Microscopic Quantification of Oxygen Consumption across Cortical Layers

Abstract

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 O 2 (pO 2 ) 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 O 2 (CMRO 2 ) based on 2-photon pO 2 measurements around diving arterioles and applied this method to estimate baseline CMRO 2 in awake mice across cortical layers. To our surprise, our results revealed a decrease in baseline CMRO 2 from layer I to layer IV . This decrease of CMRO 2 with cortical depth was paralleled by an increase in tissue oxygenation. Higher baseline oxygenation and cytochrome density in layer IV may serve as an O 2 reserve during surges of neuronal activity or certain metabolically active brain states rather than baseline energy needs. Our study provides the first quantification of microscopically resolved CMRO 2 across cortical layers as a step towards informed interpretation and modeling of cortical-layer-specific Blood Oxygen Level Dependent (BOLD) fMRI signals.

Neurophotonic Tools for Microscopic Measurements and Manipulation: Status Report

“…7.2) also enables estimation of cerebral metabolic rate of O 2 by quantifying pO 2 gradients around penetrating cortical arterioles. 574,575 Measurements sensitive to cerebral blood flow, volume and oxygenation are commonly referred to as "hemodynamic." One of the most popular hemodynamic imaging modalities is functional Magnetic Resonance Imaging (fMRI), a macroscopic noninvasive technology commonly used in humans that leverages paramagnetic properties of deoxyhemoglobin to generate signal.…”

“…32). 574,575 This method relies on 2 assumptions. The first assumption is that periarterial tissue gets all its O 2 from the arteriole.…”

“…The first assumption is that periarterial tissue gets all its O 2 from the arteriole. This is justified by the absence of capillaries within the periarteriolar space, 606 the presence of a pO 2 gradient in tissue around diving arterioles, 270,575 and a pO 2 decrease within arterioles from the vessel center to the vessel wall indicating the O 2 leaves through the arteriolar wall. This organization can be approximated by Krogh's model of O 2 diffusion from a cylinder 607 where the diving arteriole serves as a single O 2 source for the periarterial tissue.…”

Spectral fiber photometry derives hemoglobin concentration changes for accurate measurement of fluorescent sensor activity

“…One of the most notable confounders affecting fluorescence measurements is light absorption by hemoglobin (Hb) in blood vessels ( Ma et al., 2016 ; Prahl, 1999 ). Hb absorption artifacts have been studied extensively in wide-field imaging with reflective or backscattered lights ( Ma et al., 2016 ; Valley et al., 2020 ) and recently in two-photon lifetime microscopy (2PLM) measuring the partial pressure of O 2 ( Mächler et al., 2021 ). Hb absorption artifacts should be present in most photometry data because the intervascular distance of capillaries is known to be less than 60 in nearly every location in the brain ( Smith et al., 2019 ) and commonly used photometry targeted volumes are 10 5 –10 6 ( Pisanello et al., 2019 ).…”

Spectral fiber-photometry derives hemoglobin concentration changes for accurate measurement of fluorescent sensor activity

“…One of the most notable confounders affecting fluorescence measurements is light absorption by hemoglobin (Hb) within blood vessels (Ma et al, 2016; Prahl, 1999). The Hb-absorption artifact has been studied and addressed extensively in wide-field imaging with reflective or backscattered lights (Ma et al, 2016; Valley et al, 2020) and recently in two-photon lifetime microscopy (2PLM) measuring the partial pressure of O 2 (Mächler et al, 2021). The Hb-absorption artifact should be present in most photometry data because the intervascular distance of capillaries is known to be < 60 μm in nearly every location in the brain (Smith et al, 2019) and commonly used photometry targeted volumes is 10 5 –10 6 μm 3 (Pisanello et al, 2019).…”