Baseline oxygen consumption decreases with cortical depth

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 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.

Fig 1. Measurements of tissue partial pressure of O₂ (pO₂) across cortical layers of awake mice using 2-photon phosphorescence lifetime microscopy (2PLM).

(A) Imaging setup for 2PLM in awake, head-restrained mice. Ti:sapph (80 MHz)—femtosecond pulsed laser tuned to 950 nm; EOM—electro-optic modulator; D900LP and D660LP—long-pass dichroic mirrors with a cutoff at 900 and 660 nm, respectively; GaAsP—photomultiplier tubes; 900SP and 945SP—short-pass optical filters with a cutoff at 900 and 945 nm, respectively; Em525/70, Em617/70, Em736/128—bandpass emission filters. The inset in the lower right corner illustrates a phosphorescence decay; data (black) and fit (red) are overlaid. (B) Schematics of the chronic cranial window with a silicone port for intracortical injection of Oxyphor 2P. (C) An image of surface vasculature calculated as a maximum intensity projection (MIP) of a 2-photon image stack 0–300 μm in depth using a 5× objective. Individual images were acquired every 10 μm. Fluorescence is due to intravascular fluorescein isothiocyanate (FITC). Scale bar = 500 μm. (D) An example set of images tracking a diving arteriole throughout the top 400 μm of cortex (red arrowheads). Fluorescence is due to intravascular FITC. Scale bar = 50 μm. (E) A measurement plane 200 μm deep including intravascular FITC (left) and sulforhodamine 101 (SR101)–labeled astrocytes (right) for the same arteriole as in (D). Scale bar = 50 μm. (F) A square measurement grid of 20 × 20 points obtained from the imaging plane shown in (E). Left: photon counts are overlaid on the image of phosphorescence. Right: calculated pO₂ values superimposed on a vascular FITC image. Scale bar = 50 μm. (G) Histogram of pO₂ values corresponding to (F). (H) pO₂ histograms across cortical layers for all 11 arterioles from 8 animals. Each panel shows overlaid histograms from each measurement plane (gray) and superimposed average (red). (I) Quantification of the top and bottom 35% of the pO₂ distributions from (H). Error bars show standard error calculated using a mixed effects model implemented in R (p > 0.1 for the top 35% and p < 0.05 for the bottom 35%). Numerical values for (G–I) are provided in S1 Data (sheets 1G–1I).

Fig 2. ODACITI model and validation with synthetic data.

(A) Schematic illustration of the model assumptions. The center arteriole is the only source of O₂ for the pink periarteriolar region void of capillaries extending out to radius r = R_t. For r > R_t, delivery and consumption are balanced. (B) Comparison of the functional form of the Krogh–Erlang (black) and ODACITI model (green). The Krogh–Erlang model but not ODACITI forces an increase in partial pressure of O₂ (pO₂) beyond R_t. (C) Application of ODACITI to synthetic data with added Gaussian noise (σ = 2). In these data, pO₂ = P_ves for r < R_ves and pO₂ = pO₂(R_t) for r > R_t. For R_ves < r < R_t, we solved for pO₂ using the Krogh–Erlang equation. Three cases with cerebral metabolic rate of O₂ (CMRO₂) of 1, 2, and 3 μmol cm⁻³ min⁻¹ (color-coded) are superimposed. For each case, the ODACITI fit (solid line) is overlaid on the data points. (D) Simulated pO₂ data generated by solving the Poisson equation in 2D for a given geometry of vascular O₂ sources, including 1 highly oxygenated vessel (on the right) and given CMRO₂ = 2 μmol cm⁻³ min⁻¹. (E) The pO₂ gradient as a function of the distance from the center arteriole. The green line shows ODACITI fit. (F) As in (E) after adding Gaussian noise (σ = 2 SD). Numerical values for (B, C, E, and F) are provided in S1 Data (sheets 2B, 2C, 2E, and 2F).

Fig 3. Cerebral metabolic rate of O₂ (CMRO₂) estimation across cortical layers.

(A) An imaging plane 100 μm below the surface; a grid of partial pressure of O₂ (pO₂) points is overlaid on the vascular image. pO₂ values were interpolated along the radial yellow lines. The yellow shaded area extends along each direction until the first derivative becomes 0. The segmented region of interest (ROI) used for extraction of the radial pO₂ profile is shown on the right. This ROI also includes values within the low tail of the pO₂ distribution. (B) The radial pO₂ profile extracted from (A). The ODACITI fit is overlaid on the data. The fitted CMRO₂ value is 1.5 μmol cm⁻³ min⁻¹. (C) Estimated CMRO₂ for the entire dataset of 51 planes along 11 diving arterioles in 8 subjects. Measurements along the same arteriole are connected with a line. Subjects are color-coded. (D) Quantification of CMRO₂ across layers using the data in (C). Error bars show standard error calculated using a linear mixed effects model implemented in R. (E) Experimentally measured tissue pO₂ (mean ± SEM, binned at 5 μm) across cortical layers (color-coded) as a function of distance from the arteriole. Each profile represents a grand average across subjects. (F) ODACITI fit for the data shown in (E) (binned at 2 μm). Numerical values for (B–F) are provided in S1 Data (sheets 3B–3F).