mr imaging in the non-human primate: studies of function and of
TRANSCRIPT
MR imaging in the non-human primate: studies of functionand of dynamic connectivityNikos K Logothetis
Since its early development in the late 1940s, nuclear magnetic
resonance has become a powerful tool for applications ranging
from chemical analysis or the study of the structure of solids to
biomedical investigations. In the early 1990s the potential of this
technique for functional brain mapping was demonstrated,
causing unprecedented excitement in both basic and clinical
neuroscience. It was shown that by using the appropriate pulse
sequences the so-called functional magnetic resonance imaging
(fMRI) technique can be made sensitive to local magnetic
susceptibility alterations produced by changes in the
concentration of deoxyhemoglobin in venous blood vessels. This
blood-oxygenation-level-dependent (BOLD) contrast
mechanism was successfully implemented in awake human
subjects, in small animals, and recently in the non-human
primate — the experimental animal of choice for the study of
cognitive behavior. Simultaneous imaging and electrode
recordings promise new insights into the mechanisms by which
large-scale networks in the brain contribute to the local neural
activity recorded at a given cortical site. Moreover, the use of
MRI-visible tracers and of electrical microstimulation applied
during imaging proves to be ideal for the study of connectivity
in the living animal.
AddressesMax Planck Institute for Biological Cybernetics, Department of
Physiology of Cognitive Processes, Spemannstr 38, 72076 Tubingen,
Germany
e-mail: [email protected]
Current Opinion in Neurobiology 2003, 13:1–13
This review comes from a themed issue on
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Edited by Karel Svoboda and Kamil Ugurbil
0959-4388/$ – see front matter
� 2003 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.conb.2003.09.017
AbbreviationsBOLD blood-oxygen-level-dependent
CBF cerebral blood flow
CNS central nervous system
dHb deoxyhemoglobin
EEG electroencephalography
fMRI functional magnetic resonance imaging
FOV field of view
LFPs local field potentials
LGN lateral geniculate nucleus
mEFP mean extracellular field potential
MRI magnetic resonance imaging
MUA multiple-unit spiking activity
PET positron emission tomography
SNR signal to noise ratio
TE echo time
TR repetition time
V1 primary visual cortex
WGA-HRP wheat germ agglutinin conjugated to horseradish
peroxidase
IntroductionUnderstanding brain function requires comprehension of
the physiological workings of its cellular components, as
well as a detailed knowledge of their structural and
functional connectivity. Moreover, the functional plasti-
city of the brain, reflected in its capacity for anatomical
reorganization, means that a mere ‘snapshot’ of its archi-
tecture is not enough. Desirable, instead, are repeated
anatomical and physiological observations of its connec-
tivity patterns at different organizational levels. In vivoneuroimaging, especially the spatiotemporally resolved
magnetic resonance imaging (MRI), together with its
functional variant, functional magnetic resonance imag-
ing (fMRI), is therefore of obvious importance, and it is
currently one of our best tools for linking cognition and
action with their neural substrates in humans.
At the moment, brain fMRI relies mainly on the blood-
oxygen-level-dependent (BOLD) contrast mechanism,
which was first described in rat studies using high mag-
netic fields [1]. BOLD contrast is derived from the field
inhomogeneities induced by deoxyhemoglobin (dHb),
which is confined in the intracellular space of the red
blood cells that in turn are restricted to the blood vessels.
Magnetic susceptibility differences between the dHb-
containing compartments and the surrounding space gen-
erate magnetic field gradients across and near the com-
partment boundaries. Pulse sequences designed to be
sensitive to such susceptibility differences alter the signal
whenever the concentration of dHb changes.
Upon neural activation, an increase in dHb concentration
would be expected to enhance the field inhomogeneities,
reducing the BOLD signal. Yet, a few seconds after the
onset of stimulation the BOLD signal actually increases.
This enhancement reflects an increase in cerebral blood
flow (CBF) that overcompensates for the increased ox-
ygen consumption, and ultimately delivers an oversupply
of oxygenated blood [2,3]. BOLD imaging has been
successfully applied in humans [4–6] and was recently
applied in the non-human primate [7�–9�,10–12].
An example of BOLD imaging in the monkey is shown in
Figure 1a, a high resolution functional scan obtained from
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an anesthetized monkey in a moderately high field
(4.7 Tesla) scanner [11]. A gradient-recalled multi-shot,
multi-slice echo planar imaging method, optimized for
both the field and the subject-species, was used to acquire
volumes of 13 slices every 6 seconds while a polar-
transformed rotating checkerboard was presented to the
monkey through a fiber-optic system. The image shows
maps of the activation’s statistical significance (Z-scores
from a T-test) superimposed on T1-weighted anatomical
scans; whereby T1 refers to the time constant describing
the tissue’s magnetization re-growth. Significant clusters
of activation can be seen in the lateral geniculate nucleus
(LGN) and the striate cortex. The LGN of the monkey
is only about 6 mm across in the rostro-caudal direction
and approximately 5 mm in the dorso-ventral and medio-
lateral directions. The precise anatomical localization
of its activation is therefore important evidence for the
spatial specificity of fMRI in high magnetic fields.
Gradient echo sequences, like those used for the acquisi-
tion of the images in Figure 1, are sensitive to both small
and large vessels [13,14]. The significant contribution of
the large vessels can lead to erroneous mapping of the
activation site, as the flowing blood will generate BOLD
contrast downstream of the actual tissue with increased
metabolic activity. Thus, the extent of activation will
appear to be larger than it really is. The contribution of
large vessels depends on both the field strength and the
parameters of the pulse sequences [15–18], and they can
be de-emphasized in stronger magnetic fields, because
the strength of the BOLD that originates in the paren-
chyma (extravascular) increases more rapidly for small
Figure 1
dLGNV1
V1
STS
Lu
STSSTS
(a)
(d)
(b) (c)(c)(c)
STS
LuLuLu
Current Opinion in Neurobiology
Activation of LGN and visual cortex. (a) Volume imaging. The transverse section is parallel to the Frankfurt zero-plane. Measurements were made
on a vertical 4.7 T scanner having a 40 cm diameter bore (Biospec 47/40v, Bruker Medical Incorporation, Ettlingen, Germany). The system was
equipped with a 50 mT/m (180 ms rise time) actively shielded gradient coil of 26 cm inner diameter. The images were acquired using a field of view
(FOV) of 128 mm � 128 mm. T1-weighted, high resolution (256 � 256 matrix, 0.5 mm thickness) anatomical scans were obtained by using the 3D
modified driven equilibrium Fourier transform (3D-MDEFT) pulse sequence [82], with an echo time (TE) of 4 msec, repetition time (TR) of 14.9 msec, flip
angle (FA) of 20 deg, inversion time (t) of 800 msec, and 8 segments. Multi-slice fMRI (13 slices, 8 segments, 128 � 128 matrix, 1 mm thickness,
TE ¼ 20 msec, TR ¼ 740 msec, FA ¼ 50 deg, number of excitations ¼ 1) was carried out by the use of gradient-echo echoplanar imaging (GE-EPI).(b-d) Imaging with implanted radiofrequency coils. Intraosteally implanted RF coils were used to achieve sufficient signal- and contrast-to-noise ratios
for very high-resolution imaging that can be also combined with simultaneous electrophysiological measurements within the magnet. The parameters
of the EPI scan in b and c were FOV ¼ 32 � 32 mm2 on a matrix of 256 � 256 (0:125 � 0:125 mm2 resolution), slice thickness ¼ 0:720 mm,
TE/TR ¼ 20/750 msec, FA ¼ 30 deg, number of segments 8 or 16. The parameters of FLASH sequence in d were FOV 3:84 � 3:84, matrix 512 � 256,
nominal resolution 75 � 150 � 300 mm, TE 20 ms, TR 2040 ms, BW 25 kHz, 30 degree flip angle. Abbreviations dLGN, dorsal lateral geniculate
nucleus; Lu, lunate sulcus; STS, superior temporal sulcus; V1, striate cortex.
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Current Opinion in Neurobiology 2003, 13:1–13 www.current-opinion.com
vessels than it does for large ones (for review see [19]).
Thus, with a sufficiently high signal-to-noise ratio (SNR),
signals originating from the capillary bed are discernable
in strong magnetic fields and better reflect the site of
actual neural activation [20,21]. This property can be
exploited to obtain precise maps of areas defined on
the basis of functional architecture [22].
Specifically, cortex consists of specialized, separate
areas, which can be identified by their cyto- and
myelo-architecture, physiological properties and con-
nectivity (e.g. Felleman and Van Essen [23]). In the
visual system, the boundaries of some areas can be
determined by exploiting their retinotopic organization,
and several laboratories have already developed methods
for measuring visual field maps in the human brain (for a
review see Wandell [24]). Retinotopic maps were also
recently obtained in monkeys [25�], and were found to
register well with those derived from the same species
using anatomical and physiological techniques. In con-
trast to these techniques, however, non-invasive map-
ping of the retinotopic areas of individual monkeys is
likely to prove a valuable tool for several longitudinal
investigations, including studies of learning, plasticity
and reorganization.
When imaging of the entire brain volume is not the primary
interest, then small surface radiofrequency (RF) coils can
be used in either human or animal studies to obtain the
highest possible SNR in spatially resolved images. The
coils are often used as transceivers, namely both for excit-
ing the tissues and for receiving the RF signals transmitted
by them. Image quality and resolution can be further
improved by geometrically matching the coils to a specific
tissue region and, in animal experiments, by implanting
the coils in the body (for references see [26�,27]). The
small RF surface coil used in Figure 1b–d was implanted
within the bone (intraosteally). The achieved resolutions
in this case were 125 � 125 � 720 mm3, 125 � 125 � 720
mm3 and 75 � 150 � 300 mm3.
The contrast sensitivity of the image in Figure 1b is
sufficient to reveal the characteristic striation of the
primary visual cortex. The dark line shown by the white
arrow is the well-known, approximately 200 m thick Gen-
nari line formed by the axons of pyramidal and spiny
stellate cells contained in middle cortical layer (lamina
IVB). Figure 1c shows fMRI correlation coefficient maps
(in color) superimposed on the actual echoplanar (EPI)
(T�2-weighted) images. T�
2 refers to the transverse relaxa-
tion time constant in the presence of field inhomogene-
ities. The two most commonly varied MRI parameters are
the relaxation time constants T1 and T2. As mentioned
above, T1 is the tissue’s magnetization re-growth. T2, on
the other hand, refers to the so-called transverse relaxa-
tion in an ideal homogenous magnetic field. In actuality
the transverse relaxation is more rapid because of local
field inhomogeneities, including those within the imaged
tissue itself. It is then characterized by the decay constant
T�2 rather than T2. In the T�
2-weighted image of Figure 1c
both robust activation and good anatomical detail can be
discerned. Voxels of this size reflect the activity of as few
as 600–1200 cortical neurons, providing us with the
opportunity to study how neural networks are organized,
and how small cell assemblies contribute to the activation
patterns revealed in fMRI. Furthermore, they enable
direct comparisons between the imaging signals and those
signals obtained in intracortical recordings.
Intracortical recordings and fMRIThe successful application of BOLD fMRI in human or
animal brain research ultimately depends on a compre-
hensive understanding of the relationship between the
hemodynamic signal and the underlying neuronal activ-
ity. Functional MRI has already been combined with
optical imaging recordings of intrinsic signals [28] and
electroencephalography (EEG; e.g. [29]). However, the
first method also measures hemodynamic responses [30]
and thus can offer very little direct evidence of the
underlying neural activity, whereas EEG studies typically
suffer from poor spatial resolution and imprecise localiza-
tion of the electromagnetic field patterns associated with
neural current flow. Recently, combined intracortical
recordings and BOLD fMRI were successfully applied
in anesthetized and conscious monkeys. The BOLD
response was found to directly reflect an increase in
neural activity, best correlating with those electrical sig-
nals that are thought to represent synaptic inputs and
local intracortical processing [31��]. This conclusion was
derived from the detailed study of different components
of the digitized comprehensive mean extracellular field
potential (mEFP), which encompasses time-varying spa-
tial distributions of action potentials superimposed on
relatively slow varying field potentials.
Neural signals and their cellular origin
The mEFP represents the weighted sum of all sinks (e.g.
negativities caused by Naþ or Ca2þ moving from the
extracellular to the intracellular space) and sources (posi-
tivities) along multiple cells. If a microelectrode with a
small tip is placed close (within about 50 mm–100 mm) to
the soma or the axon of a neuron, then the measured
mEFP directly reports the spike traffic of that neuron, and
frequently that of its immediate neighbors as well [32].
The firing rate of such well isolated neurons has been the
usual measure for comparing neural activity to sensory
processing or behavior ever since the earliest develop-
ment of microelectrodes. A great deal has been learned
since then, and the single-electrode single-unit recording
technique still remains the method of choice in many
behavioral experiments with conscious animals. Like
any other method, however, it also has drawbacks, provid-
ing information mainly on single receptive fields but no
MR imaging in the non-human primate Logothetis 3
www.current-opinion.com Current Opinion in Neurobiology 2003, 13:1–13
access to subthreshold integrative processes or the associa-
tional operations taking place at a given site. In addition,
it suffers from an element of bias towards certain cell types
(for a review and references see [33,34]). Spikes generated
by large neurons remain above the noise level over a greater
distance from the cell than spikes from small neurons, so
microelectrodes sample their somas or axons preferentially,
a prediction supported by experimental work.
When the activity of a single neuron is not the primary
concern of an investigation, microelectrodes of lower
impedance and appropriate tip geometry can be con-
structed that are less inundated by spikes and capture
the totality of the potentials in a given region. The mEFP
recorded under these conditions is related to both local
integrative processes (dendritic events) and spikes gen-
erated by several hundred neurons.
A large number of experiments have presented data
that indicate that the two different processes can be
segregated by subjecting the mEFP to frequency-band
separation. A high-pass filter cutoff of approximately
300–400 Hertz is used in most recordings to obtain
multiple-unit spiking activity (MUA), and a low-pass
filter cutoff of about 200–300 Hz to obtain the so-called
local field potentials (LFPs).
Fast and slow components of the mean extracellular
field potential
The MUAs are a weighted sum of the extracellular action
potentials of all neurons within a sphere of approximately
140–300 mm radius, with the electrode at its center.
Spikes produced by the synchronous firings of many cells
can, in principle, be enhanced by summation and thus
detected over larger distances [33,34].
The LFPs are slow fluctuations reflecting cooperative
activity in neural populations. Until recently, these sig-
nals were thought to represent exclusively synaptic
events. Evidence for this came from combined EEG
and intracortical recordings that showed that the slow
wave activity in the EEG is largely independent of
neuronal spiking [33,34]. They also showed that, unlike
the multiunit activity, the magnitude of the slow field
fluctuations is not correlated with cell size, but instead
reflects the extent and geometry of dendrites in each
recording site. The pyramidal cells, with their apical den-
drites running parallel to each other and perpendicular
to the pial surface, form an ideal open field arrangement
and contribute maximally to both the macroscopically
measured EEG and the LFPs. The LFPs are the weighted
average of synchronized dendro–somatic components of
the synaptic signals originating from within 0.5–3 mm of
the electrode tip (e.g. [35]).
Today we know that LFPs represent both synaptic
events and other types of slow activity, including voltage-
dependent membrane oscillations (e.g. [36]) and spike
afterpotentials. The soma–dendritic spikes in the neurons
of the central nervous system (CNS) may be followed by
afterpotentials, a brief delayed depolarization, the after-
depolarization, and a longer lasting after-hyperpolariza-
tion, which are thought to play an important role in the
control of excitation-to-frequency transduction (e.g.
[37,38]). Afterpotentials, which were shown to be gener-
ated by calcium-activated potassium currents (e.g.
[39,40]), have a duration in the order of 10’s of milli-
seconds and most likely contribute to the generation of
the LFP signals (e.g. [41]).
BOLD reflects the input and local processing of a
studied brain area
To understand the contribution of such different cellular
events to the generation of the BOLD signal, we exam-
ined the correlation of LFPs, MUA and single neuron
activity with the hemodynamic response in a large num-
ber of combined imaging-physiology experiments in the
striate and extrastriate areas of the monkey visual system
[31��]. Figure 2 shows the neural and BOLD signals
recorded simultaneously from an alert, fixating monkey.
At first sight, they all seemed to be correlated with the
BOLD response, although increases in the LFP range
were consistently greater in both spectral power and
reliability (SNR). Furthermore, correlation analysis
showed that LFPs are better predictors of the BOLD
response than multiunit spiking.
The relation of the two types of signals to BOLD was best
appreciated, however, in cases of a complete dissociation
between slow waves and spikes. Sites exhibiting a strong
neural response adaptation were characterized by MUA
that returned to the baseline a few seconds after stimulus
onset, and local field potentials that remained elevated for
the entire duration of the visual stimulus. In such cases,
LFPs were the only neural signal to be associated with the
BOLD response. This striking result suggests that spikes
are a ‘fortuitous’ predictor of the BOLD signal, simply
because the firing of neurons usually happens to correlate
with the LFPs. A similar dissociation between spikes and
CBF has also been demonstrated in microstimulation
studies in the cerebellar cortex (for a review of this work
see Lauritzen and Gold [42]).
The differential contribution of LFPs and MUA to the
BOLD response can also be demonstrated by neurophar-
macological manipulations that permit the selective mod-
ulation of interneuronal and pyramidal activity. Figure 3
shows the effects of intracortical serotonin injections into
the primary visual cortex of the monkey during simulta-
neous acquisition of BOLD and neural responses (N
Logothetis, unpublished data). Using a triple pipette
(electrode, saline and the neuromodulator) 20 micro-liters
of 0.01 Mol 5HT (5-hydroxytryptamine hydrochloride;
serotonin) were injected over a period of 10 minutes. A
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couple of minutes after the injection, a profound suppres-
sion of the MUA was observed (Figure 3a). The LFP
signal showed a slight increase and returned to baseline
within a few minutes. However, no significant change was
discernible in the BOLD response. Spectrograms
obtained before and after the 5HT injection during visual
stimulation showed that the stimulus-induced spikes
were entirely eliminated, whereas LFP activity was mod-
erately increased (Figure 3b). BOLD response to the
stimuli was unaltered, indicating once again the possibi-
lity of a total dissociation between spiking activity and
hemodynamic responses. On the basis of all of these
dissociations, we conclude that the LFP signal is the
key variable for the BOLD response.
Taken together, these results suggest that the BOLD
signal mainly reflects the incoming specific or association
inputs into an area, and the processing of this input
information by the local cortical circuitry (including exci-
tatory and inhibitory interneurons). Usually, the incoming
subcortical or cortical input to an area will generate the
kind of output activity that is typically measured in
intracortical single-unit recordings, and the recorded
spike rate will be correlated to the BOLD signal. If it
does not, perhaps because the activity of projection
neurons is shunted by concurrent modulatory input,
hemodynamic responses will still be generated, but the
spiking activity measured with microelectrodes will — in
such cases — be a poor predictor of the hemodynamic
response. Several experiments demonstrate the plausi-
bility of this explanation.
In a recent study, Tolias and co-workers [43�] used an
adaptation technique to study the brain areas that process
motion information [44]. They repeatedly imaged a mon-
key’s brain while the animal viewed continuous motion in
a single unchanging direction. Under these conditions,
the BOLD response adapts. When the direction of
Figure 2
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(a)
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(c)
(d)
Current Opinion in Neurobiology
Imaging and physiology in the alert, behaving monkey. (a,b) Electrode position and BOLD activation of striate cortex. (c) Neural and hemodynamic
responses from the region of interest (green circle in b). The black trace shows the comprehensive neural signal, that is, the signal of 50 mHz to
3 kHz band-width, which includes LFP, MUA and single spikes. Superimposed is the BOLD activation (red) for the region of interest around the
electrode tip. (d) Spike raster and peristimulus time histogram (PSTH) showing the activity of a small assembly of neurons (2–3 cells). Each raster dot
signifies the occurrence of an action potential. Each bin of the PSTH is equal to the repetition time of an image (250 msec).
MR imaging in the non-human primate Logothetis 5
www.current-opinion.com Current Opinion in Neurobiology 2003, 13:1–13
motion reverses abruptly, the measured activity imme-
diately shows a partial recovery toward the initial activity
levels, or rebound. The extent of this rebound was
considered to be an index of the average directional
selectivity of neurons in any given activated area. The
results confirmed previous electrophysiological studies
that had identified a distributed network of visual areas
(V1, V2, V3, V5/MT) in the monkey that process informa-
tion about the direction of visual motion.
Surprisingly, however, strong activation was also observed
in area V4, which is only weakly involved in motion
processing (e.g. [45]). Such a discrepancy can be
explained on the basis of the arguments developed ear-
lier. Areas V4 and MT are interconnected [46]. Although
they process separate stimulus properties, each area may
influence the sensitivity of the others by providing some
kind of ‘modulatory’ input, which in itself is insufficient
to drive the pyramidal cells recorded in a typical electro-
physiology experiment. In such cases, BOLD fMRI will
reveal significant activation of an area whose output may
be only indirectly related to the stimulus or task, provid-
ing results that do not match those of neurophysiology
experiments. Similarly, attentional effects on the neurons
of striate cortex have been very difficult to measure in
monkey electrophysiology experiments [47,48]. Yet for
similar tasks strong attentional effects are readily measur-
able with fMRI in the human primary visual cortex (V1)
(e.g. [49]).
In summary, combined physiology and fMRI experiments
suggest that the BOLD signal primarily reflects the input
of neuronal information to the relevant area of the brain
and its processing there, rather than the output signals
transmitted to other regions of the brain by the principal
neurons; the cells that are most easily accessible in single
cell recordings in the behaving animal. These findings are
in agreement with several observations from experiments
on the brain’s energy metabolism and the mechanisms
that couple neural activation with metabolic demands.
Figure 3
0 10 20 30 40 0 10 20 30 40Time in seconds
BOLDresponse
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103
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Effects of serotonin on the neural and hemodynamic responses. (a) Changes in the multiunit activity, local field potentials and hemodynamic
responses during the administration of serotonin. Using a triple pipette (electrode, saline and the neuromodulator) 20 micro-liters of 0.01 Mol 5HT wasinjected over a period of 10 min, as signified by the red ramp on the x-axis of the plot. A couple of minutes after the injection a profound suppression of
the MUA was observed. The LFP signal showed a slight increase, returning to baseline in a few minutes. The lower traces show the hemodynamic
response for the site of injection (pink) and a control site on the other hemisphere. No significant change was discernible in the BOLD response.
(b) Spectrograms before and after the 5HT injection. Note the MUA activity in the control spectrogram. The activity in the same frequency band is
eliminated after the injection. (c) LFP, MUA and hemodynamic responses before and after the 5HT injection. The two lower plots show the activity
of a small number of isolated neurons.
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Selective neurovascular and neurometabolic coupling
Basics of brain energy metabolism
The brain’s demand for substrate requires adequate
delivery of oxygen and glucose through elaborate
mechanisms regulating CBF. Early experimental evi-
dence that suggested a regional coupling between these
mechanisms and neural activity [50] was later verified by
means of the deoxyglucose autoradiographic technique
that enabled spatially resolved measurements of glucose
metabolism [51].
In humans, the first quantitative measurements of regio-
nal brain blood flow and oxygen consumption were per-
formed using radiotracer techniques, which were
followed by the introduction of positron emission tomo-
graphy (PET; for a historical review see Raichle [52]).
PET showed that maps of activated brain regions can be
produced by detecting the indirect effects of neural
activity on variables, such as cerebral blood flow [53],
cerebral blood volume (CBV) [2], and blood oxygenation
[2,3]. At the same time, optical imaging of intrinsic signals
demonstrated the spectacular precision of neurovascular
coupling (the strong correlation between neural and vas-
cular changes) by constructing detailed maps of cortical
microarchitecture in both the anesthetized and the alert
animal [30].
Although the existence of a regional coupling between
neural activity, metabolism, and hemodynamic changes is
now established, the nature of the links among these
processes remain largely unknown. One important ques-
tion is whether changes in CBF are driven directly by
energy demand or by neurotransmitter related signaling
(see review by Attwell and Iadecola [54��]). If energy
demand is indeed the trigger for CBF changes, then
which are the cellular processes and sites that dominate
the energy consumption? The structural and functional
organization of the neuro-vascular system provides some
insights into these questions.
Structural neurovascular coupling
The density of the vascular network in a given region
largely correlates with its average activity. Most impor-
tantly, however, the spatial correlations reported by a
large number of investigators have been mainly between
vascular density and the number of synapses rather than
the number of neurons. For instance, on the basis of its
density the human cortical vascular network can be sub-
divided into four layers parallel to the surface [55]. These
layers systematically overlap with certain portions of the
cytoarchitectonically defined Brodmann’s laminae. Nota-
bly, the first Duvernoy layer, which consists of vessels
oriented approximately parallel to neural fibers, is entirely
within the lower part of the molecular layer (Layer I),
which in the rodent has the lowest concentration of cell
bodies and highest density of synapses [56]. Similarly, in
the primate, this layer has the lowest concentration of
neurons, the highest concentration of astrocytes, and a
high density of synapses [57]. Its vascularization is there-
fore suggestive of the contribution of these cellular types
and components in the hemodynamic response.
The spatial correlation of blood vessels and neural com-
ponents reported for the human brain were later con-
firmed in animal studies [58]. The vascularization was
found to be lowest in Lamina I and highest in IVc, with an
average IVc/I ratio (across animals) of approximately 3:1.
Interestingly, the IVc/I ratio of synaptic density in the
striate cortex of macaque is 2.43:1, that of astrocytes is
1.2:1, and that of neurons is 78.8:1. Assuming some
similarity in the distribution of neurons, synapses, and
astrocytes between squirrel monkeys and macaques, the
recent data would also support the notion that vascular
density is correlated with the density of perisynaptic
elements rather than that of neuronal somata.
Functional neurovascular coupling
The cerebral metabolic rate (CMR), is commonly
expressed in terms of oxygen consumption (CMRO2)
because glucose metabolism is about 90% aerobic and
therefore parallels oxygen consumption (e.g. [59]). The
CMRO2 varies with neuronal shape, size and firing
properties, whereby large projection neurons, which
maintain energy-consuming processes such as ion pump-
ing over a large membrane surface, may have larger
energy requirements.
High-resolution autoradiography studies showed that
the uptake of deoxy-glucose is higher in the neuropil,
with its axonal, dendritic, and astrocytic processes, than
in the cell bodies. These results were confirmed by
others, which suggests that synaptic activity dominates
energy consumption (for example, Sokoloff [60]). They
are further corroborated by microstimulation experi-
ments, in which the increase in glucose utilization was
assessed during orthodromic and antidromic stimulation,
the former activating both pre- and post-synaptic term-
inals and the latter activating only postsynaptic term-
inals. Increases were only observed during orthodromic
stimulation ([61–63]; for a review see Jueptner and
Weiller [64]), which suggests that coupling occurs pri-
marily between energy metabolism and activity in the
presynaptic terminals.
The notion that perisynaptic activity (synaptic events and
restoration of gradients disturbed by postsynaptic poten-
tials) consumes a greater proportion of the available
energy receives support from the metabolic requirements
of other cellular components of the brain. Neurons are not
the only elements contributing to the energy metabolism
of the brain; glia and vascular endothelial cells do so as
well. In fact, research suggests a tightly regulated glucose
metabolism in all brain cell types (for detailed references
see Volterra and co-workers [65]).
MR imaging in the non-human primate Logothetis 7
www.current-opinion.com Current Opinion in Neurobiology 2003, 13:1–13
An interesting case is the glial cell known as the astrocyte,
which forms a massive bridge system between neurons
and the brain’s vasculature. The structural and functional
characteristics of astrocytes make them ideal viaducts
between the neuropil and the intraparenchymal capil-
laries. It has been suggested that for each synaptically
released glutamate molecule taken up by an astrocyte
(with two to three Naþ ions), one glucose molecule enters
the same astrocyte, two ATP molecules are produced
through glycolysis, and two lactate molecules are released
and consumed by neurons to yield 18 ATPs through
oxidative phosphorylation. In other words, synaptic activ-
ity may be tightly coupled to glucose uptake through the
neuron–astrocyte system (e.g. [66,67]).
The neuron–astrocyte system is compatible with the
notion that neuronal signals, which are mediated by fast
neurotransmitters or by ions or molecules that are tran-
siently released in the extracellular space upon neural
excitation, can trigger receptor-mediated glycogenolysis
in astrocytes in anticipation of or at least parallel to
regional activation (for a review see [68]).
Atwell and Laughlin [69��] proposed that the greater part
of energy expenditure is attributable to the postsynaptic
effects of glutamate (about 34% of the energy in rodents
and 74% in humans is attributable to excitatory postsy-
naptic currents). They formed this proposal on the basis
of computations of the number of vesicles released per
action potential, the number of postsynaptic receptors
activated per vesicle released, the metabolic conse-
quences of activating a single receptor and changing
ion fluxes, and neurotransmitter recycling [54��,69��].Yet others have suggested that most of the brain’s energy
is consumed for the generation and propagation of action
potentials [70�]; according to these estimates, the cost of
an action potential would permit only 1% of the neurons
in any area to be active concurrently. This prediction
seems to be at odds with experimental and theoretical
studies that suggest that the cortical neurons operate on a
high-input regime with a well-controlled balance be-
tween excitation and inhibition [71–74].
In summary, the neurovascular coupling may be med-
iated by fast neurotransmitters, other signaling molecules
(nitric oxide, prostaglandins etc.) or by any other mechan-
ism able to sense the increasing or decreasing energy
demand rapidly enough. In the former case, research
suggests that a glutamate-evoked Ca2þ influx in postsy-
naptic neurons activates the signaling-relevant molecules
that in turn produce a vasodilation. This vasodilation
reflects both the activity of neurons presynaptic to the
cells that have released the signaling molecules and the
level of depolarization of the postsynaptic cell, which will
typically alter the Mg2þ-block of NMDA receptors and
the resulting Ca2þ influx [54��]. In the latter case, over-
whelming evidence suggests that energy demand will be
driven to a lesser degree by the need to recycle neuro-
transmitters and to a greater degree by the processes of
restoring perturbed gradients through postsynaptic cur-
rents. Both pre- and postsynaptic currents are dominant
elements of the local field potentials, which — as men-
tioned above — were in fact found to correlate best with
the hemodynamic changes in the cerebral and cerebellar
cortex.
The study of networks with MRIConnectivity studies with paramagnetic tracers
Neuroanatomical cortico–cortical and cortico–subcortical
connections have been examined mainly by using degen-
eration methods and anterograde and retrograde tracer
techniques (e.g. [23,75]). Although such studies have
demonstrated the value of the information gained from
the investigation of the topographic connections among
different brain areas, they do require fixed processed
tissue for data analysis, and therefore cannot be applied
to an animal participating in longitudinal studies, in which
consecutive studies examining an entire circuit are car-
ried out in the same subject.
MRI visible tracers that are infused into a specific brain
region and are transported anterogradely or retrogradely
along axons may enable us to study neuronal connectivity
in the living animal. Such paramagnetic tracer studies
may also be used to validate and further develop non-
invasive fiber tracking techniques, such as diffusion ten-
sor MRI, which permits the study of connectivity even in
the human brain.
Manganese (Mn2þ) is an interesting example of an MRI-
visible contrast agent. The axonal transport of its radio-
active isotope (54Mn2þ) was first studied using histologi-
cal methods [76,77]. Although these studies were carried
out with the goal of understanding the regional specificity
of Mn2þ distribution, they indicated the usefulness of
Mn2þ as an anterograde neuronal tract tracer. Mn2þ
distribution and transport has been also studied with
MRI in rats and mice [78,79]. Injection of manganese
(manganum chloride, MnCl2) in to a nostril or an eye
yields a clear signal enhancement in the olfactory and
visual pathways [78,79]. Furthermore, the possibility that
the transport of manganese may pass across synapses was
suggested by some studies [77,78]. Pautler et al. [78]
indicated that Mn2þ must have traversed a synapse to
explain the enhancements detected in the olfactory cor-
tex of the mouse following the injection of its olfactory
bulb. By contrast, Watanabe and co-workers [79] reported
that the signal enhancement they observed in their rat
study was confined to regions known to receive direct
projections from the retina, and concluded that it did not
constitute evidence for trans-synaptic crossing of Mn2þ.
An example of trans-synaptic transfer of manganese is
displayed in Figure 4 (N Logothetis, unpublished data).
8 New technologies
Current Opinion in Neurobiology 2003, 13:1–13 www.current-opinion.com
Following intravitreal (into the vitreal body of the eye
ball) manganese injection a series of anatomical scans
were acquired that illustrate the transfer of the sub-
stance along the retinogeniculate–striate pathway. Sig-
nal enhancement is clear along the optic nerve and
tract, the dorsal LGN, superior colliculus, optical radia-
tion and striate cortex. Similar results were obtained in
a recent study that provided a detailed account of both
the specificity and the trans-synaptic transfer of man-
ganese in the basal ganglia of the monkey. Injections
were made into the striatum [80��]. Its projections were
confirmed histologically by injecting wheat germ ag-
glutinin conjugated to horseradish peroxidase (WGA-
HRP) into the same sites that had previously been
injected with MnCl2. The size and location of the
projection foci in the striatal targets were comparable
to those found in both the magnetic resonance and
the histology images. By injecting WGA-HRP at the
same sites as MnCl2, we also confirmed for each animal
the absence of a direct connection from the injection
sites to various brain structures (e.g. thalamic nuclei).
In this study, manganese was actually found in several
structures receiving no direct projections from the
injected sites.
MR imaging and electrical microstimulation
Our knowledge of connectivity and functional organiza-
tion could profit a great deal from the combination of MRI
with electrical microstimulation. Microstimulation is
established as an important neurobiological tool for the
study of areal representation and the functional properties
of CNS output structures. A new method was recently
developed that combines this technique with fMRI for
the detailed study of neural connectivity in the alive
animal. Specially constructed microelectrodes were used
to directly stimulate a selected subcortical or cortical area
while simultaneously measuring changes in brain activity,
which was indexed by the BOLD signal [81]. The exact
location of the stimulation site was determined by using
anatomical scans, as well as by the study of the physio-
logical properties of neurons. Electrical stimulation was
delivered using a biphasic pulse generator attached to a
constant-current stimulus isolation unit. The compensa-
tion circuit, designed to minimize interference generated
Figure 4
OT
SC
dLGN
ONOT
OR
(a)
(b)
(c) (e)
(d) (f)
Current Opinion in Neurobiology
dLGN
Intravitreal manganese injection. 0.049 ml of MnCl2 1.2M concentration was injected over a 5 min time period. Following the injection a series of
anatomical scans were acquired (MDEFT, TE ¼ 4 ms, TR ¼ 21:3 ms, FOV ¼ 12:8 � 12:8 � 8 cm3, matrix ¼ 256 � 256 � 160, voxel size ¼ 0:5 mmisotropic, segments ¼ 4). (a) Injection site (right eye). (b) Lateral (upper) and top (lower) view of the retinogeniculate–striate pathway revealed with
the Mn2þ transfer. (c) Horizontal slice at the level of the optic tract and dLGN showing signal enhancement. (d) Coronal slices with the tracer in
the lateral geniculate bodies. (e) Chiasm and superior colliculus. (f) Nissl stained histological section showing dLGN for comparison with d.
Abbreviations: ON, optic nerve; OR, optical radiation; OT, optic tract; SC, superior colliculus.
MR imaging in the non-human primate Logothetis 9
www.current-opinion.com Current Opinion in Neurobiology 2003, 13:1–13
by the switching gradients during recording, was always
active, minimizing the gradient-induced currents in the
range of the stimulation current. Local microstimulation
of the striate cortex yielded both local BOLD signals and
activation of areas V2, V3 and MT. Microstimulation of
dorsal LGN resulted in the activation of both striate
cortex and areas V2, V3, and MT. Figure 5 shows an
example of V1 activation after microstimulation of LGN.
The findings show that microstimulation combined with
fMRI can be an exquisite tool for finding and studying
target areas of electrophysiological interest.
ConclusionsThe suitability of MRI for functional brain mapping has
become firmly established over the past decade or so.
BOLD fMRI has been successfully implemented in
awake human subjects as well as in animals such as rats,
cats, and monkeys. The use of high magnetic fields
improves both signal specificity and spatial resolution.
MRI studies, in which voxels may contain as few as 600–
800 cortical neurons, can help us to understand how
neural networks are organized, and how small cell assem-
blies contribute to the activation patterns revealed in
fMRI. The combination of this technique with electro-
physiology has fully confirmed the longstanding assump-
tion that the regional activations measured in MR
neuroimaging do indeed reflect local increases in neural
activity. In addition, it has been demonstrated that fMRI
responses mostly reflect the input of a given cortical area
and its local intracortical processing, including the activity
of excitatory and inhibitory interneurons. Finally, MRI
visible tracers and microstimulation appear to be ideal for
the study of connectivity in the living animal.
AcknowledgementsThis research was supported by the Max Planck Society. I would like tothank my collaborators M Augath, Y Murayama, A Oeltermann and J Paulsfor their contribution in the experiments, S Weber for machining work,and D Blaurock for proof reading the final version of the text.
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Figure 5
75uA/500Hz
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30uA/500Hz
Current Opinion in Neurobiology
Imaging and electrical microstimulation. The top left panel shows
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position in a coronal (left) and parasaggital (right) view.
10 New technologies
Current Opinion in Neurobiology 2003, 13:1–13 www.current-opinion.com
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neuronal connections in the Macaque monkey. Neuron 2002,34:685-700.
The authors present the first application of manganese-enhanced MRI inmonkeys. Combined injections of manganese chloride and WGA-HRPwere performed in order to evaluate the specificity of the manganesechloride by tracing the neuronal connections of the basal ganglia of themonkey. Mn2þ and WGA-HRP yielded remarkably similar and highlyspecific projection patterns. By showing the sequential transport ofMn2þ from striatum to pallidum-substantia nigra and then to thalamus,
the authors demonstrated unequivocally MRI visualization of transportacross at least one synapse in the CNS of the primate.
81. Logothetis NK, Pauls J, Oeltermann A, Augath M, Trinath T:Studying connectivity with electrical microstimulation & fMRI.Abstr Soc Neurosci 2001, 27:821-830.
82. Hochmann J, Kellerhals H: Proton NMR on deoxyhaemoglobin.Use of a modified DEFT technique. J Magn Reson 1980,38:23-39.
MR imaging in the non-human primate Logothetis 13
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