REVIEWS NATUREVol 45312 June 2008 Yet,as I have indicated above,the BOLD signal is primarily affec- in monkeys'.These studies showed that only a small fraction of VI ted by changes in excitation-inhibition balance,and this balance may cells modulate their spiking during the perceptual changes;neuroi- be controlled by neuromodulation more than by the changes in maging,on the other hand,demonstrated fMRI-signal modulations spiking rate of a small set of neurons.In fact,the BOLD signal is that were nearly as large as those obtained during the physical strongly modulated by attention,and the results of the motion alternation of stimuli".The difference,once again,reflects the fact after-effect experiments could,in principle,be due to the fact that that neuromodulatory feedback from higher areas can be easily a stimulus with illusory motion automatically draws the attention of detected by means of fMRI,but not through the measurement of a subject more compared to a situation in which there is no motion single-unit activity.Interestingly,measurements of subthreshold after-effect.This hypothesis turned out to be correct,as a later activity in another study of perceptual multistability revealed per- study-in which balance in attentional load was accomplished by ception-related modulations in LFP,despite the unaltered spike having the subjects perform a concurrent visual task-found no ratesss.Such clear spiking and BOLD signal mismatches appear even signal differences between the motion after-effect and no motion in simple experiments probing sensory processing.Simple stimuli, after-effect conditions. such as those used in the aforementioned studies,are most likely to A similar example pertains to the differences in neurophysiological generate a proportional enhancement in both the afferent and effer- and fMRI responses in the primary visual cortex during different ent activity of any sensory area.The activation of high-level asso- perceptual states.It is known that physiological signals are in general ciation areas related to cognitive processing might be more sensitive stronger when stimuli are perceived as opposed to when they are not. or even dominated by feedback and neuromodulation,whose differ- Intriguingly,in some regions the BOLD response seems to reflect this ential effect on spiking and haemodynamic responses is utterly even more sensitively than physiological measures like spikes and unknown. multi-unit activity?.An example is the pattern of fMRI activation changes in V1 during binocular rivalry (that is,the perceptual alter- Conclusions and perspectives nations experienced when the two eyes view different stimuli).This The limitations of fMRI are not related to physics or poor engineer- phenomenon has been studied extensively psychophysically and ing,and are unlikely to be resolved by increasing the sophistication also over the last two decades in a series of electrophysiology studies and power of the scanners;they are instead due to the circuitry and Va Vb 100 Figure 3 Neural and vascular contents of a voxel.The left panel right;white spots are cross-sections of vessels).The average distance between demonstrates the relative density of vessels in the visual cortex of monkeys. the small vessels(capillaries)is about 50 um.This is approximately the The dense vascular mesh is displayed by perfusing the tissue with barium distance that oxygen molecules travel by diffusion within the limited transit sulphate and imaging it with synchrotron-based X-ray microtomography time of the blood.The dense population of neurons,synapses and glia (courtesy B.Weber,MPI for Biological Cybernetics).The vessel diameter is occupy the intervascular space,as depicted in the drawing at the top right-a colour coded.Cortical surface without pial vessels is displayed at the top; hypothetical distribution of vascular and neural elements in a small section white matter at the bottom.At the left of the panel is a Nissl slice from the (red rectangle).The drawing in the background shows some of the typical same area,showing the neural density for layers II through to the white neuronal types(for example,red,large pyramidal cell;dark blue,inhibitory matter (wm).Although the density of the vessels appears to be high in this basket cells;light blue,chandelier inhibitory neurons;and grey,stellate cells) three-dimensional representation,it is actually less the 3%(see section at the and their processes. 876 2008 Macmillan Publishers Limited.All rights reservedYet, as I have indicated above, the BOLD signal is primarily affec￾ted by changes in excitation–inhibition balance, and this balance may be controlled by neuromodulation more than by the changes in spiking rate of a small set of neurons. In fact, the BOLD signal is strongly modulated by attention68, and the results of the motion after-effect experiments could, in principle, be due to the fact that a stimulus with illusory motion automatically draws the attention of a subject more compared to a situation in which there is no motion after-effect. This hypothesis turned out to be correct, as a later study—in which balance in attentional load was accomplished by having the subjects perform a concurrent visual task—found no signal differences between the motion after-effect and no motion after-effect conditions69. A similar example pertains to the differences in neurophysiological and fMRI responses in the primary visual cortex during different perceptual states. It is known that physiological signals are in general stronger when stimuli are perceived as opposed to when they are not. Intriguingly, in some regions the BOLD response seems to reflect this even more sensitively than physiological measures like spikes and multi-unit activity70. An example is the pattern of fMRI activation changes in V1 during binocular rivalry (that is, the perceptual alter￾nations experienced when the two eyes view different stimuli). This phenomenon has been studied extensively psychophysically and also over the last two decades in a series of electrophysiology studies in monkeys70. These studies showed that only a small fraction of V1 cells modulate their spiking during the perceptual changes; neuroi￾maging, on the other hand, demonstrated fMRI-signal modulations that were nearly as large as those obtained during the physical alternation of stimuli70. The difference, once again, reflects the fact that neuromodulatory feedback from higher areas can be easily detected by means of fMRI, but not through the measurement of single-unit activity. Interestingly, measurements of subthreshold activity in another study of perceptual multistability revealed per￾ception-related modulations in LFP, despite the unaltered spike rates53. Such clear spiking and BOLD signal mismatches appear even in simple experiments probing sensory processing. Simple stimuli, such as those used in the aforementioned studies, are most likely to generate a proportional enhancement in both the afferent and effer￾ent activity of any sensory area. The activation of high-level asso￾ciation areas related to cognitive processing might be more sensitive or even dominated by feedback and neuromodulation, whose differ￾ential effect on spiking and haemodynamic responses is utterly unknown. Conclusions and perspectives The limitations of fMRI are not related to physics or poor engineer￾ing, and are unlikely to be resolved by increasing the sophistication and power of the scanners; they are instead due to the circuitry and Figure 3 | Neural and vascular contents of a voxel. The left panel demonstrates the relative density of vessels in the visual cortex of monkeys. The dense vascular mesh is displayed by perfusing the tissue with barium sulphate and imaging it with synchrotron-based X-ray microtomography (courtesy B. Weber, MPI for Biological Cybernetics). The vessel diameter is colour coded. Cortical surface without pial vessels is displayed at the top; white matter at the bottom. At the left of the panel is a Nissl slice from the same area, showing the neural density for layers II through to the white matter (wm). Although the density of the vessels appears to be high in this three-dimensional representation, it is actually less the 3% (see section at the right; white spots are cross-sections of vessels). The average distance between the small vessels (capillaries) is about 50 mm. This is approximately the distance that oxygen molecules travel by diffusion within the limited transit time of the blood. The dense population of neurons, synapses and glia occupy the intervascular space, as depicted in the drawing at the top right—a hypothetical distribution of vascular and neural elements in a small section (red rectangle). The drawing in the background shows some of the typical neuronal types (for example, red, large pyramidal cell; dark blue, inhibitory basket cells; light blue, chandelier inhibitory neurons; and grey, stellate cells) and their processes. REVIEWS NATUREjVol 453j12 June 2008 876 ©2008 Macmillan Publishers Limited. All rights reserved