Negative Blood Oxygenation Level-dependent Signal Research Articles

An MRI-compatible, ultra-thin, flexible stimulator array for functional neuroimaging by direct stimulation of the rat brain.

We developed an MRI-compatible, ultra-thin, flexible stimulator array for the rat brain and performed functional MRI (fMRI) acquisition during direct electrical stimulation of the brain. This technique measured brain activity evoked by direct stimulation of the motor and the somatosensory cortex. In order to avoid MR signal loss due to interferences with the main static field and RF field in the MRI system, the stimulator array was made from a non-magnetic gold electrode of 100-nm thickness on a 2-um-thick parylene substrate. By using this stimulator array, MR images without signal loss around conducting electrode pads were acquired, and fMRI acquisition during concurrent electrical stimulation of the cerebral cortex was achieved. Neuronal activity propagated to distant brain areas from the stimulated motor cortex. Positive blood oxygenation level dependent (BOLD) signals were observed with direct stimulation of the motor cortex, while negative BOLD signals were observed with direct stimulation of the somatosensory cortex. Interestingly, the pattern of brain activity evoked by direct stimulation of the somatosensory cortex was different from that evoked by electrical stimulation of the forepaw.

Reliability of negative BOLD in ipsilateral sensorimotor areas during unimanual task activity

Research using functional magnetic resonance imaging has for numerous years now reported the existence of a negative blood oxygenation level dependent (BOLD) response. Based on accumulating evidence, this negative BOLD signal appears to represent an active inhibition of cortical areas in which it is found during task activity. This particularly important with respect to motor function given that it is fairly well-established that, in younger adults, the ipsilateral sensorimotor cortex exhibits negative BOLD during unimanual movements in fMRI. This interhemispheric suppression of cortical activity may have useful implications for our understanding of both basic motor function and rehabilitation of injury or disease. However, to date, we are aware of no study that has tested the reliability of evoked negative BOLD in ipsilateral sensorimotor cortex in individuals across sessions. The current study employs a unimanual finger opposition task previously shown to evoke negative BOLD in ipsilateral sensorimotor cortex across three sessions. Reliability metrics across sessions indicates that both the magnitude and location of ipsilateral sensorimotor negative BOLD response is relatively stable over each of the three sessions. Moreover, the volume of negative BOLD in ipsilateral cortex was highly correlated with volume of positive BOLD activity in the contralateral primary motor cortex. These findings show that the negative BOLD signal can be reliably evoked in unimanual task paradigms, and that the signal dynamic could represent an active suppression of the ipsilateral sensorimotor cortex originating from the contralateral motor areas.

Vascular Action as the Primary Mechanism of Cognitive Effects of Cholinergic, CNS-Acting Drugs, a Rat phMRI BOLD Study

Concordant results of functional magnetic resonance imaging (fMRI) and behavioral tests prove that some non-blood-brain barrier-penetrating drugs produce robust central nervous system (CNS) effects. The anticholinergic scopolamine interferes with learning when tested in rats, which coincides with a negative blood-oxygen-level-dependent (BOLD) change in the prefrontal cortex (PFC) as demonstrated by fMRI. The peripherally acting butylscopolamine also evokes a learning deficit in a water-labyrinth test and provokes a negative BOLD signal in the PFC. Donepezil-a highly CNS-penetrating cholinesterase inhibitor-prevents the negative BOLD and cognitive deficits regardless whether the provoking agent is scopolamine or butylscopolamine. Interestingly, the non-BBB-penetrating cholinesterase inhibitor neostigmine also prevents or substantially inhibits those cognitive and fMRI changes. Intact cerebral blood flow and optimal metabolism are crucial for the normal functioning of neurons and other cells in the brain. Drugs that are not BBB penetrating yet act on the CNS highlight the importance of unimpaired circulation, and point to the cerebral vasculature as a primary target for drug action in diseases where impaired circulation and consequently suboptimal energy metabolism are followed by upstream pathologic events.

Functional MRI reveals frequency-dependent responses during deep brain stimulation at the subthalamic nucleus or internal globus pallidus

Deep brain stimulation (DBS) represents a widely used therapeutic tool for the symptomatic treatment of movement disorders, most commonly Parkinson's disease (PD). High frequency stimulation at both the subthalamic nucleus (STN) and internal globus pallidus (GPi) has been used with great success for the symptomatic treatment of PD, although the therapeutic mechanisms of action remain elusive. To better understand how DBS at these target sites modulates neural circuitry, the present study used functional blood-oxygenation-level-dependent (BOLD) functional magnetic resonance imaging (fMRI) to map global brain responses to DBS at the STN and GPi of the rat. Robust activation centered in the ipsilateral motor cortex was observed during high frequency stimulation at either target site, with peak responses observed at a stimulation frequency of 100Hz. Of note, frequency tuning curves were generated, demonstrating that cortical activation was maximal at clinically-relevant stimulation frequencies. Divergent responses to stimulation were noted in the contralateral hemisphere, with strong cortical and striatal negative BOLD signal during stimulation of the GPi, but not STN. The frequency-dependence of the observed motor cortex activation at both targets suggests a relationship with the therapeutic effects of STN and GPi DBS, with both DBS targets being functionally connected with motor cortex at therapeutic stimulation frequencies.

Effects of age on negative BOLD signal changes in the primary somatosensory cortex

In addition to a contralateral activation of the primary and secondary somatosensory cortices, peripheral sensory stimulation has been shown to elicit responses in the ipsilateral primary somatosensory cortex (SI). In particular, evidence is accumulating that processes of interhemispheric inhibition as depicted by negative blood oxygenation level dependent (BOLD) signal changes are part of somatosensory processes. The aim of the study was to analyze age-related differences in patterns of cerebral activation in the somatosensory system in general and processes of interhemispheric inhibition in particular. For this, a functional magnetic resonance imaging (fMRI) study was performed including 14 younger (mean age 23.3±0.9years) and 13 healthy older participants (mean age 73.2±8.3years). All subjects were scanned during peripheral electrical median nerve stimulation (40Hz) to obtain BOLD responses in the somatosensory system. Moreover, the individual current perception threshold (CPT) as a quantitative measure of sensory function was determined in a separate psychophysical testing. Significant increases in BOLD signal across the entire group could be measured within the contralateral SI, in the bilateral secondary somatosensory cortex (SII), the contralateral supplementary motor area and the insula. Negative BOLD signal changes were delineated in ipsilateral SI/MI as well as in the ipsilateral thalamus and basal ganglia. After comparing the two groups, only the cortical deactivation in ipsilateral SI in the early stimulation phase as well as the activation in contralateral SI and SII in the late stimulation block remained as statistically significant differences between the two groups. The psychophysical experiments yielded a significant age-dependent effect of CPT change with less difference in the older group which is in line with the significantly smaller alterations in maximal BOLD signal change in the contra- and ipsilateral SI found between the two groups. Healthy aging seems to be associated with a decrease in intracerebral inhibition as reflected by smaller negative BOLD signal changes during fMRI tasks. This finding could constitute an important link between age-related neurophysiological changes and behavioral alterations in humans.

High-Resolution fMRI Reveals Laminar Differences in Neurovascular Coupling between Positive and Negative BOLD Responses

The six cortical layers have distinct anatomical andphysiological properties, like different energy use and different feedforward and feedback connectivity. It is not known if and how layer-specific neural processes are reflected in the fMRI signal. To address this question we used high-resolution fMRI to measure BOLD, CBV, and CBF responses to stimuli that elicit positive and negative BOLD signals in macaque primary visual cortex. We found that regions with positive BOLD responses had parallel increases in CBV and CBF, whereas areas with negative BOLD responses showed a decrease in CBF but an increase in CBV. For positive BOLD responses, CBF and CBV increased in the center of the cortex, but for negative BOLD responses, CBF decreased superficially while CBV increased in the center. Our findings suggest different mechanisms for neurovascular coupling for BOLD increases and decreases, aswell as laminar differences in neurovascular coupling.

A temporal analysis of the central neural processing of itch

Pruritus, or itch, is the most prevalent symptom of allergic and inflammatory skin disease. Although it is known that itch induces activation of a neural network in the brain, the temporal dynamics of the network as well as the pathophysiology and neurobiology are not well understood. The study aimed to elucidate (i) the temporal dynamics of the itch response identified in earlier studies and (ii) the relationship between central and subjective responses to itch. Using a novel time-series analysis, we performed a double-blind, placebo-controlled, randomized, within-subject functional magnetic resonance imaging (fMRI) study of the cerebral processing of histamine-induced itch in healthy volunteers (n = 16) by tracking the 8-min period following a single skin prick. Histamine-induced itch compared with saline resulted in significant area under the curve blood oxygen level dependent (BOLD) signal changes in the middle/superior temporal gyrus and right inferior frontal gyrus/insula. We observed negative itch-induced BOLD signal changes compared with saline in (i) the pregenual anterior cingulate cortex (ACC)/medial frontal gyrus, (ii) subgenual ACC/ventral striatum, (iii) bilateral temporal pole/parahippocampal gyrus and (iv) several regions within the cerebellum. We noted a trend significance in the left precentral gyrus part of the motor cortex. The BOLD signal change in several of these regions correlated with perception of itch intensity. In contrast to other fMRI studies we observed a multifocal negative signal. An improved understanding of both activated and deactivated brain regions during the itch response may in the long term facilitate development of more effective management strategies.

Is optical imaging spectroscopy a viable measurement technique for the investigation of the negative BOLD phenomenon? A concurrent optical imaging spectroscopy and fMRI study at high field (7T)

Traditionally functional magnetic resonance imaging (fMRI) has been used to map activity in the human brain by measuring increases in the Blood Oxygenation Level Dependent (BOLD) signal. Often accompanying positive BOLD fMRI signal changes are sustained negative signal changes. Previous studies investigating the neurovascular coupling mechanisms of the negative BOLD phenomenon have used concurrent 2D-optical imaging spectroscopy (2D-OIS) and electrophysiology (Boorman et al., 2010). These experiments suggested that the negative BOLD signal in response to whisker stimulation was a result of an increase in deoxy-haemoglobin and reduced multi-unit activity in the deep cortical layers. However, Boorman et al. (2010) did not measure the BOLD and haemodynamic response concurrently and so could not quantitatively compare either the spatial maps or the 2D-OIS and fMRI time series directly. Furthermore their study utilised a homogeneous tissue model in which is predominantly sensitive to haemodynamic changes in more superficial layers.Here we test whether the 2D-OIS technique is appropriate for studies of negative BOLD. We used concurrent fMRI with 2D-OIS techniques for the investigation of the haemodynamics underlying the negative BOLD at 7 Tesla. We investigated whether optical methods could be used to accurately map and measure the negative BOLD phenomenon by using 2D-OIS haemodynamic data to derive predictions from a biophysical model of BOLD signal changes. We showed that despite the deep cortical origin of the negative BOLD response, if an appropriate heterogeneous tissue model is used in the spectroscopic analysis then 2D-OIS can be used to investigate the negative BOLD phenomenon.

Negative BOLD signal changes in ipsilateral primary somatosensory cortex are associated with perfusion decreases and behavioral evidence for functional inhibition

We used functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) to study the negative blood oxygenation level dependent (BOLD) signal and its underlying blood flow changes in healthy human subjects. This was combined with psychophysiological measurements to test that the negative BOLD signal is associated with functional inhibition. Electrical stimulation of the median nerve at 7 Hz evoked robust negative BOLD signals in the primary somatosensory cortex (SI) ipsilateral to stimulation, and positive BOLD signals in contralateral SI. The negative BOLD signal in ipsilateral SI was accompanied by commensurate decreases in relative regional cerebral blood flow (rCBF). Conjunction analysis of the fMRI and PET data revealed a region in the ipsilateral postcentral gyrus showing overlap of negative BOLD signals and relative rCBF decreases. The current perception threshold (CPT) at the ipsilateral finger during concomitant stimulation of the contralateral median nerve increased significantly, suggesting augmented functional inhibition. Since the CPT in the ipsilateral hallux did not significantly change in response to median nerve stimulation, it is more likely that the CPT-increase for the finger is due to functional inhibition (Kastrup et al., 2008) than to changes in selective attention. In conclusion, our data provide evidence that stimulus-induced reductions in relative rCBF may underlie the negative BOLD signal, which in turn may reflect increments in functional inhibition.

Nonlinear hemodynamic responses in human epilepsy: A multimodal analysis with fNIRS-EEG and fMRI-EEG

Functional magnetic resonance imaging (fMRI) combined with electroencephalography (fMRI-EEG) is a neuroimaging technique based on the blood oxygenation level dependent (BOLD) signal which has been shown to be useful in the study of epilepsy for the localization of the epileptogenic focus. Functional near-infrared spectroscopy (fNIRS) combined with EEG (fNIRS-EEG) is another imaging technique based on the measurement of oxygenated and deoxygenated hemoglobin with complementary clinical potential in epilepsy, for continuous patient monitoring, language lateralization, and focus localization.In this work fMRI-EEG and fNIRS-EEG are used to quantify nonlinear hemodynamic responses in three cases of human refractory focal epilepsy, by using the Volterra kernel expansion up to second order. Prior to analyzing real data, extensive simulations are carried out to show that nonlinearities are estimable. The Volterra methodology is then applied to multimodal data recorded from 3 epileptic patients selected for their frequent spiking activity. Care is taken to account for variability of hemodynamic responses due to other causes than Volterra nonlinearities. Statistically significant nonlinearities are observed for all patients and all modalities. Good concordance between fNIRS and fMRI is found for both the amplitude of the Volterra responses, and, with limitations, in the localization of the epileptic focus and regions of inverted responses (negative BOLD signals). In one patient, Volterra nonlinearities allowed epileptic focus identification with fMRI, while analyses without nonlinearities failed to see it. In simulations when nonlinearities were included, analysis without Volterra nonlinearities performed poorly. These two observations suggest routinely checking for nonlinearities in functional imaging of patients presenting with frequent spikes.

Modeling of the Neurovascular Coupling in Epileptic Discharges

Despite the interest in simultaneous EEG-fMRI studies of epileptic spikes, the link between epileptic discharges and their corresponding hemodynamic responses is poorly understood. In this context, biophysical models are promising tools for investigating the mechanisms underlying observed signals. Here, we apply a metabolic-hemodynamic model to simulated epileptic discharges, in part generated by a neural mass model. We analyze the effect of features specific to epileptic neuronal activity on the blood oxygen level dependent (BOLD) response, focusing on the issues of linearity in neurovascular coupling and on the origin of negative BOLD signals. We found both sub- and supra-linearity in simulated BOLD signals, depending on whether one observes the early or the late part of the BOLD response. The size of these non-linear effects is determined by the spike frequency, as well as by the amplitude of the excitatory activity. Our results additionally indicate a minor deviation from linearity at the neuronal level. According to a phase space analysis, the possibility to obtain a negative BOLD response to an epileptic spike depends on the existence of a long and strong excitatory undershoot. Moreover, we strongly suggest that a combined EEG-fMRI modeling approach should include spatial assumptions. The present study is a step towards an increased understanding of the link between epileptic spikes and their BOLD responses, aiming to improve the interpretation of simultaneous EEG-fMRI recordings in epilepsy.

Negative BOLD-fMRI Signals in Large Cerebral Veins

Reductions in blood oxygenation level dependent (BOLD)-functional magnetic resonance imaging (fMRI) signals below baseline levels have been observed under several conditions as negative activation in task-activation studies or anticorrelation in resting-state experiments. Converging evidence suggests that negative BOLD signals (NBSs) can generally be explained by local reductions in neural activity. Here, we report on NBSs that accompany hemodynamic changes in regions devoid of neural tissue. The NBSs were investigated with high-resolution studies of the visual cortex (VC) at 7 T. Task-activation studies were performed to localize a task-positive area in the VC. During rest, robust negative correlation with the task-positive region was observed in focal regions near the ventricles and dispersed throughout the VC. Both positive and NBSs were dependent on behavioral condition. Comparison with high-resolution structural images showed that negatively correlated regions overlapped with larger pial and ependymal veins near sulcal and ventricular cerebrospinal fluid (CSF). Results from multiecho fMRI showed that NBSs were consistent with increases in local blood volume. These findings confirm theoretical predictions that tie neural activity to blood volume increases, which tend to counteract positive fMRI signal changes associated with increased blood oxygenation. This effect may be more salient in high-resolution studies, in which positive and NBS may be more often spatially distinct.

BOLD signal changes preceding negative responses in EEG‐fMRI in patients with focal epilepsy

In simultaneous electroencephalography (EEG) and functional magnetic resonance imaging (fMRI), increased neuronal activity from epileptiform spikes commonly elicits positive blood oxygenation level-dependent (BOLD) responses. Negative responses are also occasionally seen and have not been explained. Recent studies describe BOLD signal changes before focal EEG spikes. We aimed to systematically study if the undershoot of a preceding positive response might explain the negative BOLD seen in the focus. Eighty-two patients with focal epilepsy who underwent EEG-fMRI at 3T were retrospectively studied. Studies with a focal negative BOLD response in the region of the spike field were reanalyzed using models with hemodynamic response functions (HRFs) peaking from -9 to +9 s around the spike. Eight patients met the inclusion criteria, showing negative BOLD responses in the spike field on standard analysis. None had positive BOLD responses immediately adjacent to the areas of deactivation. Regions of deactivation were found to have congruent preceding positive responses in two cases. These early activations were seen at the combined maps of -5 to -9 s. This study indicates that in a small proportion of patients with focal epilepsy in whom the standard analysis reveals focal negative responses, an earlier positive BOLD response is probably the cause. The origin of negative BOLD signal changes in the focus as a result of an epileptic event remains, however, unexplained in most of the patients in whom it occurs.

The Effect of Opioid Receptor Blockade on the Neural Processing of Thermal Stimuli

The endogenous opioid system represents one of the principal systems in the modulation of pain. This has been demonstrated in studies of placebo analgesia and stress-induced analgesia, where anti-nociceptive activity triggered by pain itself or by cognitive states is blocked by opioid antagonists. The aim of this study was to characterize the effect of opioid receptor blockade on the physiological processing of painful thermal stimulation in the absence of cognitive manipulation. We therefore measured BOLD (blood oxygen level dependent) signal responses and intensity ratings to non-painful and painful thermal stimuli in a double-blind, cross-over design using the opioid receptor antagonist naloxone. On the behavioral level, we observed an increase in intensity ratings under naloxone due mainly to a difference in the non-painful stimuli. On the neural level, painful thermal stimulation was associated with a negative BOLD signal within the pregenual anterior cingulate cortex, and this deactivation was abolished by naloxone.

Negative Blood Oxygen Level Dependence in the Rat:A Model for Investigating the Role of Suppression in Neurovascular Coupling

Modern neuroimaging techniques rely on neurovascular coupling to show regions of increased brain activation. However, little is known of the neurovascular coupling relationships that exist for inhibitory signals. To address this issue directly we developed a preparation to investigate the signal sources of one of these proposed inhibitory neurovascular signals, the negative blood oxygen level-dependent (BOLD) response (NBR), in rat somatosensory cortex. We found a reliable NBR measured in rat somatosensory cortex in response to unilateral electrical whisker stimulation, which was located in deeper cortical layers relative to the positive BOLD response. Separate optical measurements (two-dimensional optical imaging spectroscopy and laser Doppler flowmetry) revealed that the NBR was a result of decreased blood volume and flow and increased levels of deoxyhemoglobin. Neural activity in the NBR region, measured by multichannel electrodes, varied considerably as a function of cortical depth. There was a decrease in neuronal activity in deep cortical laminae. After cessation of whisker stimulation there was a large increase in neural activity above baseline. Both the decrease in neuronal activity and increase above baseline after stimulation cessation correlated well with the simultaneous measurement of blood flow suggesting that the NBR is related to decreases in neural activity in deep cortical layers. Interestingly, the magnitude of the neural decrease was largest in regions showing stimulus-evoked positive BOLD responses. Since a similar type of neural suppression in surround regions was associated with a negative BOLD signal, the increased levels of suppression in positive BOLD regions could importantly moderate the size of the observed BOLD response.

Research priorities in epilepsy for the next decade—A representative view of the European scientific community: Summary of the ILAE Epilepsy Research Workshop, Brussels, 17–18 January 2008

Research priorities in epilepsy for the next decade—A representative view of the European scientific community: Summary of the ILAE Epilepsy Research Workshop, Brussels, 17–18 January 2008

Functional Magnetic Resonance Imaging of the Sensorimotor System in Preterm Infants

Preterm birth at <32 weeks' gestational age has a specific predilection for periventricular white matter injury. Early prediction of concomitant motor sequelae is a fundamental clinical issue. Recently, functional MRI was introduced as a noninvasive method for investigating the functional integrity of the neonatal brain. We aimed at implementing a unilateral passive forearm extension/flexion functional MRI paradigm in a routine clinical MRI setup to allow noninvasive mapping of the sensorimotor system in preterm infants and to relate the functional data to structural and behavioral data. Eight patients (median gestational age: 26.5 weeks; median birth weight: 885 g) were included. The functional MRI was performed at term-equivalent age (median: 39 weeks' postconceptional age) under chloral hydrate (50 mg/kg) sedation. In 5 of 8 patients, functional MRI data acquisition was successful. This resulted in 10 functional data sets (5 for passive stimulation of each forearm). Unilateral stimulation was associated with mainly bilateral activation of the primary sensorimotor cortex (n = 7 of 10 data sets), the prevailing hemodynamic response being a negative blood oxygenation level-dependent signal. Positive blood oxygenation level-dependent response or failure to activate the sensorimotor cortex (n = 3 of 10 data sets) were seen in those patients with aberrant structural/behavioral indices. Our data show the feasibility of passive unilateral sensorimotor stimulation during neonatal clinical MRI protocols. The bilateral activation pattern observed at this age is compatible with a bilaterally distributed sensorimotor system. Our data validate initial accounts for a raised incidence of negative blood oxygenation level-dependent responses in the primary sensorimotor cortex at this developmental stage. The negative blood oxygenation level-dependent response is likely to reflect a reduction of the oxy/deoxy-hemoglobin ratio during a maturational stage characterized by rapid formation of synapses, yet ineffective processing. Positive blood oxygenation level-dependent responses or failure to activate the sensorimotor cortex may be an early indicator of abnormal development and will have to be followed up carefully.

Non-REM sleep influences results of fMRI studies in epilepsy

Although negative blood oxygen level-dependent (BOLD) signal changes are very frequent findings in neuroimaging studies of neuronal networks underlying interictal epileptiform discharges (IEDs), the nature of negative BOLD effects in epilepsy remains unclear. To investigate the influence of sleep on BOLD responses to internal activity such as IED, hemodynamic changes associated with IED were analysed in sleep stages 1 and 2 in four children with focal epilepsies who underwent simultaneous EEG-fMRI recordings. There were significantly more voxels with negative BOLD responses and better fit of the expected with the real course of BOLD signal for the negative BOLD effect in sleep stage 2 compared to stage 1. Moreover, the increase in omega (12.0-14.0Hz) and delta (0.5-4.0Hz) power correlated with an increase in the number of deactivated voxels. This study indicates that the second stage of sleep seems to be associated with an increase in negative BOLD response to internal activity compared with sleep stage 1. An increase in inhibitory influences during sleep and decrease of sleep-associated, energy-consuming processes may be responsible for the described negative BOLD signal changes.

Behavioral correlates of negative BOLD signal changes in the primary somatosensory cortex

Functional magnetic resonance imaging (fMRI) hypothesis testing based on the blood oxygenation level dependent (BOLD) contrast mechanism typically involves a search for a positive effect during a specific task relative to a control state. However, aside from positive BOLD signal changes there is converging evidence that neuronal responses within various cortical areas also induce negative BOLD signals. Although it is commonly believed that these negative BOLD signal changes reflect suppression of neuronal activity direct evidence for this assumption is sparse. Since the somatosensory system offers the opportunity to quantitatively test sensory function during concomitant activation and has been well-characterized with fMRI in the past, the aim of this study was to determine the functional significance of ipsilateral negative BOLD signal changes during unilateral sensory stimulation. For this, we measured BOLD responses in the somatosensory system during unilateral electric stimulation of the right median nerve and additionally determined the current perception threshold of the left index finger during right-sided electrical median nerve stimulation as a quantitative measure of sensory function. As expected, positive BOLD signal changes were observed in the contralateral primary and bilateral secondary somatosensory areas, whereas a decreased BOLD signal was observed in the ipsilateral primary somatosensory cortex (SI). The negative BOLD signal changes were much more spatially extensive than the representation of the hand area within the ipsilateral SI. The negative BOLD signal changes in the area of the index finger highly correlated with an increase in current perception thresholds of the contralateral, unstimulated finger, thus supporting the notion that the ipsilateral negative BOLD response reflects a functionally effective inhibition in the somatosensory system.

Negative BOLD with Large Increases in Neuronal Activity

Blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) is widely used in neuroscience to study brain activity. However, BOLD fMRI does not measure neuronal activity directly but depends on cerebral blood flow (CBF), cerebral blood volume (CBV), and cerebral metabolic rate of oxygen (CMRO(2)) consumption. Using fMRI, CBV, CBF, neuronal recordings, and CMRO(2) modeling, we investigated how the signals are related during seizures in rats. We found that increases in hemodynamic, neuronal, and metabolic activity were associated with positive BOLD signals in the cortex, but with negative BOLD signals in hippocampus. Our data show that negative BOLD signals do not necessarily imply decreased neuronal activity or CBF, but can result from increased neuronal activity, depending on the interplay between hemodynamics and metabolism. Caution should be used in interpreting fMRI signals because the relationship between neuronal activity and BOLD signals may depend on brain region and state and can be different during normal and pathological conditions.