associated with rapid increases in neuronal activity. Inconsistent with this hypothesis, intact stimulus-induced vasodilation was observed in inositol 1,4,5-triphosphate (IP3) type-2 receptor (R2) knock-out (KO) mice, in which the primary mechanism of astrocytic calcium increase-the release of calcium from intracellular stores following activation of an IP3-dependent pathway-is lacking. Further, our results in wild-type (WT) mice indicate that in vivo onset of astrocytic calcium increase in response to sensory stimulus could be considerably delayed relative to the simultaneously measured onset of arteriolar dilation. Delayed calcium increases in WT mice were observed in both astrocytic cell bodies and perivascular endfeet. Thus, astrocytes may not play a role in the initiation of blood flow response, at least not via calcium-dependent mechanisms. Moreover, an increase in astrocytic intracellular calcium was not required for normal vasodilation in the IP3R2-KO animals.