An investigation of the relationship between BOLD and perfusion signal changes during epileptic generalised spike wave activity

In pathological conditions interpretation of functional magnetic resonance imaging (fMRI) results can be difficult. This is due to a reliance on the assumed coupling between neuronal activity and changes in cerebral blood flow (CBF) and oxygenation. We wanted to investigate the coupling between blood oxygen level dependant contrast (BOLD) and CBF time courses in epilepsy patients with generalised spike wave activity (GSW) to better understand the underlying mechanisms behind the EEG-fMRI signal changes observed, especially in regions of negative BOLD response (NBR). Four patients with frequent GSW were scanned with simultaneous electroencephalographic (EEG)-fMRI with BOLD and arterial spin labeling (ASL) sequences. We examined the relationship between simultaneous CBF and BOLD measurements by looking at the correlation of the two signals in terms of percentage signal change on a voxel-by-voxel basis. This method is not reliant on coincident activation. BOLD and CBF were positively correlated in patients with epilepsy during background EEG activity and GSW. The subject average value of the Delta CBF/Delta BOLD slope lay between +19 and +36 and also showed spatial variation which could indicate areas with altered vascular response. There was not a significant difference between Delta CBF/Delta BOLD during GSW, suggesting that neurovascular coupling to BOLD signal is generally maintained between states and, in particular, within areas of NBR.     

Identification of activated regions during a language task

Functional magnetic resonance imaging (fMRI) techniques are based on the assumption that changes in neural activity are accompanied by modulation in the blood-oxygenation-level-dependent (BOLD) signal. In addition to conventional increases in BOLD signals, sustained negative BOLD signal changes are occasionally observed in many fMRI experiments, which show regions of cortex that seem to respond in antiphase with primary stimulus. The existence of this so-called negative BOLD response (NBR) has been observed and investigated in many functional studies. Several theoretical mechanisms have been proposed to account for it, but its origin has never been fully explained. In this study, the variability of fMRI activation, including the sources of the negative BOLD signal, during phonological and semantic language tasks, was investigated in six right-handed healthy subjects. We found significant activations in the brain regions, mainly in the left hemisphere, involved in the language stimuli [prominent in the inferior frontal gyrus, approximately Brodmann Areas (BA)7, BA44, BA45 and BA47, and in the precuneus]. Moreover, we observed activations in motor regions [precentral gyrus and supplementary motor area (SMA)], a result that suggests a specific role of these areas (particularly the SMA) in language processing. Functional analysis have also shown that certain brain regions, including the posterior cingulate cortex and the anterior cingulate cortex, have consistently greater activity during resting states compared to states of performing cognitive tasks. In our study, we observed diffuse NBR at the cortical level and a stronger negative response in correspondence to the main sinuses. These phenomena seem to be unrelated to a specific neural activity, appearing to be expressions of a mechanical variation in hemodynamics. We discussed about the importance of these responses that are anticorrelated with the stimulus. Our data suggest that particular care must be considered in the interpretation of fMRI findings, especially in the case of presurgical studies.     

Non-neural BOLD variability in block and event-related paradigms

Block and event-related stimulus designs are typically used in fMRI studies depending on the importance of detection power or estimation efficiency. The extent of vascular contribution to variability in block and event-related fMRI-BOLD response is not known. With scaling, the extent of vascular variability in the fMRI-BOLD response during block and event-related design tasks was investigated. Blood oxygen level-dependent (BOLD) contrast data from healthy volunteers performing a block design motor task and an event-related memory task requiring performance of a motor response were analyzed from the regions of interest (ROIs) surrounding the primary and supplementary motor cortices. Average BOLD signal change was significantly larger during the block design compared to the event-related design. In each subject, BOLD signal change across voxels in the ROIs had higher variation during the block design task compared to the event-related design task. Scaling using the resting state fluctuation of amplitude (RSFA) and breath-hold (BH), which minimizes BOLD variation due to vascular origins, reduced the within-subject BOLD variability in every subject during both tasks but significantly reduced BOLD variability across subjects only during the block design task. The strong non-neural source of intra- and intersubject variability of BOLD response during the block design compared to event-related task indicates that study designs optimizing for statistical power through enhancement of the BOLD contrast (for, e.g., block design) can be affected by enhancement of non-neural sources of BOLD variability.     

Origins of intersubject variability of blood oxygenation level dependent and arterial spin labeling fMRI: implications for quantification of brain activity

Accurate localization of brain activity using blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) has been challenged because of the large BOLD signal within distal veins. Arterial spin labeling (ASL) techniques offer greater sensitivity to the microvasculature but possess low temporal resolution and limited brain coverage. In this study, we show that the physiological origins of BOLD and ASL depend on whether percent change or statistical significance is being considered. For BOLD and ASL fMRI data collected during a simple unilateral hand movement task, we found that in the area of the contralateral motor cortex the centre of gravity (CoG) of the intersubject coefficient of variation (CV) of BOLD fMRI was near the brain surface for percent change in signal, whereas the CoG of the intersubject CV for Z-score was in close proximity of sites of brain activity for both BOLD and ASL. These findings suggest that intersubject variability of BOLD percent change is vascular in origin, whereas the origin of inter-subject variability of Z-score is neuronal for both BOLD and ASL. For longer duration tasks (12 s or greater), however, there was a significant correlation between BOLD and ASL percent change, which was not evident for short duration tasks (6 s). These findings suggest that analyses directly comparing percent change in BOLD signal between pre-defined regions of interest using short duration stimuli, as for example in event-related designs, may be heavily weighted by large-vessel responses rather than neuronal responses.     

Visual cortex reactivity in sedated children examined with perfusion MRI (FAIR)

Sleeping and sedated children can respond to visual stimulation with a decrease in blood oxygenation level dependent (BOLD) functional MRI signal response. The contribution of metabolic and hemodynamic parameters to this inverse signal response is incompletely understood. It has been hypothesized that it is caused by a relatively greater increase of oxygen consumption compared to rCBF (regional cerebral blood flow) increase. We studied the rCBF changes during visual stimulation in four sedated children, aged 4-71 months, and four alert adults, with an arterial water spin labeling technique (FAIR) and BOLD fMRI in a 1.5T MR scanner. In the children, FAIR signal decreased by a mean of 0.96% (range 0.77-1.05) of the baseline periods of the non-selective images, while BOLD signal decreased by 2.03% (range 1.99-2.93). In the adults, FAIR and BOLD signal increased by 0.88% (range 0.8-0.99) and 2.63% (range 1.99-2.93), respectively. Thus, in the children, an rCBF increase could not be detected by perfusion MRI, but indications of a FAIR signal decrease were found. An rCBF decrease in the primary visual cortex during stimulation has not been reported previously, but it is a possible explanation for the negative BOLD response. Future studies will have to address if this response pattern is a consequence of age or sleep/sedation.     

How much luxury is there in 'luxury perfusion'? An analysis of the BOLD response in the visual areas V1 and V2

We re-analyzed the functional magnetic resonance imaging data from a study involving awake, adult, human volunteers in order to examine the influence of vascular density on the blood oxygenation level-dependent (BOLD) response. We employed a flashed and reversing stimulus paradigm where the latter stimulated twice the number of receptive fields and with it doubled the neuronal metabolic load (CMRO2) compared to the former stimulus. The blood flow increase to these stimuli was identical, so that differences in the BOLD response are due to differences in the oxygen extraction fraction. By comparing the BOLD response in human striate cortex (V1) and its neighbor, extra-striate area V2 to the two stimuli, we were able to determine the influence of the higher vascular density of striate cortex on the BOLD response. In striate cortex, the extent of activation, as measured by the number of activated voxels, was larger for the flashed than for the reversing stimulus. In extra-striate area V2, no such difference in the extent of activation was noted. Gauging the local concentration of HbR using deltaR2*, we found it to be significantly lower for the flashed than for the reversing checkerboard. We estimated the HbR concentration in extra-striate area V2 to be double that of striate cortex independent of the stimulus presented. A frequency distribution of the deltaR2* values for the flashed and reversing checkerboard revealed a shift consistent with an increase in the HbR concentration between areas V1 and V2. The metabolically most demanding stimulus, the reversing checkerboard was associated with the highest HbR concentration and with the largest number of voxels with a negative BOLD response.     

What the little differences between men and women tells us about the BOLD response

We analyzed the functional MRI signal of 15 men and 15 women. All had been presented with a flashed and a reversing, radial checkerboard stimulus. We investigated both positive and negative blood oxygenation level-dependent (BOLD) responses. The extent of activation and the change in the neuronal activity were examined. The former, by counting the number of activated voxels, the latter by using deltaR2* as an indicator of the change in the local deoxyhemoglobin (HbR) concentration. We examined both the positive and the negative BOLD response. Positive BOLD response: The flashed checkerboard gave rise to a larger number of activated voxels than for the reversing checkerboard. The mean number of activated pixels did not differ between men and women. The peak deltaR2* was significantly larger to the flashed than the reversing checkerboard, but did not reveal a gender-related difference. We noted an attenuation of the BOLD signal amplitude with time. This attenuation was larger in women than in men. Negative BOLD response: The attenuation was also larger for the flashed than the reversing stimulus and more pronounced in the chromatic contrast compared to the luminance contrast stimulus. The extent of activation was larger for the flashed than the reversing checkerboard, but did not differ between the sexes. The deltaR2* for the chromatic contrast checkerboard was larger in men than in women. No other significant differences were found. We conclude that the difference in the extent of activation between men and women is the result of our ability to detect activated pixels using statistical methods and not the result of a difference in the processing between the sexes.     

Decoding neural events from fMRI BOLD signal: A comparison of existing approaches and development of a new algorithm

Neuroimaging methodology predominantly relies on the blood oxygenation level dependent (BOLD) signal. While the BOLD signal is a valid measure of neuronal activity, variances in fluctuations of the BOLD signal are not only due to fluctuations in neural activity. Thus, a remaining problem in neuroimaging analyses is developing methods that ensure specific inferences about neural activity that are not confounded by unrelated sources of noise in the BOLD signal. Here, we develop and test a new algorithm for performing semiblind (i.e., no knowledge of stimulus timings) deconvolution of the BOLD signal that treats the neural event as an observable, but intermediate, probabilistic representation of the system's state. We test and compare this new algorithm against three other recent deconvolution algorithms under varied levels of autocorrelated and Gaussian noise, hemodynamic response function (HRF) misspecification and observation sampling rate. Further, we compare the algorithms' performance using two models to simulate BOLD data: a convolution of neural events with a known (or misspecified) HRF versus a biophysically accurate balloon model of hemodynamics. We also examine the algorithms' performance on real task data. The results demonstrated good performance of all algorithms, though the new algorithm generally outperformed the others (3.0% improvement) under simulated resting-state experimental conditions exhibiting multiple, realistic confounding factors (as well as 10.3% improvement on a real Stroop task). The simulations also demonstrate that the greatest negative influence on deconvolution accuracy is observation sampling rate. Practical and theoretical implications of these results for improving inferences about neural activity from fMRI BOLD signal are discussed.     

Coupling of simultaneously acquired electrophysiological and haemodynamic responses during visual stimulation

We investigate the relationship between the temporal variation in the magnitude of occipital visual evoked potentials (VEPs) and of haemodynamic measures of brain activity obtained using both blood oxygenation level dependent (BOLD) and perfusion sensitive (ASL) functional magnetic resonance imaging (fMRI). Volunteers underwent a continuous BOLD fMRI scan and/or a continuous perfusion-sensitive (gradient and spin echo readout) ASL scan, during which 30 second blocks of contrast reversing visual stimuli (at 4 Hz) were interleaved with 30 second blocks of rest (visual fixation). Electroencephalography (EEG) and fMRI were simultaneously recorded and following EEG artefact cleaning, VEPs were averaged across the whole stimulation block (120 reversals, VEP₁₂₀) and at a finer timescale (15 reversals, VEP₁₅). Both BOLD and ASL time-series were linearly modelled to establish: (1) the mean response to visual stimulation, (2) transient responses at the start and end of each stimulation block, (3) the linear decrease between blocks, (4) the nonlinear between-block variation (covariation with VEP₁₂₀), (5) the linear decrease within block and (6) the nonlinear variation within block (covariation with VEP₁₅).     

BOLD signal and vessel dynamics: a hierarchical cluster analysis

The aim of the present study was to analyze blood oxygenation level-dependent (BOLD) signal variation during an apnea-based task in order to assess the capability of a functional magnetic resonance imaging (fMRI) procedure to estimate cerebral vascular dynamic effects. We measured BOLD contrast by hierarchical cluster analysis in healthy subjects undergoing an fMRI experiment, in which the task paradigm was one phase of inspirational apnea (IA). By processing the time courses of the fMRI experiment, analysis was performed only on a subclass of all the possible signal patterns; basically, root mean square and absolute variation differences have been calculated. Considering the baseline value obtained by computing the mean value of the initial rest period as reference, particular voxels showed relative important variations during the IA task and during the recovery phase following the IA. We focused our interest on the signal response of voxels that would correspond mainly to white and gray matter regions and that also may be affected by the proximity of large venous vessels. The results are presented as maps of space-temporal distribution of time series variations with two levels of hierarchical clustering among voxels with low to high initial response. Furthermore, we have presented a clustering of the signal response delay, conducting to a partition and identification of specified brain sites.     

Neural activity-induced modulation of BOLD poststimulus undershoot independent of the positive signal

Despite intense research on the blood oxygenation level-dependent (BOLD) signal underlying functional magnetic resonance imaging, our understanding of its physiological basis is far from complete. In this study, it was investigated whether the so-called poststimulus BOLD signal undershoot is solely a passive vascular effect or actively induced by neural responses. Prolonged static and flickering black-white checkerboard stimulation with isoluminant grey screen as baseline condition were employed on eight human subjects. Within the same region of interest, the positive BOLD time courses for static and flickering stimuli were identical over the entire stimulus duration. In contrast, the static stimuli exhibited no poststimulus BOLD signal undershoot, whereas the flickering stimuli caused a strong BOLD poststimulus undershoot. To ease the interpretation, we performed an additional study measuring both BOLD signal and cerebral blood flow (CBF) using arterial spin labeling. Also for CBF, a difference in the poststimulus period was found for the two stimuli. Thus, a passive blood volume effect as the only contributor to the poststimulus undershoot comes short in explaining the BOLD poststimulus undershoot phenomenon for this particular experiment. Rather, an additional active neuronal activation or deactivation can strongly modulate the BOLD poststimulus behavior. In summary, the poststimulus time course of BOLD signal could potentially be used to differentiate neuronal activity patterns that are otherwise indistinguishable using the positive evoked response.     

Transient and sustained BOLD responses to sustained visual stimulation

Examining the transients of the blood-oxygenation-level-dependent (BOLD) signal using functional magnetic resonance imaging is a tool to probe basic brain physiology. In addition to the so-called initial dip and poststimulus undershoot of the BOLD signal, occasionally, overshoot at the beginning and at the end of stimulation and stimulus onset and offset ('phasic') responses are observed. Hemifield visual stimulation was used in human subjects to study the latter transients. As expected, sustained ('tonic') stimulus-correlated contralateral activation in the visual cortex and LGN was observed. Interestingly, bilateral phasic responses were observed, which only partly overlapped with the tonic network and which would have been missed using a standard analysis. A biomechanical model of the BOLD signal ('balloon model') indicated that, in addition to phasic neuronal activity, vascular uncoupling can also give rise to phasic BOLD signals. Thus, additional physiological information (i.e., cerebral blood flow) and examination of spatial distribution of the activity might help to assess the BOLD signal transients correctly. In the current study, although vascular uncoupled responses cannot be ruled out as an explanation of the observed phasic BOLD network, the spatial distribution argues that sustained hemifield visual stimulation evokes both bilateral phasic and contralateral sustained neuronal responses. As a consequence, in rapid event-related experimental designs, both the phasic and tonic networks cannot be separated, possibly confounding the interpretation of BOLD signal data. Furthermore, a combination of phasic and tonic responses in the same region of interest might also mimic a BOLD response typically observed in adaptation experiments.     

Assessment of cerebrovascular reactivity with functional magnetic resonance imaging: comparison of CO(2) and breath holding

Cerebral blood flow (CBF) and oxygenation changes following both a simple breath holding test (BHT) and a CO(2) challenge can be detected with functional magnetic resonance imaging techniques. The BHT has the advantage of not requiring a source of CO(2) and acetazolamide and therefore it can easily be performed during a routine MR examination. In this study we compared global hemodynamic changes induced by breath holding and CO(2) inhalation with blood oxygenation level dependent (BOLD) and CBF sensitized fMRI techniques. During each vascular challenge BOLD and CBF signals were determined simultaneously with a combined BOLD and flow-sensitive alternating inversion recovery (FAIR) pulse sequence. There was a good correlation between the global BOLD signal intensity changes during breath holding and CO(2) inhalation supporting the notion that the BHT is equivalent to CO(2) inhalation in evaluating the hemodynamic reserve capacity with BOLD fMRI. In contrast, there was no correlation between relative CBF changes during both vascular challenges, which was probably due to the reduced temporal resolution of the combined BOLD and FAIR pulse sequence.     

Coupling of neural activity and fMRI-BOLD in the motion area MT

The fMRI-BOLD contrast is widely used to study the neural basis of sensory perception and cognition. This signal, however, reflects neural activity only indirectly, and the detailed mechanisms of neurovascular coupling and the neurophysiological correlates of the BOLD signal remain debated. Here we investigate the coupling of BOLD and electrophysiological signals in the motion area MT of the macaque monkey by simultaneously recording both signals. Our results demonstrate that a prominent neuronal response property of area MT, so-called motion opponency, can be used to induce dissociations of BOLD and neuronal firing. During the presentation of a stimulus optimally driving the local neurons, both field potentials [local field potentials (LFPs)] and spiking activity [multi-unit activity (MUA)] correlated with the BOLD signal. When introducing the motion opponency stimulus, however, correlations of MUA with BOLD were much reduced, and LFPs were a much better predictor of the BOLD signal than MUA. In addition, for a subset of recording sites we found positive BOLD and LFP responses in the presence of decreases in MUA, regardless of the stimulus used. Together, these results demonstrate that correlations between BOLD and MUA are dependent on the particular site and stimulus paradigm, and foster the notion that the fMRI-BOLD signal reflects local dendrosomatic processing and synaptic activity rather than principal neuron spiking responses.     

Testing methodologies for the nonlinear analysis of causal relationships in neurovascular coupling

We investigated the use and implementation of a nonlinear methodology for establishing which changes in neurophysiological signals cause changes in the blood oxygenation level-dependent (BOLD) contrast measured in functional magnetic resonance imaging. Unlike previous analytical approaches, which used linear correlation to establish covariations between neural activity and BOLD, we propose a directed information-theoretic measure, the transfer entropy, which can elucidate even highly nonlinear causal relationships between neural activity and BOLD signal. In this study we investigated the practicality of such an analysis given the limited data samples that can be collected experimentally due to the low temporal resolution of BOLD signals. We implemented several algorithms for the estimation of transfer entropy and we tested their effectiveness using simulated local field potentials (LFPs) and BOLD data constructed to match the main statistical properties of real LFP and BOLD signals measured simultaneously in monkey primary visual cortex. We found that using the advanced methods of entropy estimation implemented and described here, a transfer entropy analysis of neurovascular coupling based on experimentally attainable data sets is feasible.     

Dynamics and nonlinearities of the BOLD response at very short stimulus durations

In designing a functional imaging experiment or analyzing data, it is typically assumed that task duration and hemodynamic response are linearly related to each other. However, numerous human and animal studies have previously reported a deviation from linearity for short stimulus durations (<4 s). Here, we investigated nonlinearities of blood-oxygenation-level-dependent (BOLD) signals following visual stimulation of 5 to 1000 ms duration at two different luminance levels in human subjects. It was found that (a) a BOLD response to stimulus durations as short as 5 ms can be reliably detected; this stimulus duration is shorter than employed in any previous study investigating BOLD signal time courses; (b) the responses are more nonlinear than in any other previous study: the BOLD response to 1000 ms stimulation is only twice as large as the BOLD response to 5 ms stimulation although 200 times more photons were projected onto the retina; (c) the degree of nonlinearity depends on stimulus intensity; that is, nonlinearities have to be characterized not only by stimulus duration but also by stimulus features like luminance. These findings are especially of most practical importance in rapid event-related functional magnetic resonance imaging (fMRI) experimental designs. In addition, an 'initial dip' response--thought to be generated by a rapid increase in cerebral metabolic rate of oxygen metabolism (CMRO2) relative to cerebral blood flow--was observed and shown to colocalize well with the positive BOLD response. Highly intense stimulation, better tolerated by human subjects for short stimulus durations, causes early CMRO2 increase, and thus, the experimental design utilized in this study is better for detecting the initial dip than standard fMRI designs. These results and those from other groups suggest that short stimulation combined with appropriate experimental designs allows neuronal events and interactions to be examined by BOLD signal analysis, despite its slow evolution.     

A nonlinear BOLD model accounting for refractory effect by applying the longitudinal relaxation in NMR to the linear BOLD model

A mathematical model to regress the nonlinear blood oxygen level-dependent (BOLD) fMRI signal has been developed by incorporating the refractory effect into the linear BOLD model of the biphasic gamma variate function. The refractory effect was modeled as a relaxation of two separate BOLD capacities corresponding to the biphasic components of the BOLD signal in analogy with longitudinal relaxation of magnetization in NMR. When tested with the published fMRI data of finger tapping, the nonlinear BOLD model with the refractory effect reproduced the nonlinear BOLD effects such as reduced poststimulus undershoot and saddle pattern in a prolonged stimulation as well as the reduced BOLD signal for repetitive stimulation.     

Spontaneous low-frequency blood oxygenation level-dependent fluctuations and functional connectivity analysis of the 'resting' brain

Functional magnetic resonance imaging techniques using the blood oxygenation level-dependent (BOLD) contrast are widely used to map human brain function by relating local hemodynamic responses to neuronal stimuli compared to control conditions. There is increasing interest in spontaneous cerebral BOLD fluctuations that are prominent in the low-frequency range (<0.1 Hz) and show intriguing spatio-temporal correlations in functional networks. The nature of these signal fluctuations remains unclear, but there is accumulating evidence for a neural basis opening exciting new avenues to study human brain function and its connectivity at rest. Moreover, an increasing number of patient studies report disease-dependent variation in the amplitude and spatial coherence of low-frequency BOLD fluctuations (LFBF) that may afford greater diagnostic sensitivity and easier clinical applicability than standard fMRI. The main disadvantage of this emerging tool relates to physiological (respiratory, cardiac and vasomotion) and motion confounds that are challenging to disentangle requiring thorough preprocessing. Technical aspects of functional connectivity fMRI analysis and the neuroscientific potential of spontaneous LFBF in the default mode and other resting-state networks have been recently reviewed. This review will give an update on the current knowledge of the nature of LFBF, their relation to physiological confounds and potential for clinical diagnostic and pharmacological studies.     

Mapping of cerebrovascular reactivity using BOLD magnetic resonance imaging

Blood oxygen level-dependent (BOLD) contrast MRI is a simple non-invasive method of estimating "perfusion," and combined with a vasodilatory stimulus, may allow estimation of cerebral vascular reserve. We compared BOLD carbon dioxide (CO2) reactivity in the middle cerebral artery (MCA) perfusion territory to MCA flow velocity reactivity determined using transcranial Doppler ultrasound (TCD) in 16 patients with unilateral carotid artery stenosis or occlusion. Both BOLD and TCD reactivities were calculated from measurements acquired when the subjects were breathing air, and again when breathing a 6% CO2/air mixture, and were normalized by dividing by the difference in end tidal (ET) CO2. There was a significant correlation between interhemispheric MCA reactivity difference (contralateral-ipsilateral to the stenosis or occlusion) determined by BOLD MRI and TCD (r = 0.75, p < 0.001). In contrast, treating each hemisphere individually, there was no correlation between the absolute BOLD and TCD MCA CO2 reactivities (r = 0.08, p = 0.670). This appeared to be due to a variable BOLD signal change in the non-stenosed hemisphere between subjects, with little change in the normal hemisphere of a few subjects. In one patient, focal regions of reduced reactivity were seen in non-infarcted regions of the stenosed hemisphere, in the borderzones between arterial territories. BOLD reactivity maps provide information on the whole MCA territory reactivity, and may identify small regions of impaired reactivity which are not detected using TCD. However, BOLD reactivity maps only appear to provide semi-quantitative rather than quantitative data.     

The effects of respiratory CO2 fluctuations in the resting-state BOLD signal differ between eyes open and eyes closed

Resting fluctuations in arterial CO₂ (a cerebral vasodilator) are believed to be an important source of low-frequency blood oxygenation level dependent (BOLD) signal fluctuations. In this study we focus on the two commonly used resting-states in functional magnetic resonance imaging experiments, eyes open and eyes closed, and quantify the degree to which measured spontaneous fluctuations in the partial pressure of end-tidal CO₂ (Pet_co2) relate to BOLD signal time series. A significantly longer latency of BOLD signal changes following Pet_co2 fluctuations was found in the eyes closed condition compared to with eyes open, which may reveal different intrinsic vascular response delays in CO₂ reactivity or an alteration in the net BOLD signal arising from Pet_co2 fluctuations and altered neural activity with eyes closed. By allowing a spatially varying time delay for the compensation of this temporal difference, a more spatially consistent CO₂ correlation map can be obtained. Finally, Granger-causality analysis demonstrated a “causal” relationship between Pet_co2 and BOLD. The identified dominant Pet_co2→BOLD directional coupling supports the notion that Pet_co2 fluctuations are indeed a cause of resting BOLD variance in the majority of subjects.