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F U N C T I O NA L MRI

Kmil Uluda g* G David J. Dubowitz* G Richard B. Buxton*

INTRODUCTION TO FUNCTIONAL MAGNETIC RESONANCE IMAGING (fMRI) 250 The MRI Signal is Sensitive to Changes in Blood Oxygenation 250 The Origins of fMRI 250 fMRI Has Become an Important Tool in Neuroscience Research 250 Overview of the Chapter 251 THE PHYSIOLOGIC BASIS OF fMRI 251 Neuronal Signaling and Energy Metabolism 251 The Brain is Fueled by the Oxidative Metabolism of Glucose 253 Cerebral Blood Flow Delivers O2 and Glucose and Clears CO2 253 Neuronal Activation is Followed by a Hemodynamic Response 254 Multiple Agents Mediate Neurovascular Coupling 255 The Function of Neurovascular Coupling and the Oxygen Limitation Model 255 Is Neurovascular Coupling a Feed-Forward Mechanism? 257 THE BOLD EFFECT 257 Magnetic Susceptibility Variations Distort the Local Magnetic Field and Often Create Image Artifacts 257 Magnetic Susceptibility Changes due to Blood Oxygenation Create the BOLD Effect 258 Diffusion of Water Molecules Moderates the GRE-BOLD Effect and Creates the SE-BOLD Effect 259 Intravascular BOLD Effects Make a Strong Contribution to the Net Signal Change 260

Modeling the BOLD Effect 261 Dynamics of the BOLD Signal 262 DESIGN AND ANALYSIS OF BOLD-fMRI EXPERIMENTS 263 Statistical Analysis is Required to Detect Small Signal Changes 263 The General Linear Model Provides a Statistical Framework for Incorporating All the Components of the Signal 264 Identifying Activated Voxels 265 Statistical Parametrical Maps are Used to Display Activated Voxels 266 Limitations of the General Linear Model 266 Block Designs versus Event-Related Designs 267 Detection versus Estimation 268 ARTIFACTS AND NOISE 269 fMRI is More Sensitive to Imaging Artifacts than Clinical MRI 269 Minimizing and Correcting Image Distortions 269 fMRI is Sensitive to Subject Motion 270 The fMRI Signal Includes Contribution from Physiologic Fluctuations 270 Correcting for Physiologic Noise 271 Scanner Stability and Thermal Noise 272 MEASURING CEREBRAL BLOOD FLOW, CEREBRAL METABOLIC RATE OF OXYGEN, AND CEREBRAL BLOOD VOLUME 274 Cerebral Blood Flow 274 Calibrating the BOLD Signal to Measure CMRO2 Changes 275

Assessment of Cerebral Blood Volume Using an Exogenous Contrast Agent 275 Assessment of Cerebral Blood Volume Using an Endogenous Contrast Agent 276 Magnetic Resonance Spectroscopy 277 Diffusion 277 Manganese Tract Tracing 277 EXPLORING THE HEMODYNAMIC RESPONSE TO BRAIN ACTIVATION WITH MRI 277 Are Oxygen and Glucose Metabolism Linked during Increased Neural Activity? 278 Why Does Glucose Metabolism Increase more than Oxygen Metabolism with Brain Activation? 278 What is the Underlying Neuronal Activity that Drives the fMRI Signals? 279 Spiking versus Synaptic Activity 279 Simultaneous Measurements of Electroencephalography and Event-Related Field Potentials with fMRI 280 Does Inhibition Produce a BOLD Response? 280 What is the Signicance of the Transients of the BOLD Signal? 280 Post-Stimulus Undershoot 281 Nonlinearity of the BOLD Response 281 The Physiologic Baseline Strongly Affects the BOLD Signal 282 Do BOLD Correlations Reveal Long-Range Patterns of Connectivity? 283 Spatial and Temporal Resolution 283

*The authors are supported by NIH grants NS-36722 and NS-042069.

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INTRODUCTION TO FUNCTIONAL MAGNETIC RESONANCE IMAGING (fMRI) The MRI Signal is Sensitive to Changes in Blood Oxygenation
One of the remarkable developments in recent work on MRI is the recognition that changes in the metabolic state of the brain affect the MR signal in a detectable fashion and therefore provide an intrinsic mechanism of contrast for brain activation studies. The origin of this effect is that the magnetic state of hemoglobin (Hb) depends upon its oxygenation, so that changes in oxygen saturation of the hemoglobin produce a small change in the local MR signal, the blood oxygenation leveldependent (BOLD) effect. Specically, deoxygenated hemoglobin is paramagnetic and tends to reduce the local MR signal by creating microscopic eld gradients within and around the blood vessels. If the local oxygen extraction fraction (E) always remained constant, the local oxygenation of the blood would not change, and the BOLD effect would simply be an interesting, but not particularly useful, biophysical effect. However, when combined with an unexpected physiologic phenomenon, this becomes a powerful tool for mapping brain activation. Following increased neural activity in the brain, the local cerebral blood ow (CBF) increases much more than the cerebral metabolic rate of oxygen (CMRO2), and as a result E decreases with activation. Because the local blood is more oxygenated, there is less deoxyhemoglobin present and the local MR signal increases slightly. Brain activation studies based upon BOLD contrast typically employ an experimental paradigm in which a subject alternates between periods of stimulation and rest while a rapid series of MR images is collected. The time series for each image voxel is then analyzed to determine if the signal shows a signicant correlation with the stimulus, i.e., increasing when the stimulus was applied and decreasing when the stimulus was removed. Those pixels that do show a correlation are displayed in color on a regular anatomical MR image as the areas activated by the stimulus.

The Origins of fMRI

The fact that the magnetic state of hemoglobin changes with its state of oxygenation was discovered in 1936 by Pauling and Coryell, before the discovery of nuclear magnetic resonance (NMR) itself.1 In 1982 Thulborn and colleagues demonstrated relaxation rate (T2) changes in blood samples due to the magnetic susceptibility changes caused by the presence of paramagnetic deoxyhemoglobin.2 However, it was not until the 1990s that the potential signicance of this effect for functional neuroimaging was realized.3-7 The rst demonstration that changes in blood oxygenation had a measurable effect on the MR signal in vivo was not an activation study but rather a physiologic

manipulation in which the inspired oxygen was varied. Ogawa et al imaged the brains of mice at high magnetic elds (7 and 8.4 T) with gradient-echo imaging.4 They found that the veins became noticeably darker in the MR image when the oxygen in the inspired air was reduced. The reduction of the blood signal was consistent with the earlier in vitro NMR studies that had demonstrated the effect of oxygenation on T2.2 But, in addition, Ogawa and colleagues made the key observation that the signal from the tissue surrounding the veins also was reduced, and they proposed that the cause of this effect was a change in the magnetic susceptibility of the blood. Furthermore, the effect was greatly reduced in spin-echo images. Both these observations were consistent with the source of the effect being related to magnetic susceptibility changes (to which gradient-echo images are highly sensitive) brought on by the presence of the deoxygenated hemoglobin. Ogawa and colleagues suggested that this phenomenon could form the basis for monitoring regional oxygen use in the brain, and speculated that during activation more oxygen would be removed from the blood and the deoxyhemoglobin concentration would increase. The reality of the situation turned out to be the opposite of thisdeoxyhemoglobin concentration decreases with activation because of the large CBF changebut the insight that this NMR effect could be used to measure brain function was the critical beginning of fMRI. Subsequently, Turner and colleagues imaged cat brains under the controlled conditions of anoxia and apnea with an echo-planar imaging (EPI) pulse sequence on a 2 T system.8 They also found MR signal changes that were dependent on the oxygenation of the blood. The detectability of blood oxygenation effects in these well-controlled animal experiments at least suggested the possibility that such effects might be seen in humans performing tasks that alter the oxygen utilization in the brain. Kwong and colleagues acquired images of a normal human subject during visual stimulation with a gradient-echo EPI sequence using a long echo time (40 ms) in order to enhance the susceptibility effects.3 Temporally resolved images acquired during and in the absence of stimulation showed clear differences in signal intensity, with the signal increasing during stimulation, suggesting that the deoxyhemoglobin concentration decreased with activation. In short order several other studies conrmed this nding.5-7 Other fMRI studies in the visual cortex with a high eld (4 T) scanner and the motor cortex with an echo-planar system with specially designed gradient coils also demonstrated signal increases with activation.9 Similar results were soon obtained on a conventional clinical scanner as well.10

fMRI Has Become an Important Tool in Neuroscience Research

From its origins in basic MRI research described above, fMRI has grown explosively to become a standard and indispensable tool in neuroscience research. Previously,

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positron emission tomography (PET) methods of measuring CBF change were the standard for mapping functional activity in the human brain. While PET studies are still done for a number of applications, most human brain mapping studies are now done with fMRI. Over the last decade, the sophistication of the techniques has improved enormously.For example,techniques for retinotopic mapping have become standard in studies of the visual system. The visual image produced on the retina is mapped in a spatially coherent way onto the visual cortex, and this coherent retinotopic map is repeated in many sub-regions of the visual cortex. By mapping progressive waves of activation as a subject views expanding rings or rotating wedges, the boundaries of these different functional regions of the visual cortex can be mapped.11,12 Because the functional organization does not always match up in the same way with the anatomical organization, fMRI studies provide an enormous advance in the ability to characterize the working human brain by identifying these functional sub-divisions of the visual cortex in addition to anatomical sub-divisions.

would have little impact on the diagnostic utility of clinical MR images nevertheless can severely degrade fMRI data, and some of these effects and possible remedies are discussed in Artifacts and Noise. One of the powerful features of MRI is its exibility, and other MR-based techniques have been developed to measure different physiologic aspects of brain activation that complement and enhance the standard measurements of the BOLD effect. These other methods, and how they can be combined with BOLD-fMRI, are introduced in Measuring Cerebral Blood Flow, Cerebral Metabolic Rate of Oxygen, and Cerebral Blood Volume. Finally, in Exploring the Hemodynamic Response to Brain Activation with MRI, we describe the ways in which fMRI is being used in current research on the physiology of brain activation, and some of the notable open questions.

THE PHYSIOLOGIC BASIS OF fMRI

In a typical fMRI experiment the goal is to map patterns of neuronal activation in the subjects brain while he or she performs specic tasks. However, fMRI does not measure the neuronal activity itself. Instead, the BOLD effect in response to activation is sensitive to the concentration change of deoxygenated hemoglobin, which in turn is dependent on cerebral blood ow (CBF), cerebral blood volume (CBV), and cerebral metabolic rate of oxygen (CMRO2), illustrated in Figure 9-1. A critical goal for interpreting fMRI data is to understand the underlying link between neuronal activity and the hemodynamic response. This is still an area of active research, and in this section we outline the current thinking.

Overview of the Chapter

The remainder of the chapter introduces the basic principles, mechanisms, and techniques that underlie fMRI. In The Physiologic Basis of fMRI, the physiologic mechanisms linking blood ow, oxygen metabolism, and neural activity are described. In fact, in recent years fMRI techniques have become useful tools for exploring these links. In The BOLD Effect, the biophysics underlying the BOLD effect is described, including mathematical models for how the BOLD signal depends on the local change in the O2 extraction fraction E and the venous blood volume V. In addition, because fMRI techniques measure dynamic changes, and the dynamics of E and V may have different time constants, there is the possibility for a range of transient effects in the measured BOLD response. Because the BOLD signal changes are small typically only a few percentthe design and analysis of fMRI experiments to detect these subtle effects is a critical component of fMRI; this is introduced in Design and Analysis of BOLD-fMRI Experiments. In addition, because the BOLD signal changes are small, artifacts that

Neuronal Signaling and Energy Metabolism

In the brain, neurons are maintained in a thermodynamic state far from equilibrium. The sodium ion (Na+) concentration outside the neuron is much higher than that inside the cell. Given the more negative potential inside the cell, it is a strongly downhill reaction for Na+ to move into the cell. On the presynaptic side, calcium

F I G U R E 9-1
The path of changes linking an applied stimulus to the measured local BOLD signal change in a subjects brain during an fMRI experiment. In this illustration, a ickering checkerboard stimulus triggers increased neuronal activity in the visual cortex. This is accompanied by increased blood ow, blood volume, and oxygen metabolism, and these physiologic changes combine to alter the local deoxyhemoglobin, which in turn alters the local MR signal.

Stimulus

Neuronal activity

Blood flow Blood volume Oxygen metabolism

Blood oxygenation

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ions (Ca2+) are also concentrated outside the cell, and neurotransmitters are highly concentrated in small vesicles within the presynaptic terminal waiting for release. The arrival of an action potential triggers a cascade that includes Ca2+ inux, neurotransmitter release into the synaptic cleft, binding of neurotransmitter on the post-synaptic side, and opening of ion channels for Na+ and potassium (K+) currents. This signaling process is all thermodynamically downhill, so no energy is required. The energy costs of neural activity come mainly in the recovery from this signaling: Na,+ K+, and Ca2+ must be pumped against their gradients to restore the original ion distributions, and neurotransmitter must be cleared from the synaptic cleft and re-packaged in vesicles in preparation for the arrival of the next action potential. The source of thermodynamic free energy to power these uphill processes is the pool of adenosine triphosphate (ATP) and adenosine diphosphate (ADP). The ATP/ADP system is far from equilibrium, with approximately ten times more ATP than ADP.13 For this reason, the conversion of ATP to ADP carries a large negative free energy (G) that can drive other reactions uphill. In addition to direct use of ATP, some uphill processes, such as the clearance of the neurotransmitter glutamate from the synaptic cleft, are driven by co-transport of Na+ down its gradient from the extracellular to intracellular space. The degraded Na+ gradient, in turn, is restored by the Na+/K+ pump, which pumps both Na+ and K+ against their gradients at the expense of ATP. It has been estimated14 that at least half of the energy consumed in the brain is due to the action of the Na+/K+ pump. In short, one can think of the brain as containing two stores of free energytwo batteriesthat can be used

to drive all of the energy-consuming reactions in the cell: the ATP/ADP system, and the Na+ gradient across the cell membrane. These two systems are in close communication through the Na+/K+ pump. From a biochemical perspective, the available free energy is dened by a ratio of concentrations: [ATP]/[ADP] in one case, and extracellular/intracellular Na+ in the other. That is, it is not the ATP itself that carries the energy; it is the high ratio of [ATP]/[ADP] that is far from equilibrium that endows the conversion of ATP to ADP with a large negative free energy. Recent studies have reported estimates of the energy budget for brain processing by tallying up the number of ATP/ADP conversions needed to fuel the different processes involved.15 Neurons and glia require energy to maintain their resting membrane potential and carry out other non-signaling functions within the cell. One can break down the signaling costs by considering a single action potential, which starts in one cell and travels to thousands of synapses on other cells, where presynaptic transmitter release triggers post-synaptic membrane potential changes. A key question is: how does the energy cost of generating and propagating an action potential compare with the energy costs of synaptic activity? In Figure 9-2 the energy estimates of each process per signaling event are shown (the rst number refers to rodents, the second number to primates). Post-synaptic activity, such as uptake of neurotransmitters by the neurons and astrocytes and restoration of the ionic gradients, is estimated to consume most of the energy in humans.16 The spiking itself (action potentials) only requires 10% of the total energy. In contrast, in rodents the action potentials account for almost half of the total energy. Maintaining resting potentials, presynaptic

Resting potential 10%, 2% 3Na+ 2K+ ATP Action potentials 47%, 10% Glu 3Na+ 2K+ ATP Presnaptic 3%, 7% Glu Glu Glu ATP 3Na+ 2K+ Ca2+ 3Na+ Ca2+

Glia 5%, 6% ATP

3Na+, 2K+

F I G U R E 9-2
Relative metabolic costs of each process per neural signaling event (rst number: rodents, second number: primates).16 Post-synaptic activity is estimated to consume most of the energy in humans. Action potentials only require 10% of the total energy. In contrast, in rodents the action potentials account for almost half of the total energy. Maintaining resting potentials, presynaptic processes, and the glia utilize only small amounts of energy. (Adapted, with permission, from Attwell D, Iadecola C: The neurol basis of functional brain imaging signals. Trends Neurosci 25:621-625, 2002. 2002, with permission from Elsevier.)

3Na+, H+ K+

Glu ATP Gln

Post-synaptic 34%, 75%

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processes, and the glia utilize only small amounts of energy. In Exploring the Hemodynamic Response to Brain Activation with MRI, we discuss what implications this estimation has for the interpretation of the measured fMRI signals.

The Brain is Fueled by the Oxidative Metabolism of Glucose

The ATP/ADP system that fuels the recovery from neural activity must be restored by coupling the uphill conversion of ADP to ATP to an even more downhill reaction: the oxidative metabolism of glucose and oxygen to carbon dioxide and water. The complete conversion of one molecule of glucose and six molecules of O2 generates 38 ATP molecules from ADP. The full metabolism is illustrated in Figure 9-3. This fundamental energy metabolism happens in two stages. In the cytoplasm, glycolysis converts the glucose molecule to two molecules of pyruvate, stores some of the energy in the conversion of two nicotinamide adenine dinucleotide ions (NAD+) to NADH,17 and generates two ATP molecules from ADP, all without using O2. Although the ATP yield of glycolysis is low, it is very fast. For this reason, exercising muscle relies on glycolysis to generate the ATP needed for short bursts of intense activity (e.g., sprinting), and it has been suggested that speed of production of ATP may also be important in the brain.18 Much more ATP is generated in the second stage of energy metabolism when pyruvate and O2 diffuse into the mitochondria and enter the tricarboxylic acid (TCA)

cycle. The end products of mitochondrial energy metabolism are six molecules each of H2O and CO2, and the conversion of 36 ADP molecules to ATP. As part of this net metabolism, the NADH produced by glycolysis also is shuttled into the mitochondria in exchange for NAD+, restoring the cytosolic balance. It is important to recognize that, because energy metabolism occurs in two stages with glycolysis feeding the TCA cycle, it is possible for the rate of glycolysis to exceed the rate of pyruvate metabolism in the mitochondria. In this case the cerebral metabolic rate of glucose (CMRGlc) and the cerebral metabolic rate of oxygen (CMRO2) are not matched. Fully matched oxidative metabolism requires that six O2 molecules are consumed for each glucose molecule metabolized, often described as an oxygen-glucose index (OGI) of 6.0. A typical experimental value is OGI = 5.5 at rest, but it is interesting to note that this quantity appears to decrease with activation: the CMRGlc change with activation exceeds the CMRO2 change18 (for further discussion see Exploring the Hemodynamic Response to Brain Activation with MRI). If the glucose and oxygen metabolic rates are not matched, pyruvate and NADH would accumulate in the cell and ultimately disrupt glycolysis. However, an important enzyme called lactate dehydrogenase catalyzes the conversion of pyruvate and NADH to lactate and NAD+.17 This restores the NADH/NAD+ balance but leads to the accumulation of lactate, which ultimately diffuses out of the cell and is carried away in the blood. The accumulation of lactate in the tissue, which can be measured with MR spectroscopy techniques (see Measuring Cerebral Blood Flow, Cerebral Metabolic Rate of Oxygen, and Cerebral Blood Volume and Exploring the Hemodynamic Response to Brain Activation with MRI), is thus a sign of a mismatch of CMRGlc and CMRO2.

Cerebral Blood Flow Delivers O2 and Glucose and Clears CO2

For the brain to continue functioning, glucose and oxygen must be supplied and CO2 cleared from each tissue element, and this is accomplished by blood ow. In order to understand hemodynamics, it is important to keep in mind that CBF and CBV are two distinct physiologic quantities. The CBV describes the total volume of the vasculature: the sum of the volumes of the arteries, arterioles, capillaries, venules, and veins in a volume of tissue (Fig. 9-4). The CBV is usually expressed as a dimensionless quantity, the fraction of the tissue volume occupied by blood. A typical value of CBV in the brain is 4%. In contrast, CBF is the volume of arterial blood delivered to an element of tissue in a specied time, i.e., only blood owing through arteries, arterioles, and capillaries is counted for CBF. Thus, blood owing through a volume of tissue but destined for another is not counted for CBF of this volume. The usual units are mL/100 g/min, and a typical value in the human brain is 60 mL/100 g/min. If we refer the CBF to 1 mL of tissue

F I G U R E 9-3
Non-oxidative and oxidative metabolism of glucose. The non-oxidative metabolism of glucose (glycolysis) generates two ATP molecules from ADP by converting the glucose molecule to two molecules of pyruvate. The TCA cycle and the electron transfer chain in the mitochondria metabolize the two pyruvate molecules and 6 O2 molecules to 6 CO2 and 6 H2O molecules, producing 36 ATP from ADP . In the cytosol, pyruvate and lactate are in near equilibrium, and if the glucose metabolic rate exceeds the oxygen metabolic rate, the lactate concentration will rise.

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state relationship between CBF and CBV can be described with a power law: CBV CBF (Eq. 9-2)

F I G U R E 9-4
Vascular structure of a chinchilla measured with corrosion casts. The feeding arteriole (red) delivers oxygen-saturated blood to the capillaries (orange and green). The capillaries end in draining venules (blue) having only approximately 60% oxygen saturation, that is, brain tissue extracts 40% of the oxygen from the capillaries. All these vascular compartments contribute to the cerebral blood volume (CBV). Cerebral blood ow (CBF) is the rate of blood delivery of blood to the capillary bed. (Adapted from Harrison RV, et al: Blood capillary distribution correlates with hemodynamicbased functional imaging in cerebral cortex. Cereb Cortex 12:225-233, 2002, by permission of Oxford University Press.)

where the exponent is approximately = 0.38, i.e., a CBF increase of 50% corresponds to a CBV increase of approximately 18%. This empirical relationship applies to the entire cerebral blood volume, and only after a steady state has been reached. The temporal dynamics of the total CBV will be a weighted composite of the changes in each of its compartments. For example, during functional activation the initial change will be dominated by the arterioles, but it has been postulated that the venous vessels may be slower to expand and slower to contract back to baseline after CBF has returned to normal.20,21 In general, the CBV and CBF ratio during the transients is as yet not well described, but measuring this ratio dynamically promises to provide some insights into these basic physiologic variables and into neurovascular coupling that may be altered in disease.

Neuronal Activation is Followed by a Hemodynamic Response

A large number of positron emission tomography (PET) studies, and more recently fMRI studies, have measured the changes in CBF, CBV, CMRGlc, and CMRO2 accompanying neural activity, see for example references 19 and 22 to 25. There is of course a good deal of experimental variation, but in rough numbers the basic pattern for a strong stimulus is that CBF increases dramatically (40%), CMRGlc also increases by about the same amount, CMRO2 increases much less (<20%), and CBV increases by a modest amount (15%). It is useful to summarize this complex of physiologic changes in terms of two key dimensionless numbers dened above, the oxygen extraction fraction (E) and the oxygen-glucose index (OGI). The key results are that with activation E and OGI both decrease. Trying to understand this unexpected pattern is the focus of much current research (see Exploring the Hemodynamic Response to Brain Activation with MRI). The decrease of E with activation is the primary cause of the BOLD effect. Oxygenated blood is diamagnetic and deoxygenated blood is paramagnetic, so the increase in blood oxygenation during activation changes the magnetic properties of the blood and the tissue and causes the MR signal to increase. Because the temporal resolution of fMRI is much better than with PET techniques, the BOLD signal provides a window on the temporal dynamics of the hemodynamic response. Typical experimental responses to a very short stimulation (approximately 1 second in duration), called the impulse response, and to a long stimulation (20 seconds in duration) measured with fMRI are shown in Figure 9-5. The BOLD response is typically delayed by 1 to 2 seconds and reaches its maximum after approximately 8 s (typically between 5 and 10 s). After the end of the stimulus a post-stimulus undershoot often can be seen

(with a density of about 1 g/mL), the units become mL/mL/min, or simply inverse time. Expressed this way, we could write a standard CBF of 60 mL/100 g/min as 0.01 s1. This formulation of the units emphasizes that CBF often acts like a rate constant. In particular, the metabolic rate of oxygen metabolism can always be written as: CMRO2 E Ca CBF (Eq. 9-1)

where Ca is the arterial concentration of O2, and E is the net oxygen extraction fraction. This is simply the total rate at which O2 is being delivered to tissue (Ca CBF) times the fraction of the delivered oxygen that is extracted and metabolized. In general, it is helpful to think of CBF and CBV as independent quantities: in plumbing terms, the volume of the pipes and the ow being delivered to the pipes. At some level, however, there is a connection. The CBF increase associated with neural activity is triggered by relaxation of the smooth muscle in the wall of the arterioles. The arterioles provide most of the resistance in the vascular tree and provide a way to quickly decrease the vascular resistance (by expanding). In this sense, an increased blood volume is part of the mechanism of increasing CBF (see Fig. 9-4). However, if the arteriolar volume fraction is small, this may be only a small change in total CBV. As the resistance of the arterioles decreases, the pressure drop across these vessels also decreases, raising the pressure in the capillaries and veins. These vessels may also expand due to the increased pressure, further increasing the CBV. Experimental studies19 have indicated that the steady-

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3 MR signal change (%) 2 1 0 -1 0 10 20 30 Time (s) 40 50 60

F I G U R E 9-5
Typical experimental hemodynamic responses to a very short stimulation (approximately 1 second duration, blue), called the impulse response, and to a long stimulation (20 seconds duration, red) measured with fMRI.

which lasts typically 20 s or more. More rarely, and not highly reproducibly, an initial undershoot of the BOLD signal is observed (for an overview see reference 26), called the initial dip. For further discussion and implications of the transients see Exploring the Hemodynamic Response to Brain Activation with MRI.

Multiple Agents Mediate Neurovascular Coupling

The translation of increased neuronal activity to increased CBFneurovascular couplingcan be considered from two different viewpoints: 1. what is the function served by neurovascular coupling?; and 2. what is the mechanism that accomplishes this function? Somewhat surprisingly, the mechanism is better understood than the function. A number of vasoactive agents are produced in association with neural activity, and it appears that the particular mechanisms employed to raise CBF vary in different parts of the brain.27 Some of the key vasoactive agents are nitric oxide (NO), potassium ions (K+), hydrogen ions (H+), adenosine, and carbon dioxide (CO2). This is only a partial list, but it already includes a highly diffusible gas linked to G-protein activation (NO), a key player in ion homeostasis (K+), the local pH, a neurotransmitter (adenosine), a key element of the energetic stores of the tissue (adenosine again, as the nal form of degradation of ATP), and a key product of energy metabolism (CO2). This suggests that multiple mechanisms exist to increase blood ow in association with increased neural activity. Indeed, the experimental data indicate that there is no single mechanism controlling CBF in the brain, and the mechanism of CBF control may be quite adaptive during development. At this point, there is no shortage of possible mechanisms although little is known about how the full integrated system for neurovascular coupling works.27

Two interrelated themes have received growing attention in recent years, one focused on the central role of glutamate and the other focused on the role of the non-neuronal (glial) cells in neuronal signaling and neurovascular coupling.28-31 Glutamate is the primary excitatory neurotransmitter, and the majority of synapses are glutamatergic. When glutamate binds to a postsynaptic receptor it opens a Na+ channel that allows Na+ to diffuse down its gradient, and eventually it must be pumped back by the Na+/K+ pump. For this reason, glutamate itself would serve as a good signal for the energy-consuming processes associated with neural activity, and there is some evidence that glutamate has a direct vasodilatory effect.32 But the more signicant connection is likely to be that glutamate is cleared from the synaptic cleft by astrocytes.29 The current view of the role of the glia has expanded substantially in the last few years.31,33 It has been known for some time that astrocytes are both positioned near synapses for recycling of glutamate and also have numerous projections to blood vessels by their end-feet, suggesting at least the anatomical connections for linking synaptic activity to blood ow. Recent work has indicated that astrocytes have very well-developed and essentially non-overlapping territories, further supporting a key role for the astrocytes in assessing the level of activity of the neurons and in some way communicating this to the blood vessels. In addition, astrocytes play a role in modulating neuronal activity.31 It has been hypothesized that glia provide the energy substrate for the neurons by shuttling lactate to the neurons,29 although this is controversial.34

The Function of Neurovascular Coupling and the Oxygen Limitation Model

The mechanisms of neurovascular coupling do not necessarily clarify the function served; for example, even if NO is the mechanism that triggers increased blood ow, why is the resulting CBF increase so large? For the description of the function of neurovascular coupling, the details and the pathways of the CBF regulation are less important. Because CBF increases much more than CMRO2, so that E decreases with activation, this physiologic phenomenon was originally called an uncoupling of blood ow and oxygen metabolism.35 However, it is possible that the large change in CBF is an integral part of regulating oxygen delivery, so that the decrease of E is necessary for increasing CMRO2, an idea we referred to as the oxygen limitation model.26,36-40 In the context of this model the large increase in CBF is required to support the smaller increase of oxygen metabolism (Fig. 9-6). Although this idea has by no means been proven, it is the only quantitative explanation that has been proposed for the function served by a large CBF increase. The model is based on experiments showing two effects: 1. at rest a large fraction of the delivered oxygen never leaves the capillary, but nearly all of the oxygen that does enter the extravascular tissue space is metabolized; and 2. CBF increases by increasing

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Po2 45 30
5 Mitochondria 0

Activation Rest

O2 flux

F I G U R E 9-6
Illustration of the oxygen limitation model. The oxygen concentration, measured as a partial pressure PO2, varies from approximately 100 torr in the arteries to approximately 30 torr in the veins, with a mean capillary value of approximately 45 torr. The PO2 in the mitochondria is thought to be low (approximately 5 torr). Oxygen diffuses from the high concentration in the capillary to a low concentration in the mitochondria, and to increase the O2 ux the gradient must be increased. If mitochondrial PO2 is already low, and there is no capillary recruitment to bring the blood closer to the mitochondria, then mean capillary PO2 must be increased. This requires that the oxygen extraction fraction E must be reduced, so CBF must increase more than CMRO2.

capillary velocity rather than by opening new capillaries (i.e., there is no capillary recruitment). The implications of these results can be understood from the viewpoint of diffusion down a concentration gradient, with oxygen diffusing from a high concentration in the capillary to a low concentration in the mitochondria. If the net extraction is nearly equal to the unidirectional extraction, then backux of O2 from tissue to capillary must be small, implying that the tissue PO2 is near zero. With no capillary recruitment the diffusion distance from capillary to mitochondria is xed, so to increase the O2 ux either the mean capillary PO2 must be increased or the mitochondrial PO2 must be decreased. If the mitochondrial PO2 is already near zero, then the only option is to raise capillary PO2 by raising the venous O2 content, and this means that E must decrease. At steady state, the product of E and CBF must match this increased ux from capillary to mitochondria (Eq. 9-1), so CBF must increase by a larger amount to overcome the decrease of E. The original model for the effects of limited oxygen delivery assumed the extreme form of equal unidirectional and net extraction fractions, and the prediction of that model was that the fractional change in CBF would need to be approximately 5 times larger than the fractional change in CMRO2. Measurements in the awake human brain with PET and calibrated fMRI have found this ratio to lie in the range n = 2 to 6,35,41-46 so the predicted ratio lies near the high end of the experimental results. However, this form of the model is undoubtedly too simple. Other factors can inuence the diffusibility of O2, such as capillary dilation and shifts of the binding curve of O2 and hemoglobin, and these effects should be included in the modeling.37 An increase of diffusivity through these mechanisms would soften the require-

ment for the CBF increase, but detailed modeling of these effects in this context has not been reported. Furthermore, in the original simple form of the oxygen limitation model the assumption of equal unidirectional and net extraction fractions is equivalent to assuming no backux of O2 and a tissue PO2 of zero. This is certainly an oversimplication, and is incompatible with studies of the oxidation state of cytochrome oxidase, which indicate that at rest oxygen concentration is not a limiting factor in determining the rate of oxidative metabolism in the mitochondria.47 These observations can be reconciled with the oxygen limitation model if the mitochondrial PO2 is greater than zero but still signicantly less than the average capillary PO2, and the blood ow increase serves to maintain a constant mitochondrial oxygen tension (see Fig. 9-6). Calculations indicate that a CBF/CMRO2 ratio of n = 4 is required to maintain mitochondrial PO2 at a constant value while increasing the oxygen ux.37 Other factors such as the Bohr effect and perhaps capillary dilation could reduce the value of n required. In this case the mitochondrial PO2 would be held at a high enough level that O2 availability would not become limiting in the mitochondria. In short, the tissue PO2 would constitute a buffer that is normally not used with physiologic activation, but which could come into play under some conditions at the beginning of stimulation or in hypoxia. However, oxygen delivery is still fundamentally limited, in the sense that maintenance of this mitochondrial PO2 requires a steady ux of O2 from the capillaries.Variability of this oxygen buffer (e.g., through alterations of inspired oxygen content or resting CBF) is a possible source of the variability between laboratories of detection of the initial deoxygenation26 (see Exploring the Hemodynamic Response to Brain Activation with MRI).

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Is Neurovascular Coupling a Feed-Forward Mechanism?

By the model described above, the function served by a large CBF increase following neural activity is to maintain mitochondrial PO2 at a constant value so that oxidative metabolism can proceed without being limited by oxygen availability. Two possibilities arise for how CBF is regulated: 1. a feedback model in which the amount of oxygen already available determines the increase in CBF following functional activity; 2. a feed-forward model in which the change in neuronal activity determines the change in CBF independent of oxygen availability and hence from the baseline CBF. A feedback system that responded to a drop in cytosolic PO2 would not work because the goal is to increase cytosolic PO2 above baseline in an activated state and would only produce transient changes in CBF. However, a feed-forward system is consistent with these theoretical ideas and with current experiments using vasoactive agents. That is, the neural activity starts a cascade of biochemical processes that leads to increased CBF, with no direct feedback about whether such a CBF change is needed to support the increase in CMRO2. This idea is prompted by several recent studies that measured the CBF or deoxygenation (i.e., BOLD) response to activation under conditions where the baseline CBF was altered.48-50 Baseline CBF can be increased by breath-holding, inhalation of CO2, or acetazolamide and decreased by ingestion of caffeine. The remarkable nding from most of these studies is that the increment of CBF change due to the activation is unaffected. The idea that the neurons simply call out for more oxygen without sensing whether they have enough to begin with seems inefcient. However, given the critical importance of oxygen for continued energy metabolism, the links between hypoxia and the initiation of apoptosis, and the theoretical reasons above for why oxygen itself is a poor error signal for a control system, a feed-forward system begins to seem more plausible.

ables. Specically, the oxygen extraction fraction E governs the oxygen saturation of blood leaving the capillary, but the blood volume (CBV) also affects how much deoxyhemoglobin is present in an image voxel. In the following sections we consider how local changes in deoxyhemoglobin alter both the intravascular and extravascular MR signals for gradient-recalled echo (GRE) and spin-echo (SE) acquisitions, and then use this as the basis for modeling the BOLD signal in terms of E and CBV.

Magnetic Susceptibility Variations Distort the Local Magnetic Field and Often Create Image Artifacts
The central physical idea at the heart of the BOLD effect is that deoxyhemoglobin alters the magnetic susceptibility of blood and creates magnetic eld gradients around the vessels. In clinical MRI magnetic susceptibility effects are usually familiar as a source of artifacts. In BOLD imaging we exploit these effects, and indeed choose pulse sequences that are especially sensitive to magnetic susceptibility, in order to allow us to detect the BOLD signal changes. To understand this phenomenon, it is helpful to review the basic physics of magnetic susceptibility. When any material is placed in a uniform magnetic eld B0, the intrinsic magnetic moments within the material partially align with the eld, so that the material becomes slightly magnetized. The magnitude of this induced magnetization is described by the magnetic susceptibility . The magnetization M = B0 is the equivalent dipole density of the material, and so depends on both the intrinsic dipole density and the degree of alignment of the dipoles with B0. Because the material is now magnetized, it creates an additional magnetic eld that adds to B0. It is important to note that a uniformly magnetized object generally produces a distinctly nonuniform magnetic eld that depends strongly on the geometric shape of the object. For example, a uniformly magnetized cylinder produces a uniform eld inside, but a dipole-like eld outside (Fig. 9-7). This idealized geometry can serve as a model for the eld distortions around a blood vessel containing deoxyhemoglobin. To understand the effects of microscopic eld distortions on the MR signal, it is helpful to think of the eld distortion (illustrated in Fig. 9-7) on two distinct spatial scales. On the macroscopic scale the full frame of the gure corresponds to a normal image, and the eld distortions are due to large structures such as sinus cavities or the petrous bones. The eld gradients due to these susceptibility differences between tissues add to the imaging gradients and can create image distortions. On the microscopic scale, Figure 9-7 corresponds to a single image voxel, with microscopic eld distortions around venules and small veins containing deoxyhemoglobin. In a gradient-recalled echo (GRE) MR experiment a 90 radiofrequency (RF) pulse tips the longitudinal magnetization into the transverse plane where it begins to precess with a frequency proportional to the eld

THE BOLD EFFECT

The primary MRI technique used for fMRI exploits the blood oxygenation leveldependent (BOLD) effect. The physiologic basis of the BOLD effect is that CBF increases much more than CMRO2 during increased neural activity, and as a result the oxygen extraction fraction E is reduced and the venous blood is more oxygenated. The reason this physiologic change is detectable with MRI is that the MR signal is sensitive to microscopic magnetic eld gradients, and deoxyhemoglobin and oxyhemoglobin have different magnetic properties. In this section we consider this biophysical basis of the BOLD effect in some detail, with the goal of deriving basic mathematical models that relate the underlying physiologic changes to the measured signal. This is not a simple transformation, and indeed one of the complexities involved in interpreting the BOLD signal is that it depends on several physiologic vari-

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F I G U R E 9-7
Magnetic eld distortions around a cylinder due to a magnetic susceptibility difference between the inside of the cylinder and the surrounding medium. The cylinder is oriented perpendicular to the main magnetic eld B0, creating a dipole pattern of the Bz component in the space around the cylinder.

F I G U R E 9-8

at the location of that spin. At time TE the signal is measured, and the image intensity can then be viewed as a snapshot of the net signal from the voxel at TE. If the signals from each sub-region of the voxel are out of phase due to local eld offsets, the net signal will be reduced due to the local phase dispersion. As TE increases so does the degree of phase dispersion, and so the signal is more attenuated. Microscopic eld distortions around blood vessels thus attenuate the local signal. On the other hand, if there was no microscopic variation in magnetic susceptibility, so that within a voxel all spins experienced the same eld offset, then the magnitude of the local MR signal would not be reduced but the phase of that signal would reect the eld offset of the voxel. In other words, the phase map of a GRE image can be taken as a map of large-scale magnetic eld distortions within the imaged object. The phase image in Figure 9-8 was collected in this way, and the light to dark transitions, as the phase cycles from 359 to 0, can be thought of as contour lines of the magnetic eld. Thus the local GRE signal is affected in two ways: macroscopic eld variations across the brain affect the phase of the signal, while microscopic eld offsets within a voxel reduce the magnitude of the signal. As noted above, magnetic susceptibility variations also can interfere with mapping the local signal to its proper location in space. MRI relies on using magnetic eld gradients to encode the spatial origin of the MR signal within the signal itself. Specically this means that the basic assumption of MRI is that the magnetic eld is perfectly uniform until the gradients are applied, so that any phase offsets in the signal are due just to those applied gradients. Magnetic susceptibility differences between tissues (e.g., brain, bone, and air) create broad static eld gradients that add to the applied pulsed eld gradients used in imaging. The result is that the MR signals are mismapped, creating distortions in the image. In Figure 9-8 the large-scale eld distortions due to the

Gradient-recalled echo (GRE) phase maps showing magnetic eld distortions, with magnitude images displayed on the top and phase images on the bottom: left, in a coronal section through a human head, and right, around a cylinder (as in Fig. 9-7). The local phase is proportional to the local eld offset, and the transitions from black to white (from 359 to 0) can be viewed as contour lines of the magnetic eld. Note the eld distortion in the brain due to the sinus cavity.

sinus cavities are readily apparent. In general, these largescale effects are a nuisance, distorting the image and sometimes creating signal dropouts. In addition, signals from spins lying in different elds are mapped into the same voxel.When this happens the individual signals add incoherently and so partially (or completely) cancel out. In functional brain imaging, this is most prominent in the prefrontal cortex and midbrain.

Magnetic Susceptibility Changes due to Blood Oxygenation Create the BOLD Effect
The eld distortions around a magnetized blood vessel due to deoxyhemoglobin (see Fig. 9-7) occur on a much ner spatial scale than the broad eld distortions that produce image distortions. For modeling the effect of these microscopic eld distortions, our primary goal is to understand how they produce phase dispersion within the voxel and a reduction of the net signal. Initially we will consider the GRE pulse sequence, the technique most sensitive to eld offsets. Empirically, the signal decay with increasing TE in a GRE image is described in terms of an apparent transverse relaxation time T2*. In contrast, with a spin-echo (SE) pulse sequence, a 180 RF refocusing pulse is applied at time TE/2. By reversing the phase of each local signal halfway through, the phase

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accumulation due to a constant eld offset is perfectly refocused for each local spin group. As a result, the effects of local eld offsets are compensated, and the signal decay with increasing TE is described by the true intrinsic T2, with T2 being greater than T2*. This simple picture must be modied to consider how diffusion makes the refocusing incomplete, so that the SE signal does have some sensitivity to the BOLD effect. This is discussed below, after rst describing the more pronounced BOLD effect seen with GRE imaging. The basic effect of increased deoxyhemoglobin creating eld offsets around the vessels and reducing the MR signal can be described as a reduction of T2*. With activation there is less deoxyhemoglobin present, and so the BOLD effect is really a partial lifting of this signal reduction. That is, the signal increase associated with the BOLD effect can be described as an increase in the local T2*. In the literature, the decay of the GRE signal is often written as eTER2*, where R2*, the apparent transverse rate constant, is simply the inverse of T2*. This formulation is simpler to work with in the modeling (considered in more detail below). At 1.5 T, a typical value for T2* in the brain is 50 ms (R2* = 20 s1). With activation, the increase of T2* is described as a decrease of R2*: a negative R2*. A strong activation measured at 1.5 T will create a change in R2* due to the BOLD effect that is on the order of R2* = 1.0 s1.With TE = 40 ms, the fractional change of the MR signal is then about 4%. From the basic arguments above, the eld distortions around vessels due to deoxyhemoglobin should affect the GRE signal, but not the SE signal. However, the physical picture is not yet complete; we must still consider the effects of diffusion and the changes of the intravascular signal, and these effects make the resulting GRE and SE signals more complex than the basic picture above.

Small vein r = 100 m

Venule r = 15 m

Capillary r=3 m

F I G U R E 9-9
Diffusion through magnetic eld gradients affects the GRE signal. Water molecules undergo a random walk, shown as a jagged path, through the magnetic eld gradients around a vessel. The spatial scale of the eld gradient is proportional to the vessel radius, shown as a contour map on the left. The effects of diffusion are important only if the random walk carries the spin through a range of eld offsets during the duration of the experiment TE. On the right is shown a random walk corresponding to a 40 ms duration compared with the spatial scale of capillaries, venules, and small veins. Motion due to diffusion will tend to sample the full range of eld offsets for a capillary but will have little effect for a small vein.

Diffusion of Water Molecules Moderates the GRE-BOLD Effect and Creates the SE-BOLD Effect
Water molecules continually tumble and move about due to random thermal motions, and molecules starting at the same location will spread out over time like a drop of ink in water. Specically, for pure diffusion a group of spins starting at x = 0 at t = 0 will spread into a gaussian distribution centered on x = 0 (there is no shift of the mean position) with a variance that grows with time. This variance is 2 = 2Dt, where D is the diffusion coefcient (approximately 105 cm2/s, or 1 m2/ms in brain). Note that this means that the characteristic width of the distribution, , grows only with the square root of the time. For example, in brain the characteristic distance moved due to diffusion in 10 ms is approximately 4.5 m, and in 100 ms it is approximately 14 m. This diffusion of water molecules is slow, but it does have a measurable effect on the MR signal. To understand the effect, we start by considering a simple GRE pulse sequence used to image a voxel containing magnetized vessels. An excitation pulse is applied at t = 0, and the

image data are acquired at t = TE. To keep the argument simple, imagine that the data acquisition time is very short, so that we are essentially taking a snapshot of the net signal at the time TE. The signal attenuation is directly related to the dispersion of the spin phases at time TE. If there is no diffusion each spin stays at the same location, and precesses in the same eld offset, for the entire duration TE. In this case the distribution of phases is directly proportional to the distribution of eld offsets. If we now introduce diffusion, each spin undergoes a random walk, moving around and sampling different locations (Fig. 9-9). For any one spin the nal phase will correspond to the average eld offset experienced by the spin in its random walkdiffusion effectively smooths the distribution of eld offsets. In the limiting case of very rapid diffusion, each spin wanders all around the vessel, sampling the full range of eld offsets. This reduces the phase dispersion because all the spins have similar histories, regardless of where they started out at t = 0. In this limit there is little phase dispersion and so little signal attenuation. This is called motional narrowing, meaning that the random motions have narrowed the distribution of phases at the time of data acquisition. In short, diffusion moderates the effects of the eld offsets, so the GRE signal is most attenuated when diffusion effects are negligible, and much less attenuated when diffusion effects are prominent (Fig. 9-10). The key physical parameter that denes whether diffusion is important is , the typical time required for a spin to diffuse a distance equal to the radius of the magnetized vessel ( = r 2 / 2D, where r is the radius of the vessel). For example, for a capillary with radius 3 mm, is approximately 4.5 ms in the brain, for a venule with radius 15 mm is approximately 112 ms, and for a small vein with a radius of 100 mm is approximately 5 s (Fig. 9-9 illustrates the relative scale of a random walk path for TE = 40 ms compared with different

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0.8 0.6 S1

S E C T I O N I I P HYSICS , I NSTRUMENTATION , AND A DVANCED T ECHNIQUES Motinal narrowing Static broadening

,R2*
GRE

0.4 0.2 SE 3 10 Radius ( m)

,R2*
30

F I G U R E 9-10
Change in the transverse relaxation rates R2 (for an SE experiment with TE of 100 ms) and R2* (for a GRE experiment with TE of 40 ms) of the extravascular signal as a function of the radius of the vessel creating the magnetic eld distortion. For large vessels (the static broadening regime) the gradient-echo (GRE) effect is large, but the SE effect is small because the 180 pulse is effective in refocusing the signal. For very small vessels (the motional narrowing regime) diffusion ensures that each spin samples the full range of eld offsets around the vessel, and there is little phase dispersion for either GRE or SE signals. The intermediate regime occurs when the typical distance moved due to diffusion is comparable to the size of the vessel, and only in this regime is there an appreciable SE effect. This regime happens to correspond to the capillary and small venule size scale. (Curves adapted from Weisskoff.56 Weisskoff RM: Basic theoretical models of BOLD signal change. In Bandettini PA, Moonen CTW (eds): Functional MRI. Berlin: Springer, 1999.)

vessels). If is much less than TE, so that each spin is able to sample all of the eld offsets around a magnetized vessel, then diffusion effects are important and there is little signal loss due to the eld offsets. On the other hand, if is much greater than TE, then each spin essentially samples only the relatively uniform eld near its starting location, and the larger phase dispersion between spins attenuates the signal. From these arguments we expect that for a typical TE = 40 ms, diffusion reduces the GRE-BOLD effect around capillaries but has little effect on venules and small veins. For SE measurements, the same ideas apply, but the physics is a bit more complicated because of the 180 refocusing pulse. One can think of the action of a 180 pulse as ipping the sign of the precessional phase of a spin. If a spin has acquired a phase 1 during the interval TE/2 due to eld offsets, then after the 180 pulse the phase will be 1. During the next interval TE/2, the spin will pick up an additional phase 2, so the net phase angle at the time of data acquisition (TE) is 2 1. If these accumulated phases are due just to precession with a constant eld offset, then 1 = 2 and the net phase is zeroall spins will be coherent at the time of data collection. When diffusion is added to this simple picture, the two phases 1 and 2 will not be the same, because each is the result of an independent random walk. In short, if eld distortions are on a small enough scale that diffusion becomes important, the spin-echo process does not fully refocus the signal, and so there is attenuation of the SE signal.

In general, the resulting attenuation of the SE signal is hard to model because it depends on the geometry of the eld offsets and the interaction of this geometry with the diffusion process.51-56 However, two limiting cases bracket the effect, and dene the character of the signal attenuation (see Figs 9-9 and 9-10). At one extreme is the case TE is much less than , in which the available diffusion time is much less than the time required for a diffusing spin to sample all of the eld offsets. In this static broadening regime the effects of diffusion are small, the 180 pulse works well to refocus phase offsets, and there is little signal attenuation. At the other extreme,TE is much greater than , each spin samples all of the eld offsets and acquires approximately the same phase in the two periods before and after the 180 pulse. In this motional narrowing case there is again little attenuation, although it is not because of the 180 pulse; as described above, in this regime there is little attenuation of the GRE signal either. The result is that it is only in the intermediate regime, when TE is approximately equal to , that the SE signal is attenuated. Interestingly, diffusion of water around brain capillaries appears to be in this intermediate regime, and for this reason the SE signal has often been viewed as being particularly sensitive to susceptibility changes in the capillary bed rather than larger veins. A particular concern is that a vein draining an activated region may be displaced from the actual site of activity, producing errors in localization, and that for this reason SE-BOLD activations, although weaker, may provide better localization. However, there is one more complicating factor that must be considered: the intrinsic signal change of the blood itself.

Intravascular BOLD Effects Make a Strong Contribution to the Net Signal Change
The discussion above has dealt primarily with the extravascular signal change. The blood has been treated as a uniform medium whose susceptibility depends on hemoglobin saturation. This is a reasonable model for the eld offsets produced outside the vessel, as in Figure 9-7. But the reality, of course, is that the blood is a much more complex medium. The deoxyhemoglobin is sequestered in the erythrocytes, disks a few m across, and the water molecules of blood diffuse around and through the erythrocytes. Because the intravascular water is much closer to the source of the paramagnetic inhomogeneitythe deoxyhemoglobinthe phase changes produced would be expected to be much larger than for the extravascular water. This effect is partly tempered by diffusion effects for GRE signals, but the net signal from blood is much more strongly dependent on the oxygenation of the hemoglobin. While the net signal change from extravascular spins may change by only approximately 1% with activation, the intravascular signal may change by 40%. If the venous blood volume fraction is approximately 3%, a 40% change of the intrinsic intravascular signal would contribute a change of 1.2% to the net signal change. For this reason, at least

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half of the net BOLD signal change at 1.5 T has been estimated to be due to the intrinsic change of the intravascular component.57 Recently, modeling of the SE signal of blood has suggested that most of the signal change seen with a SE experiment at 1.5 T is due to the change of the blood signal.58,59 This complicates the argument that the SE signal is more sensitive to changes at the capillary level. The largest signal change in blood should be in the veins, where the greatest change in oxygenation occurs. Because the extravascular signal change with a SE experiment is small, this suggests that the SE signal may be even more dominated by draining veins than the GRE signal. Note, however, that the contribution of the intravascular signal change becomes smaller at higher magnetic elds. As the magnetic eld increases, the T2* of venous blood becomes very small, so that even with activation the intravascular signal becomes unimportant. In this high eld limit, the SE signal should only depend on extravascular signal changes, and these in turn should depend primarily on changes in oxygenation of the capillary blood. Thus, at high magnetic elds the SE signal, although intrinsically less sensitive to the BOLD effect than the GRE signal, may nevertheless be more specic to the capillary bed and less sensitive to large draining veins.

S eR T E 1 R TE Srest R Ract Rrest

(Eq. 9-4)

The key question is: how does R depend on blood oxygenation and volume? The magnitude of the eld offset B near a magnetized vessel is proportional to B0, the magnetic susceptibility difference between the blood and the surrounding extravascular space multiplied with the main magnetic eld. Experiments indicate that , in turn, can be accurately modeled as having a linear dependence on the local deoxyhemoglobin concentration in blood, and this quantity in turn can be expressed in terms of the change in the oxygen extraction fraction E. Specically, the concentration of deoxyhemoglobin is proportional to E. Then the local eld offsets produced by deoxyhemoglobin should vary as: B B0 E (Eq. 9-5)

Modeling the BOLD Effect

Davis and colleagues introduced a model for the BOLD signal change based on reasonable approximations and the results of numerical simulations.43 Because of its simplicity, the model has proven to be a useful tool for understanding the BOLD effect in a quantitative way. The model starts from the simple picture of how the BOLD effect arises, and relates the signal change to the underlying physiologic variables and a few parameters that describe the local tissue. The MR signal is modeled in the usual way as a simple exponential dependence on the echo time TE, and can be written as: S S0 eTE R2 * * R0 R R2 (Eq. 9-3)

However, the scaling of the eld offsets does not necessarily dene the scaling of the signal attenuation. As spins evolve in an inhomogeneous eld a distribution of phase angles develops, and it is this phase dispersion at the time of data collection that determines the signal change and thus R2*. In particular, as noted above, diffusion effectively smooths the eld distribution to create a narrower spread of phases in a GRE experiment. To model this in an approximate way we follow Davis et al and assume a power law relationship between R and B, the magnitude of the eld distortions: R B (Eq. 9-6)

where S0 is the effective spin density (the signal that would be measured if TE could be reduced to zero). The transverse relaxation rate constant R2* is written as a sum of two terms: R0 is the value of R2* if no deoxyhemoglobin is present, and R describes the additional relaxation produced by deoxyhemoglobin. Note that typically R0 is much larger than R, i.e., the local T2* that describes the decay of the signal is largely determined by the intrinsic T2 and large-scale eld gradients through the voxel, and the additional effect of deoxyhemoglobin is minor. We now assume that with activation R is the only parameter that changes. Using the subscript rest to denote the resting value, and act to denote the activated value, the BOLD signal change with activation, S = Sact Srest, is then:

Numerical simulations54,60 and theoretical analyses55 suggest that when diffusion is not important, approximates to 1. Numerical simulations with diffusion suggest that approximates to 2 gives a better description around the smallest vessels when diffusion effects are signicant. In reality we are dealing with both intravascular and extravascular effects. Note that with GRE methods, the signal change is likely to be largest in the vicinity of small veins, where the change in deoxyhemoglobin is largest. For the extravascular signal we would expect that diffusion is not important, and so 1 would be appropriate. However, within the vessel diffusion is very important, and so 2 would be more appropriate for the intravascular signal change. Numerical simulations suggest that 1.5 is a good approximation for the net change in R at 1.5 T. At higher magnetic elds, the intrinsic signal of the intravascular compartment is reduced, and the BOLD signal is dominated by the extravascular effect, and should approach one. In addition to the change in E with activation, a change in blood volume also affects R. For example, even if the oxygenation of the blood did not change but the venous blood volume increased, the total deoxyhemoglobin would be increased, and we would expect this to increase R and decrease the net MR signal. Numerical

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simulations suggest that a reasonable approximation is to assume that R is proportional to V, the venous blood volume. Combining these arguments, we can model the BOLD signal as43: S Srest TE k Vact B0 Eact k Vrest B0 Erest Vact Eact A 1 V E rest rest

(Eq. 9-7)

where k is the net proportionality constant from all of the proportionalities above,V is blood volume, and A is a local scaling factor given by: A k TE Vrest B0 Erest (Eq. 9-8)

effect are likely present in real data. For example, if the repetition time TR is shorter than the T1 of blood, and the ip angle is large (e.g., 90), the increased delivery of fresh unsaturated blood due to increased CBF could increase the net signal slightly. In addition, the intrinsic signal from arterial blood is larger than the intrinsic signal of the extravascular space, so increasing the arterial blood volume fraction of the voxel also could produce a slight signal increase. Note that both of these effects are due to arterial blood changes, where deoxyhemoglobin is negligible, so these are effects in addition to the BOLD effect. In most applications these effects are thought to be small compared to the BOLD effect, especially at higher magnetic elds, but they may not be negligible.

Dynamics of the BOLD Signal

Equation 9-7 relates the BOLD signal change to the change in oxygen extraction fraction E and blood volume V. We can go one step further and try to relate these two physiologic quantities to the change in CBF. The CBF increase associated with neural activity is triggered by a relaxation of the smooth muscle in the wall of the arterioles. The arterioles provide most of the resistance in the vascular tree and provide a way to quickly decrease the vascular resistance by dilating. As the resistance of the arterioles decreases, the pressure drop across these vessels also decreases, raising the pressure in the capillaries and veins. These vessels may also expand due to the increased pressure, further increasing the CBV. As noted earlier, experimental studies have suggested that the relationship between CBF and CBV can be modeled as a power law (Eq. 9-2) with an exponent that is approximately = 0.38.19 The oxygen extraction fraction E depends on the change in CBF relative to the change in CMRO2. From Equation 9-1 at steady state, this relationship is: Eact CMRO2 act CBFrest Erest CMRO2 rest CBFact (Eq. 9-9)

This nal equation for the BOLD signal change is quite simple, depending on two physiologic changes (the change in blood volume V and oxygen extraction fraction E) and two additional parameters and A. The form of the signal equation directly describes the ceiling effect on the BOLD signal. In simple terms, A is the maximum BOLD signal change that could occur, corresponding to complete removal of deoxyhemoglobin from the voxel. The parameter should be primarily eld dependent, and we can assume that it is not a function of brain region. The parameter A, however, is a local parameter and so may vary across different voxels in the brain. Note that this parameter is proportional to the value of R at rest, the relaxation rate produced by deoxyhemoglobin in the baseline state. This means that the more deoxyhemoglobin is present at rest, the larger the BOLD signal change will be for the same fractional change in V and E with activation. We will come back to this later in the chapter when we consider the effect of the baseline condition on the magnitude of the BOLD effect. At 1.5 T, measured values of A range from 0.08 to 0.22.43,50 In practice, the observed BOLD signal change, S, in brain tissues is somewhat smaller. Signal changes of 1.9 0.7% are seen in brain parenchyma of visual cortex at 1.5 T. This increases superlinearly with increasing B0 eld strength to 3.3 0.2% in visual cortex at 4 T.61 In addition to measuring the BOLD signal, the underlying hemodynamic changes in CBF and CBV can also be measured independently. This is important for calculating the local CMRO2 (see Measuring Cerebral Blood Flow, Cerebral Metabolic Rate of Oxygen, and Cerebral Blood Volume). Additionally, these individual components of the BOLD response can have a superior spatial correspondence with the underlying neural activity than the measured BOLD signal itself (see Exploring the Hemodynamic Response to Brain Activation with MRI). Although this is a very useful model for the BOLD signal, the reader should bear in mind that it does not describe all of the effects that may contribute to the measured signal change in an activation experiment. Specically, small direct effects of CBF and CBV changes on the MR signal that are independent of the BOLD

We can adopt a more compact notation by introducing dimensionless variables in which each physiologic variable is normalized to its value at rest: f = CBFact/CBFrest, v = Vact /Vrest, and m = CMRO2act /CMRO2rest. Then for the resting state f = v = m = 1, and with activation all three variables increase to different degrees, and Equation 9-2 becomes v = f . The BOLD signal can then be expressed as: S m S A 1 v f

(Eq. 9-10)

Equation 9-10 can be used to illustrate the possible dynamics of the BOLD effect. Each of the physiological parameters v, m, and f is a function of time. When a stimulus is applied the state of the brain changes to a new steady state, characterized by particular relationships between these physiologic variables. Empirically, these relationships are:

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vf

f 1 n m1

(Eq. 9-11)

where the rst equation is simply Equation 9-2 in our notation with normalized physiologic variables. The second equation represents the empirical nding that CBF and CMRO2 increase together, but the fractional change in CBF is larger than the fractional change in CMRO2 by a factor of n. Most experimental studies have found n = 2 to 3, although larger values also have been reported.43,44 Equations 9-11 can be taken as steady-state relationships between the physiologic variables. However, during the transitions between one steady state and another the variables may change at different rates, even though they eventually settle into the relationships dened by Equations 9-11. This possibility for different dynamics of the underlying physiologic variables can introduce transient features into the BOLD signal response (Fig. 9-11). For example, a common observation is a dip of the BOLD signal below baseline after the end of the stimulus, referred to as a post-stimulus undershoot, that can take a long time to resolve (more than 30 s). In block design experiments in which the rest period is not sufciently long to allow the undershoot to resolve, the effect appears as an apparent lowering of the baseline after the rst stimulus block. When the CBF response itself is measured with arterial spin labeling techniques, the post-stimulus undershoot is smaller (or not evident) and resolves more quickly. For this reason, the poststimulus undershoot of the BOLD signal cannot be explained in terms of an undershoot of CBF. Two similar models, the balloon model20 and the windkessel model,21 have been proposed to explain this phenomenon as a slow return of CBV to baseline after the stimulus. These models were motivated by the observa-

tion of such a slow return of CBV in a rat model,62 corresponding to the duration of the BOLD poststimulus undershoot, and occurring despite a more rapid return of CBF to baseline. The basic idea is that if CBF and CMRO2 return to baseline values, the oxygenation of venous blood also returns to normal. However, if the total venous blood volume remains elevated, there is more deoxyhemoglobin present, so the signal dips below baseline. Figure 9-11 illustrates this phenomenon with balloon model calculations. A less common nding is a brief initial dip of the BOLD signal prior to the larger signal increase.63-68 The effect is small and not always present,26 but it has stirred interest because it may reect a rapid increase of CMRO2 prior to the CBF increase,69 and this phenomenon may be better localized to the area of increased metabolism (that is, the CBF increase may cover a wider area). Optical studies have found a brief initial increase of deoxyhemoglobin, consistent with the initial dip of the BOLD signal.69-72 This was interpreted as an increase of CMRO2 that precedes the increase in CBF, creating a short increase in the oxygen extraction fraction E. In principle, however, an initial increase in deoxyhemoglobin also could occur due to a rapid rise in blood volume V. Early experimental studies usually showed an increase in deoxyhemoglobin, but no corresponding decrease of oxyhemoglobin, suggesting a combined change of E and V. A more recent study, however, found a corresponding initial decrease in oxyhemoglobin in conjunction with an increase in deoxyhemoglobin, more clearly suggesting a change in E.73 Figure 9-11 shows how a delay of 1 s between the CMRO2 response and the CBF response can produce an initial dip in the BOLD signal.

DESIGN AND ANALYSIS OF BOLD-fMRI EXPERIMENTS Statistical Analysis is Required to Detect Small Signal Changes

1.4 1.2 1 20 1

CBF CMRO2 CBV

1.4 1.2 1 20 1

CBF CMRO2 CBV

40 BOLD signal

40 BOLD signal

0 20 40 Time (s)

0 20 40 Time (s)

F I G U R E 9-11
Dynamic curves for CBF, CMRO2, CBV, and the resulting BOLD signal, showing left, a simple response in which the physiologic variables co-vary with similar time courses, and right, transients due to a CBV change that is slow to return to baseline, creating a post-stimulus undershoot. In addition the CBF response lags behind the CMRO2 by 1 s, producing an initial dip.

The basic data analysis done in fMRI is to examine the time course from each voxel of the image and identify those voxels in which the signal intensity increases when the stimulus is on. The intrinsic difculty with this analysis is that the effect is subtle, with a CBF change of 30% causing a BOLD signal change of only approximately 1% at 1.5 T. Even without any stimulation the MR signal varies, due to a number of physical and physiologic perturbations. Because the BOLD signal change is small, it is necessary to take these perturbations into account when performing a statistical analysis. The key challenge is to distinguish the signal changes due to neural activity in response to the stimulus from all the other random and systematic sources of variation of the MR signal. Hence, the general idea of the statistical analysis is to estimate whether a response to the stimulus pattern accounts for a sufciently large fraction of the measured signal variance to reach statistical signicance. Because the

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statistical analysis is an integral part of an fMRI experiment, in this section a short introduction to the general principles is presented. The MR signal always contains random thermal noise, and this adds random uctuations to the signal that are also on the order of 1%. A primary goal of MR technology development is to improve the intrinsic signal-to-noise ratio (SNR), either by moving to higher magnetic elds or by improvements in RF coil design, such as multichannel coils. However, random thermal noise is not the only source of signal variability, and indeed often is not the primary problem. A number of systematic effects also create signal uctuations, including instrumental effects, such as coil heating, but also physiologic fluctuations. The most important physiologic uctuations are due to heartbeat (approximately 1 Hz), respiration (approximately 0.3 Hz), and the so-called slow oscillations (approximately 0.1 Hz, and discussed further in Exploring the Hemodynamic Response to Brain Activation with MRI). In addition, the appearance of these uctuations in the MR signal is complicated because the data sampling rate (1/TR) is typically not fast enough to resolve the cardiac frequency, and so these uctuations are aliased to lower frequencies in the data. For these reasons, the observed uctuations in the MR signal are a composite of physical and physiologic perturbations. The required statistical analysis is essentially analysis of variance. The signal exhibits uctuations around a mean value, and the variance of these uctuations has several sources: 1. the BOLD uctuations driven by the stimulus, the effect of interest; 2. systematic physiologic and instrumental uctuations, described as confounding effects; and 3. random errors that are taken to be gaussian noise. The aim of the statistical analysis is to determine if there is sufcient evidence to say with condence that the contribution of the effect of interest is different from zero. This is done by calculating a statistic, such as a correlation coefcient, a t-statistic, or a z-score, that essentially measures the ratio of the signal variance that can be attributed to the effect of interest (stimulus-driven BOLD signal changes) to the variance due to noise, after taking into account the variance due to the confounding effects. Note that in this formulation we have specically separated systematic effects from gaussian noise. This is essentially a physics approach, aimed at identifying and measuring all systematic uctuations in order to remove them from the data. For example, key physiologic variables such as the cardiac and respiratory cycles can be measured during data acquisition, and their contributions to the total signal variance can be included in the analysis (see Artifacts and Noise). A more general statistical approach is to consider all of the error signals together as one source of error, but with a non-gaussian distribution. Then the data can be analyzed to empirically derive the statistics of the noise, or smoothed (temporally and/or spatially) to force the distribution closer to gaussian. The combination of these two approaches, the physical and the statistical, provides the best promise for achieving the maximum sensitivity for detecting subtle activations, by carefully removing as many physiologic

sources of variance as possible, and then analyzing variance that remains to determine the distribution. For the rest of this section, which is meant to be simply an introduction, we will assume that the total error consists of random gaussian noise and low-frequency systematic variations of the MR signal.

The General Linear Model Provides a Statistical Framework for Incorporating All the Components of the Signal
The most used statistical method in analyzing fMRI data is the general linear model (GLM).74 Essentially, the GLM regards the measured signal as the weighted sum of different model functions, with unknown weights, and random gaussian noise. The analysis then amounts to tting the collection of models to the data to estimate the weighting factors, or more generally, the unknown parameters that dene the model functions. An illustration of the GLM is shown in Figure 9-12, with model functions shown for the stimulus response, a systematic cardiac signal, and a linear drift. The stimulus response function is usually obtained by convolving the stimulus waveform with an assumed ideal impulse hemodynamic response, which can be represented by one or more of the so-called basis functions. As an example, in Figure 9-13 a gamma-variate hemodynamic response function it with a full width at half maximum (FWHM) of about 4 s and its derivative are shown. The mathematical expression of the gamma-variate h(t) is: ht 1 k h k 1! t
h k

et /

(Eq. 9-12)

Typical values used are k = 3, and h = 1.2. In addition, to cover the delay of the hemodynamic response, a time lag of 1 s is used. Ideally, this delay is simply another parameter of the model function, and should be estimated along with the amplitude. However, the signal does not depend on this parameter in a linear way, so it cannot t into the GLM analysis. A shift of the hemodynamic response function can be approximated in a linear way by including the derivative of it as a second model function. Then to a rst approximation, a nonzero weight for this additional model function shifts it in time. That is, the weighted sum of h(t) and its derivative is similar to a shifted hemodynamic response function (HRF), so by using both functions hemodynamic responses with different delays can be modeled. This is very useful because it is known that the HRFs in different brain areas vary in their delays.75 In mathematical terms, the total MR signal time course from a voxel can be modeled as: Y Y
1 hemodynamic response gaussian noise 1 2

heartbeat (Eq. 9-13a)

X1

X2 e

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Stimulus response

F I G U R E 9-12
Illustration of the general linear model (GLM). Model functions such as an ideal stimulus response, physiologic noise, and scanner drift are tted to measured data assuming that the residual is purely random noise. For each voxel the GLM analysis provides estimates of the weights for each model function that give the best t to the data. The GLM analysis tests whether the estimated amplitude of the stimulus-driven model function is signicantly different from zero for the estimated level of noise.

Measured data 0 50 100 150 200 250 Time (s)

Physiologic noise 0

Random noise 50 100 150 200 250 Time (s)

Scanner drifts 0 50 100 150 200 250 Time (s)

The term Y denotes BOLD signal, the terms Xi the model functions, their weightings, and e the residual noise assumed to be gaussian. In a more compact way this equation can be written in matrix terms: Y X e (Eq. 9-13b)

The matrix X is called the design matrix, the vector Y the observed variable, the term is the weighting vector, and e the noise vector. For each voxel the GLM analysis provides estimates of the weights for each model function that give the best t to the data. The best t is dened in a least-squares sense, such that the sum of the squared residuals, obtained after the model t is subtracted from the data, is minimized. Provided that the noise has a gaussian distribution, the least-squares solution to Equation 9-13 is: X T X 1 X T Y (Eq. 9-14)

activity whatsoever in the voxel, the GLM analysis could produce a non-zero amplitude for the stimulus model function just because of the randomness of the noise. The key question is whether the estimated amplitude is sufciently larger than the range of amplitudes we would expect due to just noise alone. For this purpose the signal contribution due to the stimulus response has to be compared with the noise level. The higher the signalto-noise ratio, the more likely it is that the voxel is truly activated. This can be quantied in terms of a t-statistic,

HRF

The superscript T denotes the transpose of the matrix.

0 5

Derivative 10 Time (s) 15 20

Identifying Activated Voxels

The statistical goal is now to determine if the voxel time course shows a stimulus response, by testing whether the amplitude of the stimulus-driven model function estimated from Equation 9-14 is signicantly different from zero. That is, even if there was no stimulus-driven

F I G U R E 9-13
A gamma-variate hemodynamic response function (HRF) and its derivative. The ideal stimulus response function used in the GLM formalism is usually obtained by convolving the stimulus waveform with an assumed ideal impulse hemodynamic response. A shift of the HRF can be approximated in a linear way by including the derivative of HRF as a second model function.

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whose distribution is known, so that the probability that the tted amplitude could result from chancethe p-valuecan be estimated. For example, a p-value of 0.05 means that by chance there is a 5% probability that this t-value or larger is obtained. The higher the t-value, the higher the signicance and the less likely that this is a chance event, and thus the lower the p-value. An alternative way of describing the signal-to-noise value is the correlation between the model stimulus response and the measured response.76 The correlation coefcient r varies between 1 and 1, with the value 1 being a total correspondence between the reference function and the measured response and the value 1 corresponding to a perfect inverse correlation between the responses. The value r = 0 means that there is no correspondence between the model response and the data. Analyzing the data in terms of either a t-value or a correlation coefcient is equivalent, because r can be transformed to a corresponding t-value using the formula: t v r 1 r2 (Eq. 9-15)

GLM analysis with sine waves as the model functions. The particular advantage of sines and cosines is that they are derivatives of each other, and a shifted sine wave, representing a delayed response, is precisely modeled as a linear combination of a sine and cosine wave with the same frequency. For the more general shape of response described above, the inclusion of the derivative function as another linear model function is only an approximate way to include a shift of the response. For sine waves it is exact.

Statistical Parametrical Maps are Used to Display Activated Voxels

After the time course of each voxel has been analyzed to determine a t-value (or correlation coefcient), the data are displayed as a statistical parametric map. This is typically done by choosing a threshold for the statistic and overlaying the map of voxels passing the threshold on a high-resolution, black and white anatomic image. The colored image is then presented as a map of the brain activation pattern associated with the stimulus. However, it is important to note that this approach to deciding whether a voxel is activated or not is based on a signal-to-noise evaluation, not a pure signal evaluation. For example, it is conceivable that two voxels in different parts of the brain would exhibit the same neural activation and BOLD signal change (say, 1%), but the other sources of signal uctuations are much higher in one voxel than the other. Because the statistical test for whether a voxel is activated measures the signal-tonoise ratio, one voxel could pass the threshold and be identied as activated while the other does not, despite identical levels of BOLD signal change. To put this more precisely, this statistical parametric mapping approach identies voxels in which the signal time course shows a uctuation due to the stimulus that is sufciently large that it is unlikely to happen randomly due to the noise uctuations of the voxel. Thus this is a test that is designed to protect against false positives, but not against false negatives. For this reason, the correct interpretation of a statistical parametric map is that we can say with condence that the colored voxels were activated, but we cannot make that statement for the other voxels. In other words, we cannot conclude from this analysis alone that the other areas were not activated unless we know that the noise level is similar across the brain.

The variable represents the degrees of freedom (dof), which is the number of time points reduced by the number of model functions. The source of this term is that we begin with N independent measurements in the time series, and from these data we estimate k model parameters. This leaves = Nk measurements to estimate the variance of the random gaussian noise (i.e., the variance of what remains after subtracting out the best-t model functions). For this reason the signicance of a given correlation coefcient depends on the degrees of freedom; the higher the dof, the higher is the statistical signicance. If the stimulus is presented in a periodic fashion, another approach for determining if there is a statistically signicant activation is to work in the frequency domain and compare the amplitude at the stimulus frequency with the amplitudes at other frequencies. A useful feature of this approach is that the Fourier transform also provides a phase for each frequency component, and this phase translates directly into the time delay between the frequency components. For a periodic design the main frequency representing the stimulation is the inverse of the time of the stimulation cycle. Hence, the magnitude of this component can be statistically compared with the magnitude of the other frequency components using the t-statistic as described above in order to determine the contribution of the stimulus to the total variance. An additional feature of this frequency analysis is that it can give information about other neuronal populations that have an atypical response to the stimulus pattern. For example, neurons that respond while the stimulus is on will show a strong response at the fundamental frequency of the stimulus, while neurons that respond to a change of stimulus, ring at the beginning and end of the stimulus, will show a strong response at twice the fundamental frequency. While the implementation of the Fourier approach seems different from the GLM approach, it is really just a

Limitations of the General Linear Model

Potentially, there are several problems with the GLM approach deriving from the basic assumptions, which are not necessarily valid for fMRI data obtained on the living human brain. The assumptions and prerequisites of the GLM are: 1. Model functions have to be specied. This requires prior knowledge about which functionshemodynamic response and confoundsshould be included in the design matrix. The hemodynamic response is

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still incompletely understood (see Exploring the Hemodynamic Response to Brain Activation with MRI) and may vary across the brain and with age and disease.75 In order to be exible with the assumed response, two or more hemodynamic model functions (see above) can be used enabling modeling of different hemodynamic responses.74 The other model functions used in the analysis are usually linear and quadratic drifts due to scanner instabilities. The physiologic confounds are more difcult to treat, and it is still an area of active investigation to determine how they translate into uctuations in MR signal time series (see Artifacts and Noise). Several correction schemes have been proposed for removing cardiac and respiratory uctuations (for an overview, see reference 77). 2. The residual noise is assumed to be gaussian, although real fMRI data show a non-gaussian distribution. One approach to deal with this problem is to whiten the noise into a gaussian form by ltering.78 Another approach is to use the data themselves to estimate the distribution. 3. The physical and physiologic noise levels are nonuniformly distributed over the brain areas. Because the statistical signicance is essentially determined by the signal-to-noise ratio, the GLM favors areas in which the noise level is low and the signal sensitivity is high. Smoothing the data spatially reduces the nonuniform distribution of the noise. However, this leads to an effective lowering of the spatial resolution. As a recommendation, if a hypothesis about the activity of one area is specied or discussed, a close look at its time series and averaging data over time and space should be done; this is also the case when the statistical analysis fails to result in signicant voxels in this area. This is especially important when different groups are compared, because their spatial noise distribution and noise level might differ. 4. The statistical analysis is performed voxel-wise. Because of the huge number of voxels (64 64 24 approximates to 100,000 voxels in a typical experiment) some voxels can be active by chance. For example, if p = 0.01 was chosen as the threshold, approximately 1000 voxels would be classied as activated by chance, referred to as the multiple comparison problem. The most conservative approach is to take the multiple comparisons into account with the Bonferroni correction, choosing a much more conservative p-value to determine the threshold. By dividing the desired p-value (e.g., p = 0.01) by the number of voxels, the probability of detecting a false activation anywhere in the brain is reduced to the desired p-value. Because the resulting threshold is usually too strict, several other correction schemes have been proposed. However, the multiple comparison problem cannot be regarded as solved. One way of reducing the problem is to look at clusters of activated voxels, because it is less probable that several voxels are activated by chance than that only one voxel is activated by chance. The bigger the cluster size, the lower is the probability of recording a false activation. A drawback of this approach is that it biases

the analysis to regions where the expected activity is more spatially distributed, such as sensory areas, than to regions of more focal activation,such as small nuclei.

Block Designs versus Event-Related Designs

In nearly all fMRI experiments the hemodynamic response is treated as a linear smoothing lter. The simplest experimental design is to block the stimuli in 20 to 60 s blocks alternated with equal blocks of the rest condition, and repeat this pattern for several cycles. As described above, the typical data analysis strategy is to convolve the stimulus pattern with an assumed impulse response function, and then correlate this estimated response with the time course of the signal from each voxel.76 The BOLD impulse response function is often taken as a gamma-variate function with a width of 4 to 6 s. However, for a block design the assumption of the shape of the impulse response is not critical: if the block length is signicantly longer than the width of the impulse response, then the amplitude of the BOLD response on the plateau of a block will depend just on the area under the impulse response, and not on details of the shape (Fig. 9-14). Most of the fMRI studies done to

Hemodynamic responses

10

15

20

10 20 30 40 50 60 70 80 90

10 20 30 40 50 60 70 80 90 Time (s)

F I G U R E 9-14
Block designs and event-related designs of fMRI experiments. Top, Two possible hemodynamic response functions (HRFs), constructed to have the same area. The lower gures show the resulting time courses when the HRFs are convolved with a block design (middle) and a randomized eventrelated design (bottom). The block design is more efcient for detecting a response from the variance of the signal, while the event-related design is more efcient for estimating the shape of the HRF.

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date have used block designs, and so the measured quantity that is used for mapping activation has been primarily the area of the impulse response. For most of these studies the goal was to detect whether a local piece of tissue was activated, not to characterize the shape of the BOLD response. For many applications a block design is not feasible, or at the very least introduces an articial quality into the stimulus that makes the results difcult to interpret. A trial-based (or event-related) design signicantly broadens the types of neural processes that can be investigated.79-88 A block design by denition presents similar stimuli together, which makes it difcult to study processes where predictability of the stimulus is an important consideration. For example, studies of recognition using familiar stimuli and novel stimuli are hampered if all of the familiar stimuli are presented together. A trial-based design allows randomization of different stimuli and a more sophisticated experimental design. To analyze event-related data one could use the same correlation approach used for block designs, by convolving the stimulus pattern with an assumed hemodynamic response and correlating the resulting model curve with each voxel time course. However, for an event-related design the resulting model curve is much more dependent on the exact shape of the assumed BOLD response, as illustrated in Figure 9-14. Two impulse response functions were constructed to have the same area but different shapes, and then convolved with a block of stimuli and a random presentation of stimuli. For the block design the two predicted responses are similar, but for the event-related design there is a greater difference. The reason for this is that the expected BOLD response depends strongly on the shape of the impulse response only during transitions (e.g., the beginning and end of a block), while the steady state (e.g., the plateau of a block) depends only on the area of the impulse response.With long blocks there are long steady-state periods but only a few transition periods, but with an event-related design there are many transition periods and typically no steady-state periods. The introduction of event-related designs into fMRI experiments has signicantly expanded the eld, but it has also made our lack of understanding of the hemodynamic response a more acute and signicant problem. A fruitful approach for dealing with the uncertainty about the exact shape of the hemodynamic response is to estimate the response from the data.82 Specically, instead of assuming a given shape for the impulse response, with only a single unknown amplitude, the impulse response is treated as set of unknown amplitudes at different time lags after the initiation of a stimulus. That is, the amplitudes of the response 1 s after the stimulus, 2 s after the stimulus, etc., are treated as separate model functions within the GLM. For example, if the impulse response is assumed to last 15 s and it will be estimated at 1 s intervals, this is equivalent to including 15 model functions corresponding to each of the time lags. A hemodynamic response is then estimated for each voxel, and the full response (or a specied range) is then examined to determine if it differs signicantly from zero.

Detection versus Estimation

An interesting feature that emerges from consideration of block and event-related designs is that there are actually two distinct criteria that can be used to evaluate different experimental designs: the detection sensitivity and the estimation sensitivity.89 If we are only interested in detecting whether a response is present, and do not care what the shape of the response is, then the primary measure of efciency is the signal variance produced by the stimulus. For example, Figure 9-14 illustrates two distinct patterns for presenting the same 48 stimuli. For each one we can convolve the pattern with a simple assumed response and then calculate the variance of the resulting model curve. The statistical analysis will then compare this variance to the variance produced by other noise sources to determine if the voxel is activated, so the larger this intrinsic variance the greater the probability of detecting the response. As illustrated in Figure 9-14, blocking the stimuli creates a variance approximately four times larger than the variance for the randomized pattern, and so creates a more detectable response. However, if the goal is to estimate the shape of the response itself, a different criterion is needed to evaluate different stimulus patterns. Dale and colleagues introduced the gure of merit for this purpose; it is calculated just from the pattern of stimuli, and is independent of the shape of the hemodynamic response.82 In the example in Figure 9-14, this gure of merit is approximately four times larger for the random design than for the block design, so the random design is much more efcient for estimating the impulse response. The differences in detection efciency and estimation efciency can be traced to the width of the impulse response, which creates overlap of responses from different events. Such overlap can be good or bad, depending on the type of sensitivity of interest. The variability of the estimation efciency comes primarily from differences in the degree of systematic overlap of responses from different stimuli, such as the poor efciency of a periodic closely spaced single trial design with perfectly regular overlap of responses. The variability of the detection power can be traced to the constructive effects of overlapping responses to different events,as in a block design, where the addition of responses to closely spaced events serves to increase the nal variance of the signal. Both of these sources of variability in the sensitivity of stimulus designs would be removed if we restricted the minimum time between events to be greater than the width of the impulse response. In that case there would be no overlap of responses from different events, and all designs with the same number of events would have the same detection efciency and estimation efciency. Because this is not the case, the design of fMRI experiments is a richer and more complex eld. In general, event-related designs are a powerful and robust approach for fMRI experimental design. Many experiments do not t into a block design, and the variability of the hemodynamic response across brain regions, subjects, and experimental conditions is an important factor to take into account. Event-related

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0.4

0.1 Head neutral 0 -0.1 -0.2

ARTIFACTS AND NOISE fMRI is More Sensitive to Imaging Artifacts than Clinical MRI
Functional MRI of brain activation is one of the more demanding applications for an MRI scanner. It relies on detecting MR signal changes of only a few percent (or less) over a relatively prolonged measurement period, typically 20 to 40 minutes. There are several sources of noise in fMRI; the primary sources are noise introduced by the MRI scanner or associated hardware, and physiologic noise introduced by the subject (e.g., changes in the MR signal due to thermodynamic or electrical noise, or changes in MR signal due to cardiac or respiratory motion). In addition to noise, distortions in the image (which degrade image quality) as well as susceptibility dropouts will also reduce the available SNR. To detect the small perturbations in the MR signal which are associated with fMRI it is critically important that the noise and scan-to-scan variability are well below the signal changes being investigated. This section addresses some of these sources of artifacts and noise and how their inuence can be minimized.

Chin up

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F I G U R E 9-15
Mean registered B0 maps calculated following synthetic linear shimming from ve subjects with head position: chin down (upper panel), head neutral (middle panel), and chin up (lower panel). The improvement in homogeneity as the head tilts up is most evident in the inferior regions of the frontal and temporal lobes, as well as in occipital cortex. (Modied from Tyszka JM, Mamelak AN: Quantication of B0 homogeneity variation with head pitch by registered three-dimensional eld mapping. J Magn Reson 159:213-218, 2002.)

Minimizing and Correcting Image Distortions

MRI relies on a homogeneous magnetic eld to correctly encode the gradient-induced distribution in precessional frequencies of nuclear spins into an image. Inhomogeneities in the static magnetic eld will result in distortions and signal loss in the nal MR image. fMRI utilizes rapid gradient-echo pulse sequences that are particularly sensitive to the microscopic susceptibility perturbations induced by deoxyhemoglobin. This is particularly evident in single-shot EPI or spiral gradientecho sequences, where the read-out window is of the same order as the echo time; these sequences will also be sensitive to bulk susceptibility changes. There is a greater need in fMRI than in most conventional clinical MRI applications to minimize these macroscopic inhomogeneities in the magnetic eld. Functional MRI studies frequently rely on the use of additional materials for monitoring subjects (eye tracking, EEG electrodes, headphones) or to stabilize the head (bite bar, plastic pads). Even though these materials are non-metallic they may have susceptibility different from the adjacent tissues

and cause local eld distortions.93 Materials used inside the MR scanner during fMRI studies must thus be chosen carefully. Even without extraneous materials in the scanner, the patient or subject alone will cause inhomogeneities in the magnetic eld. The close juxtaposition of air, fat, bone, and tissue in the human head is not conducive to maintaining a homogeneous magnetic eld. The susceptibility distortions around the sinuses and petrous parts of the temporal bones are often the most evident and can be difcult to correct, resulting in signal loss in the prefrontal cortex and temporal lobes. The use of highorder shims to minimize bulk susceptibility effects is particularly important, particularly with the use of higheld MRI (3 T and above). In addition to shimming, the position of the head within the static magnetic eld is also important. Tyszka and Mamelak demonstrated that the normal anatomic position used for radiographic positioning for almost a century might actually worsen the inhomogeneities around the prefrontal and temporal cortices. By tipping the head upwards so that the position of the sinus is moved more cranially, the orientation of the induced gradients relative to the B0 eld lines tends to reduce the effects on local eld inhomogeneity.94 Figure 9-15 shows the dependence of B0 homogeneity on head position.94 In addition to externally applied shims, other approaches have been suggested to minimize the local magnetic eld inhomogeneity around the paranasal sinuses.Wilson et al 95,96 proposed the use of local shims in the mouth to reduce the local eld inhomogeneities in inferior frontal lobes from the paranasal sinuses and skull base. Pyrolytic graphite is strongly diamagnetic and can

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designs allow one to do this in a systematic way, and the efciency of potential designs can be evaluated numerically to optimize the experiment in advance.81,82,90-92 Finally, block and random eventrelated designs represent two ends of a spectrum, with one of the key differences being predictability of the stimulus. An intermediate design, with some pseudo-random portions and some pseudo-block portions, can make the stimulus less predictable while retaining some of the detection efciency of a block design.86,89

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Improved B0 homogeneity (upper panel) and image quality on GRE echo-planar images (lower panel) from strategic placement of intraoral local shims (made from continuously nucleated pyrolytic graphite). The B0 range is 0.8 ppm (white) to +0.8 ppm (black). Note the improved B0 homogeneity in the inferior frontal cortex, and corresponding reduced distortion and improved gray matter/white matter interface on the EPI images. (Modied from Wilson JL, Jenkinson M, Jezzard P: Protocol to determine the optimal intraoral passive shim for minimisation of susceptibility artifact in human inferior frontal cortex. Neuroimage 19:1802-1811, 2003. 2003, with permission from Elsevier.)

be used to change the local magnetic eld to minimize local susceptibility-induced gradients. Figure 9-16 shows the improved B0 homogeneity and image quality from strategic placement of local shims against the hard palate. In addition to signal loss, susceptibility differences between tissues also cause distortion in the image as the spatial mapping of the MR signal assumes the magnetic eld is homogeneous. The mislocalization of a particular pixel is predictable if the local magnetic environment is known. An additional measurement made of the actual local eld (the eld map) acquired at the same time the EPI or spiral acquisition is made can then be used to reposition pixels and reduce distortions.97,98

best approach to removing motion artifacts in the MR data is to try to prevent them at the outset. The comfort of the subject in the MR scanner is of paramount importance. Subjects should be dissuaded from drinking excessively prior to the examination, and allowed to empty their bladder immediately before the examination. Although minor discomfort (insufcient padding around the head, knees not well supported, hard examination table, neck over or under extended) may seem trivial at the beginning of the examination, after 30 minutes it may no longer be tolerable, resulting in voluntary and involuntary movement. Many groups also employ a bite-bar or a vacuum pad to further stabilize the head. Although very effective, this requires careful set-up and subject cooperation or it too can become uncomfortable part way through the examination. Subject motion results in displacement or rotation of the image in successive acquisitions. Small degrees of motion can be corrected with image registration. The most common of these are based on the automated image registration (AIR) algorithm.99,100 Slow patient drift within the magnet may appear as a linear drift in the MR time series and can also be compensated for in the general linear model during post-processing. Subject motion that is most difcult to remove is motion that is temporally correlated with the stimulus. This most often occurs with poorly trained subjects or with strong stimuli that produce a startle response coincident with the onset of stimulation (e.g., loud noises, bright lights, or painful somatosensory stimuli). A careful examination of the raw data is needed to ensure that apparent BOLD activation is not biased by patient motion. Excessively large signal changes out of proportion to the expected fMRI response, and apparent activation at contrast and tissue boundaries or in areas not expected to be associated with fMRI activation (e.g., the scalp) are signs of patient motion rather than true activation. Changes on the statistical map that are out of phase on either side of a tissue boundary (i.e., positively correlated signal change on one side of the head, and negatively correlated signal change on the other side of the head) are also signs of gross subject movement.

The fMRI Signal Includes Contribution from Physiologic Fluctuations

A signicant source of artifact in fMRI results from the periodic modulation of the MRI signal by physiologic processes, particularly from respiration and cardiac pulsations.101,102 These periodic modulations increase noise and reduce the statistical signicance of the activation signals,103 and are collectively referred to as physiologic noise. Artifacts due to physiologic effects are well recognized in clinical MRI and a number of strategies have been suggested to try to minimize them, in particular breath-holding, novel k-space trajectories,104 reordering k-space to time lock it with respiration or cardiac motion,105 navigator echoes,106 cardiac and respiratory gating, and ow compensation. However, not all the techniques used in clinical imaging are applicable for

fMRI is Sensitive to Subject Motion

Functional MRI studies are extremely sensitive to subject motion. This is most evident at tissue or contrast boundaries where even sub-pixel movement can generate large perturbations in the MR signal, frequently several times greater than the underlying BOLD signal itself. The

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functional MRI. Use of navigator acquisitions in fMRI requires some additional modication since there is only a short time available between excitation and data acquisition.107 Additionally, gated acquisitions are difcult to implement in fMRI as this results in a variable TR. This introduces additional signal uctuations unless the TR is sufciently long.108 Also signal variations related to motion, such as respiration, are incompletely addressed with ow compensation.107 The problem of physiologic noise requires some additional techniques more specically suited to fMRI. We present here an overview of the effects of this physiologic noise on the MR signal and T2*-weighted images, and consequently on the fMRI statistical power. Strategies to reduce its impact are also addressed. Physiologic motion that occurs during the mapping of MR signals into k-space will result in phase offsets and ghosts in the T2*-weighted images. This is most evident for multi-shot techniques (fast low-angle shot [FLASH] imaging, multi-shot EPI). The ghosts can be identied during a careful inspection of the raw images prior to making statistical maps. Physiologic variation that occurs between successive repetitions of snapshot EPI or spiral acquisitions will tend to lead to periodic variations in signal intensity synchronous with the cardiac or respiratory cycle. The most effective way to observe these effects is by analysis of the frequency distribution by Fourier transform of the fMRI time-series data (the power spectrum). Figure 9-17 shows a power spectrum acquired during a visual task with a stimulus repetition rate of 0.016 Hz. The fundamental frequency of the stimulus is clearly seen in the spectrum. Additionally, periodic variations in MRI signal are also seen at 0.85 to 0.95 Hz and at 0.15 to 0.25 Hz, corresponding to cardiac and respiratory components. A number of processes lead to this periodic variation in the MR signal. For respiratory pulsations, the dominant effect is from susceptibility changes originating in the chest due to the cyclical change in the proportion of water and air. This leads to a magnetic gradient in the z-direction, and results in small amounts of intra-slice dephasing and minor misregistration of slice position (for axial images) or distortions (for sagittal and coronal images).77 In addition to the periodic modulation of the eld homogeneity, there is also evidence that the brain physically moves between 110 and 950 m during the respiratory and cardiac cycle.109 Changes in intrathoracic pressure are transmitted to the head as changes in intravascular and CSF pressure, with secondary cyclical effects on cerebral blood ow. Since the volume of the calvaria is xed, this results in displacement of brain parenchyma. In addition, depending on the positioning of the subject, movement in the anterior chest wall may be directly transmitted as a pitching head motion. Cardiac-induced susceptibility changes are signicantly smaller, and the dominant transmission of cardiac artifacts to the brain is a direct result of uctuation in cerebral blood volume and oxygenation (and consequently brain and CSF volume) within the calvaria. An additional source of physiologic noise that has received increasing attention in recent years is slow oscillations, synonymously referred to as vasomotion, V-signal,

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Frequency distribution of the MR signal from a 4 mm3 region in primary visual cortex during a visual functional MRI experiment (alternating 20 s ickering checkerboard, 40 s darkness). Note spectral power in the 0.15 to 0.25 Hz band corresponding to respiration, in the 0.85 to 0.95 Hz band corresponding to cardiac pulsations, and at 0.016 Hz corresponding to the fundamental frequency of the stimulus. Slow oscillations are seen around 0.1 Hz. (The relative power at the different frequency bands varies with choice of region of interest and subject.)

spontaneous oscillation, and low-frequency waves (see reference 110 for overview). These oscillations are centered on 0.1 Hz, and their origin is still under debate (metabolic, vascular, or neuronal). Even lower frequency drifts have been described, and these may also represent physiologic noise or patient drift. However as these effects are also seen in cadaveric studies,111 they can be very difcult to distinguish from signal drifts originating within the MRI hardware.

Correcting for Physiologic Noise

Strategies to reduce the effects of physiologic noise can be broadly divided into methods to remove the effects beforehand, and methods to remove the effects using post-processing. Training subjects to maintain steady breathing and to avoid excessive, large excursions in chest motion will help reduce some of the larger signal changes from respiratory motion. Careful positioning and head restraint (ideally using a bite-bar) will also help to minimize pitching motions of the head during thoracic wall motion. Since the predominant effect of physiologic noise is to cause a phase shift in successive image acquisitions, Hu and Kim107 proposed applying navigator echoes to track these induced phase variations. Acquiring an additional line at the center of k-space (for gradient-echo [FLASH] acquisitions) or an additional data point at the center of k-space (for EPI acquisitions) before the phase-encode gradient allows the phase offset due to motion or B0 uctuation to be quantied and thus

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accounted for. This can reduce the spectral power due to physiologic noise by 30% to 50% (depending on exact location). Imaging sequences that collect additional measurements of the center of k-space can also be used to remove the phase offsets from periodic noise. For example, a rosette trajectory that over-samples the center of k-space allows a 25% to 62% reduction in the standard deviation of the noise.104 Removing respiratory and cardiac motion with gated acquisitions is not widely used in fMRI as this induces a variation in TR, introducing signal modulation due to variable longitudinal relaxation and thus increasing the variance in the fMRI signal. For imaging the brainstem, where there is signicant brain motion with the cardiorespiratory cycle, Guimaraes et al proposed a combination of prospective cardiac gating and retrospective correction for the variable longitudinal relaxation based on recordings of the actual imaging TR for each acquisition.112 Several strategies have also been proposed to remove physiologic noise retrospectively. An examination of the power spectrum (see Fig. 9-17) shows that the physiologic noise often falls into well-dened frequency bands. It would appear that a simple approach to remove physiologic noise is selective digital ltering of a specic frequency band. The sampling rate for the physiologic fluctuations is determined by the imaging TR. For a typical MR experiment this is typically 1 to 2 s, corresponding to a temporal resolution of between 0.5 and 0.25 Hz. As the cardiac rate is around 1 to 2 Hz, this will be undersampled at this TR and the signal will be aliased.113 The aliased signal will no longer appear in the same welldened frequency band and may also be superimposed at the fundamental frequency of the stimulus/response, making it more difcult to selectively lter. Several approaches have been proposed to address these aliased noise signals. By repeating the task at a different rate, the effective sampling rate is changed and the degree of aliasing may be reduced or the position in the frequency spectrum where physiologic noise signals become superimposed can be shifted. Frank et al proposed a technique that utilizes the fact that physiologic noise is spatially correlated across large areas of the brain and treats adjacent slices as phase-shifted additional samples, increasing the effective sampling rate of physiologic noise and allowing previously aliased signals to be retrieved and removed.114 Biswal and colleagues measured the cardiac and respiratory contributions separately by resampling contemporaneous pulse oximeter waveforms recordings at the same sampling rate as the imaging TR, time-locked to the fMRI signal acquisition.113 The frequency location of aliased physiologic signals could then be determined, allowing them to be selectively ltered. The relative under-sampling of noise data is a signicant limitation to identifying and characterizing physiologic noise. An alternative approach is to record physiologic and cardiac signals as a separate time series using a photoplethysmograph and pneumatic belt to record cardiac pulsations and respiratory excursions respectively. The cardiac and respiratory changes can then be sampled at a considerably higher rate, and their contribution to the MRI signal estimated and removed.

Mitra and Pesaran propose that since cardiac and respiratory effects are approximately periodic in nature, they can be modeled in terms of slow amplitude- and frequency-modulated sinusoids.115 The contributions to the MR signal can be removed from the data in the frequency domain. Other techniques take a similar approach and characterize the cardiorespiratory contribution from a separate time series, but remove the noise using image-based techniques and are thus less reliant on the periodicity of the noise. A second-order Fourier series is expanded in terms of cardiac and respiratory phases (calculated from the separate cardiac and respiratory activity measured during the fMRI study). The weights of the Fourier terms can be estimated with a general linear model and are then applied to the image116 or the k-space data.108 Another approach involves decomposition of the imaging data into its principal or independent components115,117,118 This differs from the image and frequency domain techniques described so far in being data driven (rather than being constrained to a particular model, waveform, or time course). However, removing selected components with such a decomposition of the data must be done cautiously as the noise component is not necessarily uniquely contained in any single component.115 No single technique will remove all the physiologic noise from every data acquisition. Thus, whichever approach is employed, it remains imperative to carefully examine the raw data visually (looking at the raw images, or a movie of the images and the power spectra of the data series) both as a crude check of the quality of data collection and to direct further analysis.

Scanner Stability and Thermal Noise

The delity and stability demanded for good fMRI are not typically included in the quality assurance (QA) specications of a routine clinical MR scanner. However, with the more widespread use of fMRI in clinical and neuroscience applications, it is now more important than ever to address the performance capabilities of the MR instrument and hardware chain. Ensuring homogeneous scanner performance also provides quality assurance for longitudinal investigations, studies across multiple subjects, or comparison of measurements from different institutions. A number of techniques have appeared in the literature in recent years to address MR scanner stability and baseline performance, but their implementation has traditionally been guided by the needs of individual scanner operators. A more widespread initiative is required to standardize these measures and facilitate comparison of scanner performance from different manufacturers, from different sites, or obtained at different time points. The Biomedical Informatics Research Network (BIRN) (http://www.nbirn.org) is an initiative sponsored by the National Institutes of Health (NIH) and National Center for Research Resources (NCRR) that facilitates large-scale biomedical science collaborations. Standardization of MR scanner performance has been one of the foci of attention of BIRN. As well as addressing stability of a

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particular scanner, BIRN also proposes to standardize levels of performance for fMRI across centers, allowing studies from different sites to be compared. To address the noise and variation introduced into an fMRI study by the MR instrument itself, four basic measures have been identied by BIRN to measure scanner performance and ongoing stability: 1. the root mean square (RMS) signal stability over a region of interest (typically 20 20 pixels) expressed as percent uctuation; 2. the signal-to-uctuation-noise ratio (SFNR) calculated for each pixel and averaged over the same region of interest; 3. the correlation diameter (rdc); and 4. the drift in the signal over time, calculated per pixel and expressed as a percent change. The basis of these tests,and a suitable protocol for making the measurements, are presented here. Quality assurance measurements are done on a 17 cm spherical phantom lled with agar gel using a standard head coil. It is recommended that the tests be performed at least weekly (ideally, daily) to quickly identify adverse trends that may affect scanner performance. The imaging parameters for the test are similar to those used in a typical fMRI study. Thirty-ve contiguous 4 mm slices are acquired axially through the phantom using a single-shot EPI or spiral gradient-echo imaging sequence. The acquisition is repeated for 200 time frames with eld of view (FOV) of 22 cm, matrix of 64 64,TR of 3000 ms,TE of 30 ms (at 3 T) or 40 ms (at 1.5 T), and ip angle of 90. The bandwidth (BW) is 100 kHz or higher. Acquisition time is typically 10 minutes. Through-time measurements of MR signal variation in the central slice are used to calculate RMS stability, drift, mean signal, and SNR. The rst two time points are ignored to allow longitudinal magnetization to reach steady state. The RMS stability is measured as the uctuation over time of the average signal from a 20 20 pixel region of interest (ROI). Trends are removed by tting a secondorder polynomial. The residual uctuation is expressed as a percentage. Typically, this should be less than 0.15%. This provides a measure of the uctuation over time of the average signal across 400 pixels. This is useful to detect global uctuations in the MR signal, for example, due to instabilities in the RF ampliers, receiver gain instabilities, shim instabilities, or timing errors in the effective echo time. The SFNR is calculated from maps of signal mean and signal standard deviation across the 198 time points. For each pixel within a 20 20 ROI the ratio of mean to standard deviation is calculated and the average value of this ratio is reported as SFNR. The SFNR should be 200 or greater. The SFNR is an important metric for fMRI. It describes the variance in a single pixel independent of any functional activation and thus directly inuences the statistical power of any activation-induced changes. The RMS and SFNR measures are both used to examine spatially varying effects induced by the MR scanner hardware. SFNR is different from the RMS measurement in that this is the average of the uctuations in 400 pixels (rather than the uctuation of the average). Effects that act globally such as hardware instabilities will tend to inuence both measures. Effects that act more locally such as random noise in the image will be more evident in the SFNR than the RMS measurement.

The rdc (correlation diameter) is a measure of the spatial auto-correlation of the noise in the MR signal over the 20-pixel diameter of the sampled ROI. If the variation in the MRI signal was purely thermodynamic noise (i.e., uncorrelated noise), then this could be reduced by simple averaging (the basis for improving imaging SNR in clinical scanning by increasing the NEX value). In this case, averaging adjacent pixels together would decrease the relative noise standard deviation in proportion to the square root of the number of pixels averaged (i.e., a fourfold increase in pixels would decrease the relative noise standard deviation two-fold). Other sources of correlated noise, such as uctuations in amplier performance or spikes from the gradients, will tend to affect large numbers of pixels similarly. Thus averaging across several pixels will not reduce this spatially correlated noise.119 For an ideal scanner, a plot of noise standard deviation against number of pixels would follow an inverse square root relationship. Any deviation from this relationship indicates the presence of correlated noise. The point at which the plot of noise versus ROI width deviates from this ideal negative square root relationship is a quantitative indication of the degree of correlated noise present in the MR system. This is expressed as a pixel diameter the correlation diameter or rdc. For an ideal scanner, this value will be 20 (or higher if larger ROI measurements were taken) indicating that even up to a diameter of 20 (or more) pixels, there is still an SNR benet from pixel averaging (i.e., presence of uncorrelated noise). A poorly performing scanner will have a low value of 1 or 2 pixels, indicating that the noise is strongly correlated. A well-performing scanner should have a rdc value of 5 or higher; a value lower than this is an indication of larger correlated noise contributions from the system hardware. Deterioration in the rdc value indicates potential instabilities in subsystem hardware components such as gradients, RF ampliers, or resistive shims. Drifts in the MR signal over time are also important. These may be caused by problems in the ampliers or shims, or may indicate drifts of the B0 eld itself. Problems in the gradients, which cause excessive heating, can also be seen as drifts in the MR signal and the heating may also change the temperature of the resistive shims, exacerbating any drifts in signal. Drift is calculated on a pixelby-pixel basis and expressed as a percent change over a dened time interval. Figure 9-18 shows plots of signal uctuation and frequency spectra for a clinical 3 T scanner while operating at a baseline level for that scanner, and subsequently when image quality was degraded by large noise contributions. EPI acquisitions are prone to ghosting artifacts, an apparent displacement of some of the image intensity by half of the eld of view (FOV). Ghosting in EPI images is another source of noise in fMRI, and needs to be tightly controlled (typically less than 2% to 3% signal power in the ghosts). An analysis of the frequency spectrum of any scanner noise or ghosting is valuable in determining its origin and signicance. Thermodynamic noise is uncorrelated and has equal amplitude at all frequencies. Hardware noise often occurs at a particular frequency and will thus appear as individual spikes in the frequency spectrum (see Fig. 9-18).

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Two examples of measurements of scanner stability taken on the same 3 T MR scanner on different days (day 1: A and B; day 2: C and D). On day 1, signal uctuation (A) is less than 0.15% (0.08%) and there is minor drift in the baseline signal over the 200 acquisitions (600 s). SFNR is greater than 200, and the spectrum (B) shows the noise power is low and white. The RDC measure was 2.2 pixels (data not shown). These numbers can then be used as the baseline indicators for scanner performance on subsequent days. On day 2, signal drift (C) over 600 seconds has now increased (6.58), and the noise uctuation is now greater than 0.15% (0.25%). The reason for this is apparent on the noise power spectrum (D), which is no longer white and shows large spectral power at 0.02 Hz (excessive low-frequency oscillatory noise). The RDC value was lower (1.2 pixels), indicating the noise is more spatially correlated across pixels than previously.

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Additional measurements that should be recorded in the regular quality assurance are the transmitter gain and receiver gain used for the EPI measurements on the standard phantom and the center frequency of the scanner. Minor statistical uctuations in all of these parameters are expected on a day-to-day basis, however the ability to monitor trends can identify emerging problems with scanner delity even before image quality is adversely affected.

MEASURING CEREBRAL BLOOD FLOW, CEREBRAL METABOLIC RATE OF OXYGEN, AND CEREBRAL BLOOD VOLUME Cerebral Blood Flow
Cerebral blood ow (CBF) studies using 15O-labeled water as the contrast agent have been used extensively for functional mapping with positron emission tomography (PET).120 The magnetic resonance counterpart to this

PET technique is to magnetically label (spin label) arterial water with an applied RF pulse.121 In a typical implementation, the magnetization of blood is inverted on the proximal side of the slice, and after a delay of 1 to 1.5 s to allow labeled spin to ow into the slice, the image is acquired. The experiment is then repeated without the inversion, and a subtraction of these tagged and non-tagged images then yields a net signal proportional to the arterial spin delivered to the voxel. For this reason the difference image directly reects CBF. Arterial spin labeling (ASL) can be used to measure the baseline CBF, as well as activation-dependent changes in CBF. Because most of the water molecules delivered to a capillary bed in the brain are extracted, and because the inow time in these experiments is very short, few if any of the tagged spins reach the venous side of the vasculature. For this reason, the ASL signal reects delivery rather than clearance, so the ASL signal is localized in parenchyma rather than draining veins. Images of CBF using the ASL technique result in improved spatial correlation with brain parenchyma compared with conventional BOLD fMRI in both human122 and animal studies.123 ASL also provides a quantitative means to study the mechanisms underlying

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the BOLD technique itself.50,124 A number of ASL techniques have been developed; they can be broadly divided into continuous and pulsed tagging techniques. In pulsed techniques the inversion is done with a slice selective 180 pulse, and with continuous labeling the inversion is done adiabatically as the blood moves through a gradient eld during a continuous RF pulse. Both classes of ASL have been used for measuring CBF changes in humans following neuronal activation. The durations of CBF and BOLD signals are similar20 (see Fig. 9-11) and the same stimulus design can be adapted for both BOLD and ow activation measurements.125

sign to that of the BOLD signal, and thus causes a small underestimation of the BOLD signal. Conversely, the BOLD effects can also contaminate perfusion measurements. This effect is smaller than the effect of ow on the BOLD signal. To a rst approximation, the BOLD effect causes a simple scaling of the MR signal on the order of 2% to 4% during activation, which is just at the limit of detectability.122

Assessment of Cerebral Blood Volume Using an Exogenous Contrast Agent

The earliest implementations of functional MRI used contrast based on cerebral blood volume (CBV) rather than blood oxygenation. These utilized the intensity of signal change during the rst-pass passage of an intravenous bolus of paramagnetic contrast agent through the brain and tracer kinetics analysis to estimate changes in CBV.127 The passage takes approximately a minute to complete, and additional time is required for clearance of the contrast agent before the measurement can be repeated. Temporal resolution is thus limited, and this approach provides only a snapshot measure of cerebral blood volume. These rst-pass measurements of dynamic susceptibility contrast using gadolinium can be useful for assessing resting blood volume or abnormal CBV/CBF associated with tumor neovascularization or cerebral ischemia,128 but they lack the temporal resolution necessary to examine CBV changes during neuronal activation. Greater temporal resolution can be achieved with intravascular contrast agents that maintain a prolonged steady-state concentration. Depending on the type of contrast agent used, the sensitivity to CBV can alter the T1 or T2 relaxation time; however, the use of T2 agents appears to give better sensitivity to CBV changes than does the use of T1 agents.62 Dextran-coated monocrystalline iron oxide nanoparticles (MION) affect T2 relaxation and have a prolonged blood half-life (ranging from 3 to 15 hours in monkeys129,130), making them very suitable as susceptibility agents for measuring CBV. At a sufciently large dose the contribution to blood magnetization from the contrast agent far exceeds that from deoxyhemoglobin, so T2*-weighted MR sequences are rendered insensitive to changes in deoxyhemoglobin concentration. Flow changes also have no effect (the concentration of paramagnetic agent is essentially the same in the arterial and venous circulation, so increased ow does not affect susceptibility). The imaging contrast is thus only sensitive to the concentration of paramagnetic agent in the imaging voxel. As local CBV increases, the vascular proportion of the voxel (and hence the observed amount of contrast agent) increases. Since T2 agents shorten T2 and T2*, the effect of increased blood volume (and increased iron concentration) is to reduce the signal in the voxel, thus neuronal activation is associated with a decrease in MR signal for these CBV-weighted images.129 The signal advantage of the CBV measurement over BOLD for fMRI is most evident at low B0 eld strengths. Several factors contribute to this. First, the effect of the

Calibrating the BOLD Signal to Measure CMRO2 Changes

As noted above, ASL techniques have been useful for unraveling the possible sources of transients in the BOLD experiment. But if we now return to the consideration of steady-state changes, Equation 9-10 can be used as the basis for calibrating the BOLD effect and estimating the CMRO2 change with activation. Consider a block design experiment in which the response reaches a plateau steady-state value and both the BOLD signal change S/S and the CBF change ( f ) are measured for each voxel. If we assume that v is related to f by the empirical relationship of Equation 9-11 there are then only two unknown quantities in Equation 9-10: A, the local scaling factor, and m, the normalized change in CMRO2. The innovation of Davis et al43 was to show that the local scaling factor A can be measured with a separate experiment, and the combination of measured values of A, S/S, and f then provides an estimate of the change in CMRO2 (m) through Equation 9-10. The trick for measuring A is to make use of the fact that inhalation of CO2 causes a pronounced increase in CBF, but apparently no change in CMRO2.126 The experiment is then to use the same ASL/BOLD imaging with CO2 as a challenge. Equation 9-10 is then applied with the assumption that m = 1 (no CMRO2 change), so the measured BOLD signal and CBF changes with CO2 allow calculation of the local value of A. This calibrated BOLD approach has been used very effectively to show that the CBF increase with activation is 2 to 3 times larger than the CMRO2 increase.43,44 The limitation of this technique is that, currently, only a limited number of slices can be imaged at a time, and the slices chosen need to be as orthogonal as possible to the feeding artery for optimal spin-tagging.122 Although the magnitude of the physiologic change is higher, the MR signal, and hence the available SNR, is lower for quantitative CBF measures.122 The CBF and BOLD signals from this combined technique are not without some crosstalk. The T1 of blood is known to be longer than that of tissue, so there is some residual negative ow weighting in the average due to exchange of blood water with tissue water. Typically, the ow signal in the average of tag and control images is approximately 18% of the ow signal in the difference signal between tag and control states. This ow contribution has the opposite

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BOLD signal (which increases with functional activation) is opposite to the CBV signal (which reduces with activation). A BOLD signal is still present, but the larger negative signal change from CBV measurement dominates any positive signal change due to BOLD. BOLD contamination of the CBV signal is more pronounced at higher B0 eld strength for a given dose of contrast agent.62 Second, as the effect of the contrast agent is to decrease the MR signal, the injected dose of contrast agent needs to be reduced at large B0 elds, or the MR signal becomes too small to measure above the noise. Finally, the maximum CBV signal change is constrained by the actual physiologic CBV change (which, unlike BOLD, is eld independent). The signal boosting effect of contrast agent will be less when the BOLD effect is highest (at high B0 eld), and the maximum improvement in SNR is thus seen at lower eld strengths.62 Threefold increases in signal at 1.5 T and 3 T have been reported compared to BOLD studies.129,130 Since the BOLD signal and contrast-enhanced CBV signal have opposite sign, enough contrast agent must be used to overcome the BOLD signal. Using high-dose contrast and short echo times at 1.5 T, vefold increases in SNR have been observed.131 This increased SNR allows higher resolution imaging, and voxel dimensions of 2 mm 2 mm 2 mm are achievable at 1.5 T with this method.131 Signals from large veins relax very quickly and do not contribute signicantly to the image, so the CBV signals are thus better spatially correlated with brain parenchyma than BOLD fMRI.62 In addition to providing improved SNR for functional MRI studies, this method also allows the actual CBV change to be calculated, and like CBF measures of functional activity, the CBV change provides quantitative information. By measuring the iron concentration in venous blood it is possible to calibrate the R2* relaxation rate, and thus get an absolute measure of CBV change with neuronal activation (the CBV can be calculated from the slope of R2* change with [Fe]blood (for derivation see references 62 and 129). S a 1 1n TE S r CBV CBV r R2 * r R2 * 0

Assessment of Cerebral Blood Volume Using an Endogenous Contrast Agent

In recent years, noninvasive techniques have been proposed using the blood itself as the contrast agent to determine the CBV. These are based on nulling the blood pool signal using ow-weighting gradients132,133 or inversion recovery.134 Determining the activation-induced change in CBV from the nulled blood pool signal is not trivial. The ow-weighting method is based on the observation that the application of ow-weighting gradients leads to a greater attenuation of blood signal than of brain parenchyma because of the intravoxel dephasing of randomly oriented vessels in the microvasculature. Thus, the difference of images acquired with and without ow-weighting gradients yields an image that is proportional to CBV. Confounding factors include slight diffusion weighting of brain parenchyma, artifacts due to the pulsatility of CSF, and activation-related changes in the T2 of venous blood. To address these confounds, Liu et al132 used a T2-FLAIR preparation sequence135 followed by a spin-echo sequence with a spiral readout to null out the CSF signal and compensate for blood T2 changes. The diffusion weighting of parenchyma was minimal and could be compensated for by a multiplicative term based on the average diffusion coefcient for brain tissue. A remaining confounding factor is the increased attenuation of blood signal due to the activation-related increase of microvascular blood velocities, which can result in an overestimate of the CBV change. Calculations based on current knowledge of the microvasculature architecture indicate that this factor is small; however, a comparison of the owweighting method with CBV measurements from the MION measurement described above would provide an independent validation of the accuracy of the method. The vascular space occupancy (VASO) method proposed by Lu et al134 uses a nonselective inversion pulse to null out the blood signal. Because the T1 of blood has been shown to be independent of oxygenation, this pulse nulls out blood in all compartments of the microvasculature. The remaining VASO signal is composed of brain parenchyma and CSF. As CBV increases, the VASO signal should decrease as parenchyma is displaced by blood. In experiments with healthy human volunteers, Lu et al found that the VASO signal decreases with hypercapnia, increases with hypocapnia, and decreases with visual stimulation. The temporal dynamics of the VASO signal with visual activation were consistent with a rapid increase in arteriolar volume upon onset of activation and a slow decrease in venous volume at the end of activation. Although the VASO signal provides an indication of the dynamics of CBV, it does not currently provide a quantitative measurement of either the percent change in CBV or the absolute values of CBV in the rest and active conditions. A comparison of the VASO signal to CBV measurements obtained with MION has the potential to provide a calibration method for the VASO method. At present these techniques for fMRI based on CBV change are less widely used for human subjects than BOLD and CBF studies.

(Eq. 9-16)

R2* is the relaxation rate at rest (r), during activation (a), and without MION prior to the start of the experiment (0). For photic stimulation from a 6-second duration 8 Hz ickering checkerboard, the CBV change in macaque primary visual cortex using the iron oxide contrast agent method is 32%. This value corresponds closely to the CBV changes measure in humans127 using the rst-pass gadolinium contrast agent method. Iron oxide contrast media enter the normal iron stores in the body and are excreted by the liver. The large dose needed to overcome the signal from BOLD (approximately 5 to 7 mg Fe/kg at 1.5 T) would rapidly lead to overload of the normal iron stores. This has limited these iron oxidebased methods to studies in animal models.

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Magnetic Resonance Spectroscopy

MR methods other than fMRI can be utilized to measure functional activity and also provide measures of additional aspects of cerebral physiology. A number of studies have demonstrated stimulus-related changes in cerebral metabolites, in particular reduced glucose and elevated lactate levels, following visual,136,137 somatosensory,138-140 motor,141 auditory,142 and cognitive stimulation.143,144 Recent functional MR spectroscopy studies have improved the temporal resolution using time-resolved145,146 or EPIbased techniques.142,147 These show good spatial concordance of spectroscopic changes with BOLD fMRI.147 The SNR for spectroscopic techniques is lower than with BOLD, and thus there is reduced spatial and/or temporal resolution. However, they allow investigation of additional cerebral parameters associated with neuronal activation, and thus provide further insight into control of cerebral metabolism.

5 mm

Diffusion
Diffusion tensor MRI (DTI) provides quantitative maps of natural microscopic displacements of water molecules that occur in tissues as part of the physical diffusion process. During typical diffusion times of 50 to 100 ms, unrestricted water molecules move approximately 15 m.148 Measurement of any restriction in water diffusion reveals microscopic architectural details about tissue structure. Although more widely used to diagnose cerebral ischemia149-151 or to map white matter tract architecture,152 functional changes in diffusion anisotropy may also be used to image cerebral activation.153 Direct optical measurements in animals demonstrate an early change in photon scattering following neuronal activation. Changes in neuronal volume, particularly at the axon hillock, have been proposed as a possible mechanism for this observation.154,155 Small changes (less than 1%) seen in the apparent diffusion coefcient (ADC) following visual stimulation may reect this change in neuronal and glial cell shape.156 This MRI technique thus provides another way of interrogating changes in cellular physiology associated with neuronal activation.

F I G U R E 9-19
T1-weighted axial-oblique image of the brain of a mouse at 7 tesla following injection of 1 L of manganese chloride into the anterior chamber of the right eye. Manganese is a T1 contrast agent that is transported both antegradely and retrogradely along activated neurons. The globe, retina, and optic tracts are enhanced by the contrast agent. (Image courtesy of Miriam Scadeng and James Lindsey.)

tracts and crosses synaptic junctions. Synaptic transport of manganese parallels neuronal activity,161 thus providing images based on actual functional connectivity. Figure 9-19 shows retinal and optic tract enhancement in a mouse after injection of manganese chloride into the anterior chamber of the eye. At present the technique is limited to animal studies due to the need to deliver the manganese directly into the tract under investigation and also toxicity associated with manganese. With further improvements in mechanisms for delivery and safety, this technique holds promise for studying functional connectivity in normal human subjects and patients.

Manganese Tract Tracing

As described above, measures of diffusion anisotropy can provide architectural detail of white matter tracts. The ber maps generated using this DTI technique impart structural connectivity information based on diffusion of water, but do not necessarily detail functional connectivity. Manganese tracking is an emerging MRI technique that maps functional white matter connectivity by tracking the transport of manganese chloride contrast agent.157-160 Manganese is a T1 contrast agent which acts as a calcium analog. It is transported both anterogradely and retrogradely along neuronal

EXPLORING THE HEMODYNAMIC RESPONSE TO BRAIN ACTIVATION WITH MRI

In the previous sections we introduced and reviewed the current thinking about fMRI signals, their physiologic basis, and the analytic methods typically employed. In this section we discuss concepts and issues that are currently being debated and researched. By its nature, the selection of the topics discussed is somewhat arbitrary.

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Are Oxygen and Glucose Metabolism Linked during Increased Neural Activity?
As mentioned earlier (see The Physiologic Basis of fMRI), recent evidence suggests that CBF and CMRO2 change in a graded fashion with different neural activity states, the CBF change being approximately twice as large as the CMRO2 change. These data provide some support for the general idea that neural activity leads to increased energy demand and that CBF increases to provide the oxygen and glucose for energy metabolism, and suggests that neuronal activity changes are appropriately reected by CBF changes. However, the link between oxygen metabolism, glucose metabolism, and neuronal activity is yet not well established. The primary expenditure of energy is required to restore the ion gradients degraded during neural activation. The intracellular/extracellular Na+ gradient is far from equilibrium, so pumping Na+ against this gradient is a strongly uphill reaction in a thermodynamic sense. For this reason, the most costly aspect of neural activity is likely to be excitatory synaptic activity in which glutamate opens sodium channels. Indeed, the action of the Na+/K+ pump is thought to consume a large fraction of the ATP energy budget in the brain.14 In a recent animal experiment, blocking voltage-dependent sodium channels substantially reduced the CBF response,162 supporting the idea that the dominant energy-consuming process in the brain is recovery from excitatory activity. Glucose metabolism has attracted less attention than oxygen metabolism from the MRI community, although 13 C spectroscopy techniques show considerable promise for addressing critical questions.163,164 The delivery of glucose is not as limited as that of oxygen (see The Function of Neurovascular Coupling and the Oxygen Limitation Model). In fact, almost half of the glucose extracted from the blood vessels is not metabolized and diffuses back into the venous blood. This means that, at least in principle, an increase in glucose metabolism can be accomplished without a concomitant increase in CBF. In animal experiments in which the CBF response to activation was blocked, the glucose metabolic rate nevertheless increased by the same amount as it did when the CBF change was not blocked.165 Thus, within certain physiologic limits, the glucose metabolism change might be functionally independent of the CBF. Despite this independence during activation, at baseline CBF and CMRGlc appear to be well matched. The glucose transporters Glut1 and Glut3 are mainly responsible for the transport process in the brain by facilitated diffusion.165 As reported by Duelli and Kuschinsky, the density of these transporters matches the local glucose utilization measured with the 2-[14C]deoxyglucose method. In addition, it was found that the transporter density was highly correlated with the capillary density. The transporter density can be regulated upward or downward by a chronic (approximately a few days) increase or decrease in glucose metabolism. This nding, together with the hypothesis of Harder et al166 that astrocytes sense neuronal activity and lead to angiogenesis as observed in cell cultures, leads to an intriguing speculation about the function of

hyperemia following stimulation: that the CBF increase is needed rather for the formation of new capillaries than for nutritional support. While angiogenesis following stimulation is most likely true for chronic hyperemia, it is, however, unlikely that this could serve as a general explanation for the large transient CBF changes associated with the short stimulation periods (less than a few minutes) used in fMRI studies. The close correspondence in the baseline state between the enzymes associated with glucose metabolism and CBF is similar to the situation for oxygen metabolism. Stains for cytochrome oxidase, a key enzyme in the mitochondrial respiratory chain, correlate closely with capillary density.167 In addition, Raichle and colleagues167a reported that from many PET studies the oxygen extraction fraction E is nearly uniform across the brain with a value of approximately 40%. This suggests a close coupling of CBF and CMRO2 in the baseline state. Also, the oxygen glucose index (OGI), the ratio of CMRO2 to CMRGlc, is typically approximately 5.5 at rest, close to the theoretical ratio of 6 for full oxidative metabolism of glucose.18 In short, a number of measures support a close association of CBF, CMRO2, and CMRGlc at rest, reected by a uniform value of E and an OGI near 6. What is then surprising is that this pattern is not followed with activation. As we have described in detail, E decreases with activation, and produces the BOLD effect. The OGI also decreases with activation, meaning that CMRGlc increases more than CMRO2.

Why Does Glucose Metabolism Increase more than Oxygen Metabolism with Brain Activation?
As noted above, the OGI is approximately 5.5 during rest and deviates from the expected value of full oxygen metabolism of 6 (see The Physiologic Basis of fMRI).18 In addition, the OGI even decreases to a lower value during activation.168 It is currently not known why this OGI pattern during rest and activation occurs. Some possible explanations are: 1. Glucose is metabolized non-oxidatively to pyruvate and then lactate, which is partly cleared by venous ow and is lost for full oxidative metabolism. MR spectroscopy can be utilized to measure the increase in lactate concentration in the brain137,169,170 and thus allows the study of the fate of the glucose used. 2. Glucose is used to produce other chemical products (e.g., glutamate, glutamine) and hence serves other functions than metabolism alone.171 The products are then metabolized after stimulation ends, replacing glucose and reducing the glucose extraction fraction until the resting concentrations are reached. An MR method to measure these metabolites is [13C]-MR spectroscopy.28 3. Another hypothesis for the extensive use of glucose is that the non-oxidative metabolism is needed for fast processes. Glycolysis generates ATP much faster than full oxidative metabolism.170 Shulman and colleagues

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proposed a glycogen shunt model in which glucose is metabolized to glycogen during rest to ll up the glycogen pool in astrocytes, which in turn during stimulation is metabolized quickly within milliseconds in order to deliver the energy needed to recycle the neurotransmitters released during neuronal activity. However, this model would only explain transient changes in OGI. 4. In some structures in the brain oxidative metabolism may not be possible, e.g., due to missing mitochondria in some dendritic spines.172 However, energy metabolism is still required. No estimate has been presented of how much of the glucose metabolism needs to be non-oxidative and if this mechanism can explain the experimental ndings during rest and stimulation. Future studies using MR spectroscopy combined with fMRI are needed to resolve these issues and will be very useful for investigations of the physiologic basis of fMRI signals.

What is the Underlying Neuronal Activity that Drives the fMRI Signals?
With these uncertainties in mind we come back to the question of what neuronal processes are mainly reected by CBF and the BOLD signal. Synaptic activity may be excitatory or inhibitory, and this synaptic activity may or may not produce spiking activity. Different fMRI signals that differ between brain areas or between subjects might be due to different types of neuronal activity or differences in neurovascular coupling (or both). This clarication is important for two reasons. First, as pointed out earlier, the hemodynamic response is tightly coupled to oxygen metabolism. The neurovascular coupling translates very different neuronal and astrocytic processes into one dimension of CBF response, most likely reecting just the total oxygen metabolism of these processes. Thus, different activities of neurons and astrocytes can lead to the same CBF change. Second, the location of the hemodynamic response may differ from the location of the neuronal activity not only because the hemodynamic response is controlled in a coarse manner from feeding arterioles, but also because local processing and input from another brain area may elicit different responses (see below).

Spiking versus Synaptic Activity

The current debate about precisely which aspect of neural activity drives the CBF change is usually framed in terms of synaptic activity versus spiking activity. Synaptic activity includes neurotransmitter release and recycling, post-synaptic potential changes, and ion uxes initiated by the neurotransmitter, while spiking activity refers to the generation and propagation of action potentials. Rees et al and Heeger et al have demonstrated a linear relationship between spiking activity in monkeys and BOLD signal in a similar cortical area in humans; in the former study 9 spikes per second per neuron in V5

and in the latter study 0.4 spikes per second per neuron in V1 was found to correspond to 1% BOLD signal change.173,174 One of the difculties in interpreting these studies is that the spiking and post-synaptic activity are usually strongly correlated. Two studies have successfully dissociated synaptic and spiking activity. In one study, Lauritzen and colleagues stimulated either the parallel or the climbing bers in the rat cerebellum, which have either inhibitory or excitatory effect on the Purkinje cells in (for a review see reference 175). With this method they were able to minimize the spiking activity while synaptic activity continued, and this correlated with a CBF increase due to stimulation despite zero spiking activity. A second study comparing BOLD signal with high-frequency electrical activity or multi-unit activity (thought to reect spiking activity) and the mean local eld potential (thought to reect synaptic activity) found a slightly higher correlation between BOLD signal and local eld potential (LFP).176 Recently, by injecting serotonin, the same group succeeded in suppressing the multi-unit activity while leaving the LFP unchanged.177 The BOLD response also remained unchanged, enforcing the strong connection between LFP and BOLD signal. However, the relationship between electrical activity of neuronal ensembles and CBF might be even more complicated. In a recent study from Caesar et al, using topical application of GABAA (muscimol) receptor agonist in the cerebellum of the rat, the baseline CBF did not change whereas the spiking activity and the CBF response to climbing ber stimulation were reduced.178 However, the agonist did not affect the LFP. One conclusion might be that the correlation of LFP and CBF is valid for excitation circuits but not for inhibition circuits (see Does Inhibition Produce a BOLD Response?). In summary, the experimental results favor synaptic activity rather than spiking activity as the prime driver of CBF changes. This is reinforced by theoretical estimates that for the primate brain 70% or more of the required energy metabolism is due to synaptic activity (see Fig. 9-2). These ndings might have important implications in interpreting CBF and BOLD changes regarding the spatial information encoded by these signals. Input signals to one area or in one neuron are mainly represented by synaptic activity, whereas spiking activity represents the output signals.176 Therefore the current suggestion is that CBF represents the input to an area rather than the output. This would have the important implication that BOLD and CBF changes in one area can be caused remotely by input signals of another area and hence that these changes may spatially misrepresent the regions with increased spiking activity. However, local processing in one area evokes both spiking and synaptic activity because of the many local connections, so it is reasonable to assume that most of the neuronal signals causing CBF changes are within the area measured. Thus, mapping CBF changes to infer neural activity changes should only miss activated areas that are spiking with minimal synaptic activity. In addition, subthreshold activity in one area has a broader spatial extent than spiking activity, such as LFP, and thus CBF should have a broader spatial distribution than the spiking activity.

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Simultaneous Measurements of Electroencephalography and Event-Related Field Potentials with fMRI

Event-related eld potentials (ERP) and electroencephalography (EEG) measured with fMRI have been utilized to investigate noninvasively the relationship between CBF and underlying neuronal activity. ERP and LFP have a close relationship because both are assumed to be a weighted sum of post-synaptic currents. In contrast, EEG oscillations reect mainly synchronized neuronal activity. Arthurs and colleagues have found a linear correlation between somatosensory evoked potentials and BOLD response in humans.179 Using two-pulse stimulation with different inter-stimulus intervals up to 100 ms, Ogawa et al showed in rats that neuronal interaction measured as suppression of the ERP magnitude to the second stimulus is reected by the BOLD signal.180 By the way, this experiment showed that although the hemodynamic response evolves in seconds, the magnitude of the hemodynamic response could encode events and interactions on a millisecond time scale. In a recent study intradural ERP in patients and BOLD signal in healthy volunteers during the same visual stimulation were measured.181 ERP and BOLD signal did not correspond coherently for all areas. Furthermore, additional analysis revealed no consistent correlation between the EEG and BOLD signal. In summary, the connection between ERP and BOLD signal clearly needs further investigation. In the near future, the main problems in combining both measures (e.g., a non-magnetic EEG device, EEG time course artifacts caused by switching MR gradients, etc.) will be solved and this will lead to routinely combined fMRI-EEG measurements in healthy volunteers and in disease populations. In the last few years many groups have made combined measurements with fMRI and EEG. As an example from the increasing literature, several groups have correlated BOLD baseline uctuations (resting either with eyes open or eyes closed) with -power in EEG and have found negative correlations in the visual cortex and positive correlations in the thalamus.182 This nding is supported by the hypothesis that the generator of the -rhythm is the thalamus creating a subsequent local deactivation in the visual cortex. However, in our opinion the -power (or a complex composite of EEG oscillations) cannot be regarded as a general predictor of the CBF time course. Synchronized activity of the pyramidal cells reected in the EEG oscillations does not have to be more (or less) energy demanding than non-synchronized activity and, thus, can cause similar CBF and BOLD response. That is, in other areas other EEG oscillations are expected to correlate with CBF changes. Therefore the positive nding of these studies should be considered as a special case of high correspondence of CBF and EEG. However, to understand the neural basis of the fMRI signals it is not only the EEG signals that correlate with the BOLD signal that are important but also the EEG signals that do not correlate. Clearly more work is needed to explore the

relation between changes in the fMRI signals and the underlying electrophysiologic signals, which can be measured directly by invasive means or indirectly noninvasively with EEG and ERP.

Does Inhibition Produce a BOLD Response?

In the cortex the number of excitatory neurons is approximately ve times larger than the number of inhibitory neurons.183 Release of the main excitatory transmitter glutamate triggers increased metabolism in astrocytes,184 which in turn has been shown to correlate to neuronal oxidative metabolism.28 In addition, the coupling between CBF and CMRO2 has been shown to be the same during increased and decreased activity within the range of physiologic manipulations.185 Thus, the association of excitatory synaptic activity with increased energy metabolism is clear. Does the main inhibitory neurotransmitter, GABA, produce the same increase (or even a decrease) in metabolism as glutamate? Excitation and inhibition share common energy consuming processes in synapses, such as clearing neurotransmitter from the synaptic cleft and repackaging neurotransmitter in vesicles. For both types of activity ionic gradients must be restored, although the energy requirements may be different because the ions are different. Excitatory activity opens sodium channels, while inhibitory activity opens chloride and/or potassium channels. The sodium distribution across the cell membrane is the farthest from equilibrium, and so the thermodynamic energy cost may be higher for pumping sodium than chloride or potassium. Therefore, it is reasonable to assume that inhibition also is energy demanding, but it is not clear whether it is as demanding as excitatory activity. That is, like excitation, inhibition should cause an increase in CBF, which in fact was found in the hippocampus186 and in rat cerebellum.187 However, in cell cultures no increase in metabolism caused by GABA release was found in astrocytes.188 In support of this nding, no change in CBF during inhibition was observed using a go/no-go event-related paradigm and combined transcranial magnetic stimulation and fMRI measures.189 Interpreting these results in a conclusive way is difcult, because inhibition might also cause a decrease in CBF by suppressing the excitatory activity in the subsequent neuronal circuit: for example, a decrease in CBF is often found during sensorimotor stimulation in the ipsilateral cortex area due to transcallosal inhibition (see, e.g., reference 190). In summary, the net effect of inhibition might be an increase, no change, or a decrease of CBF (for a review see reference 191).

What is the Signicance of the Transients of the BOLD Signal?

The evoked hemodynamic response has some typical characteristics. Notable features of experimental measurements of the BOLD response are the transients, an

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better localized than the primary BOLD effect (see below), and because it may represent a transient uncoupling of CBF and oxygen metabolism.

Post-Stimulus Undershoot
The most frequently observed transient is a post-stimulus undershoot that often lasts for tens of seconds. A possible explanation for this phenomenon is that the CBV change resolves more slowly than the CBF change at the end of the stimulus. This effect has been seen in a study in a rat model,21 and two similar theoretical models have been proposed to explain the effect (the delayed compliance model21 and the balloon model20). Both of these models are based on the distensibility of the venous vessels, and the long time constant for CBV to adjust is then treated as a biomechanical property of the vessels. Note that this view involves some subtlety with regard to the relationship between CBV and CBF. In order to increase CBF, the vascular resistance must be decreased by dilating the arterioles. This active volume change on the arterial side, the direct cause of the CBF change, is thought to be a small fraction of the total CBV change. The venous change, however, is thought to be a passive response to the increased CBF and corresponding pressure changes. However, much of this picture is still speculative, although the resulting model curves often describe experimental BOLD data quite well. A recent nding supports this basic picture: using a sequence sensitive to blood volume changes (called VASO), Lu and colleagues reported that the early change in VASO signal is parallel to the increase in CBF, consistent with the idea that the early increase in CBV is caused by the arterioles, which in turn leads to the CBF increase.134 However, the VASO signal after the stimulus ended showed a much slower recovery to baseline than CBF, consistent with the idea that the late part of the VASO signal is dominated by venous blood volume changes. Other explanations of the post-stimulus undershoot are still under discussion: 1. CMRO2 recovers to its baseline value more slowly than CBF,136 hence oxygen is extracted even after CBF recovered to baseline producing transient hypo-oxygenation; 2. CBF drops below baseline after the stimulus ending, presumably due to neuronal inhibition and a corresponding decrease of neural activity in the post-stimulus period.177 Only the rst explanation is not compatible with a consistent coupling of CBF and CMRO2 and requires a transient uncoupling. However, as pointed out above, experimental data suggest that the post-stimulus undershoot is a result of the temporal mismatch of CBF and CBV and enhanced by a CBF undershoot. However, the temporal dynamics of the post-stimulus undershoot and its dependence on the baseline CBF value are not well understood and are still under investigation.

"Dip" Post-stimulus undershoot

F I G U R E 9-20
A typical BOLD response for a block design stimulus. Transients are an occasional brief initial dip at the beginning, an initial overshoot, and a prolonged post-stimulus undershoot.

occasional brief initial dip at the beginning or the more common prolonged post-stimulus undershoot.20,26 The positive BOLD response reaches its maximum after approximately 5 to 8 seconds. For a block design an initial overshoot is observed if a post-stimulus undershoot is present in the response to an event. A typical BOLD response is shown in Figure 9-20. The poststimulus undershoot often appears as an apparent lowering of the baseline after the rst stimulus block, when the undershoot has not fully resolved before the next stimulus block begins. In general, to clearly distinguish the undershoot, the rest block should be longer than the stimulus block. In the balloon model,20 such transients have two distinct sources. The initial dip is modeled as a slight delay (approximately 1 s) of the CBF response compared to the CMRO2 response. The post-stimulus undershoot arises in the model because CBV is slower to recover than CBF and CMRO2. Then if the oxygen extraction fraction returns to baseline at the end of the stimulus, but the venous blood volume remains elevated, total deoxyhemoglobin will be higher than baseline, reducing the BOLD signal. In the original version of the balloon model20 it was noted that an initial dip could result from a rapid rise in blood volume. A number of optical studies found that the initial dip period corresponded to an increase of deoxyhemoglobin but without a dip in deoxyhemoglobin, suggesting a combined change in CBV and E.69,192 A more recent study, however, found a corresponding initial decrease in oxyhemoglobin in conjunction with an increase in deoxyhemoglobin, more clearly suggesting a change in E.73 Some recent data show that in fact the tissue oxygenation decreases shortly after beginning of the stimulation.193 However, whether this tissue deoxygenation is translated into vascular deoxygenation has still to be proven. Although more data are clearly needed to resolve the experimental inconsistencies, recent experimental work suggests the change in E as the source of the initial dip. Despite its elusive nature,26 it has excited great interest because it appears to be

Nonlinearity of the BOLD Response

Although the standard statistical analysis assumes a linear response, such that the net response to several stimuli is simply the sum of the individual responses to a single

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stimulus, the hemodynamic response is often nonlinear. The BOLD response typically exhibits a temporal nonlinearity, such that an appropriately shifted and added response to a brief stimulus over-predicts the true response to an extended stimulus.85,194-199 This temporal nonlinearity is most pronounced when the brief stimulus is less than about 4 s and the extended stimulus is longer than 6 s. Comparing short and long duration stimuli that are both longer than approximately 4 s, the temporal nonlinearity is reduced. A nonlinearity such as this could arise in several ways. In the step from the stimulus to the evoked neural activity, adaptation can produce an initial sharp rise in activity that decreases to a lower plateau level, even with a constant stimulus. In addition, the step from neural activity to CBF response could be nonlinear, for example through a ceiling effect on CBF change. Friston et al suggested that a large part of the nonlinearity of the BOLD response arises from the transformation of a CBF response to the BOLD signal change.85 In a subsequent study they showed that the Volterra kernel characterization of experimentally observed nonlinearities could be accounted for with the balloon model plus a linear transformation up to the CBF response, again supporting the idea that the primary nonlinearity is in the transformation from the CBF to the BOLD response.200 The two sources of nonlinearity (neural or vascular) can be distinguished experimentally by whether the nonlinearity is present in just the BOLD response or in both the BOLD and CBF responses. For example, Miller et al199 found that both visual and motor cortices exhibited a nonlinear BOLD response, but only the visual cortex showed a nonlinear CBF response. This source of nonlinearity in the step from CBF to BOLD can be explained as a BOLD ceiling effect. Even an innite CBF change could still produce only a nite BOLD response, corresponding to removing all deoxyhemoglobin from the voxel. This effect produces an overprediction of the amplitude of a long duration stimulus from a short duration stimulus when the ow change due to the short stimulus is substantially weaker than the flow change due to the longer stimulus. That is, the BOLD ceiling effect should introduce a nonlinear response when the shorter stimulus is narrower than the width of the CBF response, which is approximately 4 s, in good agreement with experimental data. In addition to the nonlinearity of the amplitude, there are nonlinearities of the timing of the response, such as latency or response width. A recent proposal by Behzadi et al201 and Baird and Warach202 is a promising approach for accounting for these effects. Instead of treating CBF as a linear convolution with the neural activity, it is assumed that neural activity releases a vasoactive agent that alters CBF, as in the model described in reference 200. The difference is that the agent is modeled as acting on the compliance of the vessel. The compliance in turn is treated as a combination of the smooth muscle tension, which can be controlled by the agent, and a xed elastic component that becomes a more dominant factor in determining compliance when the vessel is expanded. In this way, the same concentration of the

agent will have a greater effect on CBF when the ow is lower, and potentially a different effect on the nonlinearity of the response.

The Physiologic Baseline Strongly Affects the BOLD Signal

The fact that the BOLD signal depends on a combination of changes in CBF, CBV, and CMRO2, and also on the baseline physiologic state, makes it difcult to interpret the magnitude of the BOLD signal change unambiguously without further experimental information. For this reason, the BOLD effect has been used primarily as a mapping tool, based on detecting signal changes, rather than as a probe of the underlying physiology based on a detailed analysis of the BOLD response. However, combined measurements of BOLD and CBF can be used to model the effects of the physiologic baseline on the BOLD response. Experiments have found that when baseline CBF is increased by breathing CO2 or administering acetazolamide, the BOLD response is reduced. For example, Brown et al49 found that acetazolamide raised baseline CBF by 20% and the BOLD response in the motor cortex with nger tapping was reduced by 35%, but the CBF change (CBF) with activation stayed the same. With CO2 inhalation CBF increases but it is thought that CMRO2 remains the same. This means that at this new baseline the oxygen extraction fraction E must be smaller than it was at the previous baseline, and this implies that there is less deoxyhemoglobin present at the new baseline. Thus, even if the fractional changes of the physiologic quantities were the same, the BOLD signal would be reduced. However, in addition, the experimental data suggest that CBF remains constant despite the raised baseline CBF, so the fractional CBF change is smaller at the elevated baseline, and this will further reduce the BOLD response. As a numerical example, consider an activation that produces a 30% change in CBF and a 10% change in CMRO2 from the initial baseline state, giving a BOLD signal change of 1.36% (calculated with Equation 9-10 and A = 0.1). If the subject now breathes CO2 that produces a 20% increase of baseline CBF with no change in CMRO2, the raw BOLD signal baseline will increase by 1.82%. If the same activation on top of this new baseline state produces the same absolute changes in CBF and CMRO2, we can calculate the net effect by rst considering that if this net physiologic change had occurred in the original baseline state, the relevant changes would have been a 50% change in CBF and a 10% change in CMRO2, which would have given a BOLD signal change of 2.61% from the original baseline. Subtracting the BOLD baseline shift of the new state, the BOLD signal change from the new baseline is 0.79%. This is a 42% reduction of the BOLD response to the same activation due just to the 20% increase of the CBF baseline, and is in reasonable agreement with the experimental data. The implication of this sensitivity of the BOLD signal to the baseline state is that, potentially, many factors could alter the baseline state of a patient group (e.g.,

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anxiety or vasoactive medications) that could make their BOLD responses signicantly different from a healthy population even if the neural responses in the two groups were identical. For this reason, the CBF measured with ASL techniques may prove to be a much more robust approach for quantitative fMRI studies. While these considerations show how changes in the baseline CBF can affect the BOLD response, we have not dealt with the issue of what determines the baseline CBF. A recent theory, which requires further development, is that CBF is regulated to maintain a constant ratio of O2 to CO2 at the mitochondria, in order to preserve the thermodynamic free energy available from oxidative metabolism of glucose.203 The O2 concentration at the mitochondria ([O2]) is determined by E, so to increase the diffusive ux of O2 from capillaries to mitochondria, while maintaining [O2], requires E to decrease with activation. In addition, increasing CO2 in the blood increases CO2 in the tissue as well, degrading the [O2]/[CO2] ratio, and this is again restored by decreasing E. Thus, the model predicts that CBF should increase with CO2, and that an additional increase is required to meet the needs of increased CMRO2. As commonly used, the terms activation and deactivation are relative terms, so the choice of an appropriate task to dene the neural activity of the baseline condition plays a critical role in an fMRI experiment.204 The uncritical use of the notion of baseline may lead to erroneous interpretation of fMRI data. Essentially, the brain is always intrinsically active as the high value of resting metabolism shows. The brain has a weight of approximately 2% of the weight of the whole body, but accounts for 20% of the whole metabolism. Strictly speaking, in stimulation experiments, two different active states are compared with each other and one is referred arbitrarily to be the baseline state. This is especially problematic for complex brain functions and thus the baseline state has to be chosen carefully.

should create temporally correlated uctuations in BOLD signal. However, although this is certainly true, until now no estimation exists of the magnitude and the frequency of these BOLD uctuations. Therefore, results of this approach should be interpreted cautiously.

Spatial and Temporal Resolution

The transformation of neuronal signals to vascular dynamics may lead to information loss: 1. Qualitatively: Different dimensions of neuronal processes like inhibition and excitation or presynaptic and post-synaptic activity are transformed into vascular changes. Clever experimental designs are needed in order to recover this information and distinguish these processes from measured vascular changes. 2. Time domain: The vascular response is on the order of one second, whereas the neuronal changes are on the order of milliseconds. Thus, neuronal changes are averaged and temporally blurred in the vascular domain. However, as noted above, Ogawa et al have shown that using two-pulse stimulation of 10 ms duration the amplitude of the vascular changes can encode neuronal changes which are separated by only a few milliseconds.180 This idea needs further elaboration and more experiments are required to elucidate the temporal limits of fMRI. In particular, interaction analysis and nonlinearities seem to provide a versatile tool to improve the temporal limits of fMRI. 3. Spatial domain: It is not clear what the minimum spatial scale is for the vascular changes to occur. In the olfactory cortex it was shown by Chaigneau and colleagues in 2003 with two-photon optical imaging that capillaries 100 m remote from the activated area are not showing any stimulus-correlated change.206 In a recent review, Iadecola pointed out that this nding is an argument against diffusion-controlled changes in CBF and an argument for direct control of CBF by neurons. This direct control can be employed by interneurons, which integrate the neuronal activity in local neuronal circuits.207 Harrison et al showed that vascular imaging signals correspond well with the underlying vascular structure as shown in Figure 9-21.208 However, the translation of the neuronal activity to CBF response introduces spatial blurring. It is often assumed that CBF changes are actively controlled by the arterioles. The feeding region of an arteriole is on the order of a millimeter,207 i.e., changes of neuronal activity within different neuronal populations in the receptive eld of the same arteriole cannot be spatially separated by fMRI signals. Thus, fMRI signals reect activity of a population of neurons, and a subpopulation of these neurons cannot be spatially resolved by fMRI. In addition, due to intravascular signaling, arterial blood vessels upstream from the location of the neuronal activity also dilate. This effect is called retrograde vasodilation,207 introducing further spatial blurring.

Do BOLD Correlations Reveal Long-Range Patterns of Connectivity?

As pointed out earlier (see Design and Analysis of BOLD-fMRI Experiments and Artifacts and Noise), the BOLD signal varies due to physiologic uctuations. Beside heartbeat and respiration related changes the so-called slow oscillations (approximately 0.1 Hz) are observed.110 It is currently debated whether these oscillations are vascular or neuronal in origin. The vascular hypothesis refers to brain blood pressure autoregulation, with 0.1 Hz as the mean frequency of the autoregulation, so that the vascular system acts as a frequency lter at 0.1 Hz. Thus, according to this view, correlations of these oscillations reveal only vascular symmetry. However, a growing number of studies are now using BOLD baseline uctuations in order to nd modular neuronal networks in the brain.205 The basis for this is the assumption that neuronal connectivity is reected by BOLD signal uctuations. Neuronal connected areas have simultaneous uctuations in baseline activity and thus

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cortical columns, suggesting fairly tight spatial control of CBF.213 In sum, the limits of the spatial resolution of the CBF and BOLD response are not well understood and need further investigation.

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F I G U R E 9-21
Vascular structure of a chinchilla measured with corrosion casts and region of changes in the imaging signal (top right, inset). Vascular imaging signals correspond well with the underlying structure of the vasculature. (Adapted from Harrison RV, et al: Blood capillary distribution correlates with hemodynamic-based functional imaging in cerebral cortex. Cereb Cortex 12:225-233, 2002, by permission of Oxford University Press.)

Beyond the CBF response the initial dip (see What is the Signicance of the Transients of the BOLD Signal?) has been suggested as a way to improve the spatial resolution of imaging signals. The dip is thought to be produced by initial oxygen consumption, and so might be more precisely located than the ensuing CBF and BOLD changes. Kim and colleagues have measured cortical columns in the visual cortex of cats with a functional distance of the columns of approximately 1 mm.209 However, Logothetis, who has shown that the early fMRI signal was also found in the sagittal sinus, challenged the interpretation of this study.210 Studies in monkeys176,211 found correlation between neuronal activity and BOLD in parenchyma, but this decreased close to large vessels. For typical fMRI in humans with imaging resolution 3 mm 3 mm 3 mm, the inclusion of small draining veins in the imaging voxel does not unduly skew the representation of neuronal activity. As resolution is increased, steps need to be taken to reduce the inuence of large vessels. At higher elds, the use of spin-echo acquisition123 or diffusion weighting during a BOLD experiment reduces the signal contribution from large veins. In a different study, Kim et al measured the spatial correlation of single- and multi-unit activity with BOLD signal and found a linear relationship between these signals when the voxel size is greater than approximately 3 3 mm2. Because most studies utilize voxel size greater than this value, the BOLD signal reects accurately the underlying neuronal signal.212 Finally, a recent study using ASL techniques was able to resolve

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