Low-frequency calcium oscillations accompany deoxyhemoglobin oscillations in rat somatosensory cortex

Abstract

Spontaneous low-frequency oscillations (LFOs) of blood-oxygen-level-dependent (BOLD) signals are used to map brain functional connectivity with functional MRI, but their source is not well understood. Here we used optical imaging to assess whether LFOs from vascular signals covary with oscillatory intracellular calcium (Ca(2+)i) and with local field potentials in the rat's somatosensory cortex. We observed that the frequency of Ca(2+)i oscillations in tissue (∼0.07 Hz) was similar to the LFOs of deoxyhemoglobin (HbR) and oxyhemoglobin (HbO2) in both large blood vessels and capillaries. The HbR and HbO2 fluctuations within tissue correlated with Ca(2+)i oscillations with a lag time of ∼5-6 s. The Ca(2+)i and hemoglobin oscillations were insensitive to hypercapnia. In contrast, cerebral-blood-flow velocity (CBFv) in arteries and veins fluctuated at a higher frequency (∼0.12 Hz) and was sensitive to hypercapnia. However, in parenchymal tissue, CBFv oscillated with peaks at both ∼0.06 Hz and ∼0.12 Hz. Although the higher-frequency CBFv oscillation (∼0.12 Hz) was decreased by hypercapnia, its lower-frequency component (∼0.06 Hz) was not. The sensitivity of the higher CBFV oscillations to hypercapnia, which triggers blood vessel vasodilation, suggests its dependence on vascular effects that are distinct from the LFOs detected in HbR, HbO2, Ca(2+)i, and the lower-frequency tissue CBFv, which were insensitive to hypercapnia. Hemodynamic LFOs correlated both with Ca(2+)i and neuronal firing (local field potentials), indicating that they directly reflect neuronal activity (perhaps also glial). These findings show that HbR fluctuations (basis of BOLD oscillations) are linked to oscillatory cellular activity and detectable throughout the vascular tree (arteries, capillaries, and veins).

Keywords: cerebral hemodynamic; neuroimaging; neuronal calcium; resting-state functional connectivity; spontaneous low-frequency brain oscillations.

Fig. 1.

OFI for imaging of LFOs in CBFv, HbO₂, and HbR from cerebrovascular vessels and cortical tissue. (A) Absorption spectra of HbO₂ and HbR to illustrate the principle of OFI. (B and C) Spectral images at λ₂ and λ₃. (D) LSI at λ₄ to image relative CBFv. (E) Ca²⁺ fluorescence with excitation at λ₁. (F and G) Quantitative 3D ODT of CBFv and separation of AF (red arrows) and VF (blue arrows). (H) Extraction of LFOs in an AF and a VF highlighted in F. (Top) time-lapse ODT images to show CBFv LFOs. (Middle) LFO with DC removed. (Bottom) Time-varying LFO by STFT.

Fig. 2.

LFO signals observed from multichannels of OFI and ODT modalities. Power spectra and spectrograms of cortical artery, vein, and parenchymal tissue of (A) CBFv (observed by ODT, λ = 1,300 nm or LSI, λ₄ = 830 nm). (B) Total hemoglobin (tHb) or cerebral blood volume (CBV) (observed by OFI, λ₂ = 570 nm). (C) Raw HbR by λ₃ = 630 nm. With λ₂ it can be used to separate HbO₂ from HbR. (D) Rhod₂-Ca²⁺_i fluorescence (emitted at 590 nm while excited at λ₁ = 530 nm). Location of the vessels is demonstrated in 3D ODT.

Fig. 3.

Characteristics of arteriolar LFOs in rat cortex. (Left) LFO power spectra of CBFv (A₀), HbO₂ (B₀) and HbR (C₀) and their spectrograms (Insets) under normocapnia. (Center) LFO power spectra of CBFv (A₁), HbO₂ (B₁), and HbR (C₁) and their spectrograms (Insets) under hypercapnia. Dashed curves are traces from selected ROIs (m = 6–8) for each rat. Bold curves are averaged traces from the rat. (D and E) Comparison of mean LFO peak frequency and mean amplitude within its oscillation band between normocapnia and hypercapnia across the animals (n = 10). Asterisks indicate statistical significance (P < 0.05).

Fig. 4.

Characteristics of venular LFOs in rat cortex. (Left) LFO power spectra of CBFv (A₀), HbO₂ (B₀), and HbR (C₀) and their spectrograms (Insets) under normocapnia. (Center) LFO power spectra of CBFv (A₁), HbO₂ (B₁), and HbR (C₁) and their spectrograms (Insets) under hypercapnia. Dashed curves are traces from selected ROIs (m = 6–8) for each rat. Bold curves are averaged traces from the rat. (D and E) Comparison of mean LFO peak frequency and mean amplitude within its oscillation band between normocapnia and hypercapnia averaged across animals (n = 10). Asterisks indicate statistical significance (P < 0.05).

Fig. 5.

Characteristics of tissue LFOs in rat cortex. (Left) LFO power spectra of CBFv (A₀), HbO₂ (B₀), HbR (C₀), and Ca²⁺_i (D₀) and their spectrograms (Insets) under normocapnia. Mid (Center) LFO power spectra of CBFv (A₁), HbO₂ (B₁), HbR (C₁), and Ca²⁺_i (D₁) and their spectrograms (Insets) under hypercapnia. Dashed curves are traces from selected ROIs (m = 6–8) for each rat. Bold curves are averaged traces from the rat. (E and F) Comparison of mean LFO peak frequency and mean amplitude within its oscillation band between normocapnia and hypercapnia averaged across animals (n = 10). Asterisks indicate statistical significance (P < 0.05).

Fig. 6.

Cross-correlation between Ca²⁺_i and HbR/HbO₂ LFOs. (A) Time traces of Ca²⁺_i (black), HbR (blue), and HbO₂ (red) LFOs acquired from a rat. (B and C) A close view of LFOs in A and the normalized traces of B. (D) Cross-correlation between the time traces of Ca²⁺_i-HbR (blue) and Ca²⁺_i–HbO₂ (red). (E) Statistical results (n = 10) of correlation coefficients of Ca²⁺_i-HbR and Ca²⁺_i-HbO₂ LFOs without shifting (Δt = 0) and after shifting (time lag − Δt).

Fig. 7.

(A) Frequency distribution of CBFv LFO mostly oscillated at ∼0.1 Hz. (Inset) LFO profile and its FWHM bandwidth of 0.08–0.15 Hz (red box). (B) Distribution of LFP spontaneous firing frequency, indicative of neuronal firing rate also around 0.1 Hz. (C) Least-squares fitting of CBFv and LFP activity curves, indicating a corresponding linear increase (r = 0.93) in the low frequency range (<0.15 Hz) followed by rapidly decays with different offsets. (D) Correlation of LFP firing rates with CBFv LFOs (r = 0.83), implying that the spontaneous hemodynamic fluctuations are of neuronal origin.