Introduction
Functional magnetic resonance imaging (fMRI) is a unique window to the brain, enabling
scientists to follow changes in brain activity in response to hormones, ageing, environment,
drugs of abuse and other stimuli. There are two features that make fMRI unique when
compared with other imaging modalities used in behavioural neuroscience. First, it
can be entirely noninvasive: each animal can serve as its own control over the natural
course of its life, vital for following neuroadaptation and other developmental processes
critical to understanding behaviour. Second, fMRI has the spatial and temporal resolution
to observe patterns of neuronal activity across the entire brain in less than a minute.
Although fMRI does not have the cellular spatial resolution of immunostaining, nor
the millisecond temporal resolution of electrophysiology, synchronised changes in
neuronal activity across multiple brain areas seen with functional MRI can be viewed
as functional neuroanatomical circuits coordinating the thoughts, memories and emotions
for particular behaviours. Thus, fMRI affords a systems approach to the study of the
brain, complementing and building from other neurobiological techniques to understand
how behaviour is organised across multiple brain regions. In this review, we present
a general background to fMRI and the different imaging modalities that can be used
in fMRI studies. Included are examples of the application of fMRI in behavioural neuroscience
research, along with discussion of the advantages and disadvantages of this technology.
What are the different fMRI techniques and how do they work?
Blood oxygen level dependent (BOLD) technique
Functional MRI indirectly detects neural activity in different parts of the brain
by comparing contrast in MR signal intensity prior to and following stimulation. What
is responsible for the change in MR signal intensity? Areas of the brain with increased
synaptic and neuronal activity require increased levels of oxygen to sustain the heightened
metabolic activity involved. Enhanced brain activity is accompanied by an increase
in metabolism followed by local increases in blood flow and blood volume (1–4). The
enhanced blood flow usually exceeds the metabolic demand, which exposes the active
brain area to a high level of oxygenated haemoglobin. This is important for BOLD imaging
because oxygenated haemoglobin increases the MR signal intensity, whereas deoxygenated
haemoglobin decreases MR signal intensity. The relationship between metabolism, haemoglobin
oxygen saturation, blood flow and MR signal intensity is shown in Fig. 1 and is called
the blood oxygen level dependent technique for functional imaging or BOLD (5).
FIG. 1
Schematic diagram showing conditions contributing to blood oxygen level dependent
(BOLD) signal changes (31).
BOLD depends on the different MR signal intensities associated with oxygenated versus
deoxygenated haemoglobin. So how does haemoglobin oxygenation affect he MR signal?
To understand this we first need to deal with the basis of the MR signal. Mobile protons
associated with hydrogen atoms in water and fat are the primary source of MR signal.
The hydrogen nucleus with its single charged proton spins creating a surrounding electromagnetic
field with the characteristics of a magnetic dipole. When placed in an external magnetic
field, quantum mechanics tells us that this hydrogen nucleus can have two ‘spin’ states
or energy levels. Most of the nuclei prefer the lower energy state rather than the
higher. When electromagnetic radiation in the radiofrequency range (MHz) is applied,
nuclei can absorb energy and be elevated to the higher energy state. The energy given
off as these nuclei ‘relax’ back into their lower state is the source of the MR signal.
A more intuitive but less accurate description of nuclear magnetic resonance is provided
by classical mechanics. Consider each hydrogen nucleus as a spinning top with a random
orientation in a non magnetic environment as shown in Fig. 2. When placed into a magnetic
field, the hydrogen nuclei align in parallel with the field lines of the magnet. The
net magnetisation from all of these aligned nuclei is shown as a vector parallel to
the main external magnetic field B0. These aligned nuclei are the source of potential
energy for MR signal. Increasing the strength of the magnetic field recruits more
hydrogen protons for imaging. The differences in hydrogen proton density between tissues
like cerebrospinal fluid, grey and white matter are one reason for signal contrast
in neuroanatomical imaging.
FIG. 2
Schematic diagram showing the behaviour of hydrogen nuclei in magnetic and nonmagnetic
environments (31).
The spinning hydrogen nuclei ‘wobble’ or show an angular precessional frequency when
placed in the main magnetic field as depicted by the circles in Figs 2 and 3. This
precessional frequency is directly related to the field strength of the magnet. Animal
magnets with field strengths of 4.7, 7.0, 9.4 and 11.7 Tesla create precessional frequencies
for hydrogen nuclei of 200, 300, 400 and 500 MHz, respectively. Brief radiofrequency
(RF) pulses can be used to create a magnetic field B1 perpendicular to the main field
B0 as shown in Fig. 3. If these RF pulses have the same frequency as the precessing
hydrogen nuclei then the resonant energy is absorbed, flipping the nuclei into the
transverse x-y plane where they continue to precess. When initially flipped, all of
the hydrogen nuclei precess at the same frequency and are said to be in-phase. This
‘in-phase’ precession in the transverse plane sets up an oscillating MR signal that
can be picked up by a special antenna called an RF receiver or probe. However, this
oscillating signal rapidly decays lasting only 20–50 ms. Interactions between these
nuclei and inhomogeneties in their surrounding microenvironment cause some to precess
slower than others resulting in ‘de-phasing. The BOLD technique enhances the MR signal
because deoxygenated haemoglobin is paramagnetic and creates its own micromagnetic
field, promoting de-phasing and thus reducing MR signal (Fig. 1). However, oxygenated
haemoglobin has very little magnetic susceptibility. Because active brain areas have
enhanced blood flow and are high in oxygenated haemoglobin, phase coherence is promoted
resulting in a stronger MR signal.
FIG. 3
Schematic diagram showing behaviour of hydrogen nuclei flipped into the transverse
x–y plane with a radiofrequency (RF) pulse. B0 is the main magnetic field and B1 is
the magnetic field created in the transverse plane by the RF pulse (31).
Following an RF pulse, hydrogen nuclei undergo two simultaneous events. They precess
in the transverse plane generating MR signal for as long as they remain in-phase and
they relax back into the orientation of the main magnetic field returning to their
lowest energy state. Both time courses are exponential functions. The time constant
for relaxation is called T1 whereas the time constant for de-phasing is called T2.
T1 is measured in seconds and T2 is measured in milliseconds. A third time constant
called T2* is shorter than T2 and reflects de-phasing caused by conditions in the
microenvironment contributing to magnetic susceptibility. Changes in T2* are the bases
behind BOLD signal changes in functional imaging.
It is important for MR imaging to note that, for as long as there is net magnetisation
in the transverse plane, there is potential for generating a series of MR signals.
Spin echo and gradient echo pulse sequences have been developed to bring the hydrogen
nuclei back into phase or have them ‘echo’ repeatedly until the system comes back
to equilibrium.
In a spin echo pulse sequence, the initial 90° pulse flips the hydrogen nuclei into
the transverse plane as shown in Fig. 4. Afterward, a refocusing 180° pulse is applied,
which reverses the direction of the precessional spins. The faster spinning hydrogen
nuclei that have been turned around by these pulses are now facing the slower spinning
nuclei. The MR signal waxes and wanes as the faster nuclei catch up and pass the slower
nuclei. The 180° refocusing pulse can be applied several times while there is transverse
magnetisation. Usually, after 1.5–2.5 s, the sequence is repeated (time to repeat
or TR) and another 90° pulse is applied to flip the hydrogen nuclei into the transverse
plane and the echo series begins again.
FIG. 4
Schematic diagram showing the behaviour of precessing hydrogen nuclei in a spin echo
pulse sequence. Net magnetisation (red arrows) in the main magnetic field (B0) slowly
relaxes back to equilibrium over successive 180° pulses following the initial 90°
pulse. Time to repeat (TR) is the repetition time between 90° pulses (31).
One of the major advantages of fast spin echo pulse technique, particularly in imaging
fully conscious animals, is its tolerance to artifacts from physiologic motion (e.g.
cerebrospinal fluid movement) or magnetic susceptibility (e.g. distorted signal at
air/liquid interfaces) (Fig. 5). However, magnetic susceptibility caused by deoxygenated
haemoglobin is a key component in the BOLD signal. Hence, the fast spin echo technique
is less sensitive to changes in BOLD signal than other techniques (see description
of gradient echo, below). This problem of sensitivity can be addressed with higher
field strengths and better RF electronics as discussed later.
FIG. 5
Shown are magnetic resonance images highlighting the advantages and disadvantages
of spin echo and gradient echo pulse sequences. All images were collected from the
same animal over the same imaging session. Susceptibility artifact is very pronounced
in the substantia nigra (SN); and ventral tegmental area (VTA). S/N, Signal-to-Noise
ratio (28).
In a gradient echo pulse sequence, the initial RF pulse only partially flips the hydrogen
nuclei into the transverse plane as shown in Fig. 6. Because these nuclei are only
partially flipped, they rapidly lose their transverse magnetisation as they return
to equilibrium in the main magnetic field. Hence, a series of short TRs usually between
50 and 75 ms are necessary to collect multiple MR signals. The main purpose of the
gradient echo technique is to increase the speed of the scan. The rephasing event
required for echo generation is accomplished by reversing the local magnetic field
with special gradient coils. Gradient echo is particularly sensitive to magnetic susceptibility
and, for this reason, is commonly used in functional imaging, where the differential
magnetic susceptibility of oxygenated versus deoxygenated haemoglobin provides the
signal. However, this advantage of increased sensitivity is also the major disadvantage
of gradient echo imaging because of susceptibility artifacts, most noticeably seen
in neuroimaging at the air/tissue interfaces associated with sinuses. Gradient echo
is also very sensitive to shimming (improving the field homogeneity), making it difficult
to collect undistorted slices across the entire brain.
FIG. 6
Schematic diagram showing the behaviour of precessing hydrogen nuclei partially flipped
into the transverse plane at an angle of 30°. During the short-lived event, the gradient
coils are used to reverse the magnetic field (black horizontal line) to refocus the
precessing nuclei (31). TR, Time to repeat.
Cerebral blood flow (CBF) technique
Changes in image intensity due to the BOLD effect are the result of complex interactions
between blood flow, blood volume, haemoglobin oxygenation and neurovascular coupling
in metabolically active areas. However, a robust increase in BOLD signal can be obtained
without a change in neuronal activity. A good example of this ‘uncoupling’ is a simple
carbon dioxide (CO2) challenge. Inhalation of CO2 causes direct cerebrovascular dilation,
increasing blood flow and BOLD signal intensity to brain tissue independent of metabolism
(6). Indeed, theoretically, there is no change in cerebral metabolism with CO2 challenge.
Consequently, it is not always true that a localised change in BOLD image intensity
is a reflection of a change in brain activity. In these situations, MRI techniques
which permit the noninvasive measurement of absolute or relative changes in CBF enable
direct observation of haemodynamic changes without the confounding influence of deoxygenated
haemoglobin.
Noninvasive MRI techniques for measuring CBF use the protons in water molecules in
the blood as an endogenous tracer, whereby the state of blood magnetisation is modified
to produce a measurable change in signal intensity downstream in the tissue into which
this blood flows (7). Two types of these arterial spin labelling techniques are commonly
used in research, continuous arterial spin labelling (CASL) and pulsed arterial spin
labelling (8). The CASL technique (Fig. 7) is commonly used in animal research applications,
and requires two brain scans to be performed. In the first scan, an RF pulse is applied
for a period of several seconds in a slice containing the carotid arteries. Blood
flowing through these arteries at this location experiences complete inversion, and
flows into the brain where these labelled water molecules in the blood exchange with
water molecules in the brain. An image of the brain is then acquired in the presence
of these labelled water molecules. In the second scan, the same arterial spin labelling
pulse is applied to a location symmetrically opposed to the imaging slice, away from
the feeding arteries. The same image is acquired in the absence of any labelled water
molecules from arriving blood, and the difference between the control and labelled
images yield a measure of local perfusion.
FIG. 7
Schematic diagram showing arterial spin labelling procedure and image processing.
(a) Control image. (b) Image acquired with arterial spin labelling. (c) Label/Control
− 1. (d) False coloured overlay of blood flow. Note the enhanced blood flow to the
cortex as compared to subcortical areas. Legend scale 0 ± 12%.
The CBF technique is widely used in imaging studies such as ischemic stroke to assess
blood flow to the different areas of the brain; however, it has not displaced other
imaging techniques widely used by behavioural neuroscientists to assess functional
changes in brain activity. Because of the small amount of contrast (typically only
a few percent of total image intensity between the control and labelled images), the
sensitivity of arterial spin labelling CBF measurements is inherently lower than that
of several other functional MRI modalities (9). Additionally, multislice CBF measurements
using the CASL technique require that an additional coil, located near the carotid
arteries supplying the brain, be used to transmit the labelling pulse (10). The limited
sensitivity and complex hardware requirements of ASL experiments explain the comparatively
infrequent use of this technique in the literature. However, CBF measurements can
provide valuable information under certain circumstances. For example, when BOLD and
CBF changes are measured simultaneously during the presentation of a stimulus, a calibrated
calculation of changes in Cerebral Metabolic Rate of Oxygen consumption (CMRO2) can
be performed, providing potentially enhanced inference into changes in the underlying
neural metabolism than is possible using either measurement alone (11). In our own
laboratory we are using CBF to assess baseline conditions prior to and following an
imaging experiment because studies have shown that changes in CBF will alter the magnitude
of the BOLD response (12–14). Evoked cortical responses produce greater changes in
BOLD signal with low CBF and lesser changes with higher CBF. By assessing CBF over
the course of a BOLD imaging study, we can better evaluate the quality of the data.
Cerebral blood volume (CBV) technique
In fMRI, there is always a demand to increase sensitivity in MR signal above background
noise. Two terms used in the MRI field that relate to signal sensitivity are signal-to-noise
ratio (SNR) and contrast-to-noise ratio (CNR). SNR provides a quantitative evaluation
of the MRI image before the start of an imaging session. A simple and practical way
to measure SNR is to draw two identical region-of-interests (ROI), one over the object
and another in the surrounding field of view (Fig. 5). The SNR should be comparable
across experiments, a measure of system stability. We routinely perform this calculation
in our laboratory at the beginning of each imaging session. CNR is of vital importance
because it reflects signal contrast between two functional states. Specifically, CNR
is the difference in signal intensity between a stable window of baseline activity
and a functional challenge (e.g. drug administration, odour presentation, visual stimulation).
Standardised measurements of CNR can be assessed at the beginning of each imaging
session by simple block design of (off–on–off–on–off) of 5% CO2 inhalation at 1-min
intervals.
Anything that will increase SNR and CNR in fMRI will increase spatial and temporal
resolution. A simple but costly way of increasing MR signal in functional imaging
is to increase the field strength of the main magnet. As an approximation, signal
intensity increases linearly as you move up in field strength (15). This hardware
solution is not always feasible because ultra-high field scanners are still few in
number and not readily available to behavioural neuroscientists as research tools.
Consequently, exogenous contrast agents have been developed to achieve high sensitivity
even at low field strength scanners. For example, monocrystalline iron oxide nanocolloid
(MION) is a contrast agent that, when given intravenously, will dramatically change
the microenvironment of spinning hydrogen nuclei causing rapid dephasing (16). As
noted earlier, an increase in tissue metabolic activity causes a localised increase
in blood flow. Because cerebral blood vessels have some compliance, there is an accompanying
increase in blood volume in the area of neuronal activity. The increase in CBV raises
the relative concentration of MION, reducing the MRI signal. The magnitude of the
change in signal contrast is far greater than the endogenous contrast imaged with
BOLD or CBF techniques. Indeed, the major advantage of CBV over BOLD and CBF techniques
is its use in fMRI at magnetic field strengths under 3.0 T. However, to take full
advantage of the CBV technique, gradient echo pulse sequences must be used which distort
the image quality and complicate data interpretation in brain areas sensitive to susceptibility
artifacts (Fig. 5).
CBV has been used most often in pharmacological MRI to assess brain activation to
a sustained drug challenge. With its enhanced CNR, it is possible to assess dose–response
and pharmacokinetic relationships with changes in MR signal for discrete brain areas.
Presently, work is under way in the laboratory to examine the effect of ethanol on
brain activity (17). Using a nonlinear pharmacokinetics model (18), it is possible
to fit the raw CBV data to an ethanol-induced response curve, calculate the area under
the curve (AUC), and compare this change with a vehicle control (Fig. 8). Three important
pharmacokinetic parameters can be extracted using this model: (i) absorption rate;
(ii) peak to peak response (maximum response); and (iii) elimination rate. In the
data shown, there are no significant differences in the response to ethanol challenge
between cortex and striatum in terms of absorption and maximum response; however,
a much slower elimination rate was observed in cortex compared to striatum leading
to CBV signal with a greater AUC in cortex.
FIG. 8
Shown is an activation map for the cerebral blood volume (CBV) response (percent change
in area under the curve) in a conscious rat in response to intravenous ethanol (0.75
g/kg). Below are raw time course data from the four contiguous voxels located in each
of the black boxes depicted in the activational map. The CBV signal in each voxel
was fitted to a nonlinear model using the Analysis of Functional Images (AFNI) software
program (29). From these data, it is possible to assess the pharmacokinetic profile
of ethanol's action on the brain.
Methodological considerations when imaging conscious animals
Controlling for motion artifact
Motion artifact is a considerable problem in fMRI studies. Any minor head movement
distorts the image and may also create a change in signal intensity that can be mistaken
for stimulus-associated changes in brain activity (19). In addition to head movement,
motion outside the field of view caused by respiration, swallowing and muscle contractions
in the face and neck are other major sources of motion artifact (20, 21). Motion artifact
can be reduced by the use of general anaesthesia. Indeed, historically, the primary
reason for imaging fully anaesthetised animals is the reduction in noise leading to
improved signal resolution. However, this is not a feasible solution for neuroscientists
wanting to understand the neurobiology of behaviour as it precludes the study of brain
activity involving cognition and emotion. Furthermore, anaesthetics depress neuronal
activity reducing MR signal (6, 22, 23). Consequently, different methods have been
developed to restrain animals during an imaging session to minimise motion artifact.
Surgery is one option, in which the animal has a head-stabilising post or cranioplastic
cap fixed to the skull. During imaging, this skull implant is locked into a holder
in the bore of the magnet (24–26). In place of surgery, animals can be paralysed with
curare-like drugs and artificially ventilated to minimise motion artifact and to control
for the levels of blood gases (27). One entirely noninvasive system developed by Insight
Neuroimaging Systems (Worcester, MA, USA) can be used to set up an animal in just
a few minutes (28). In brief, just prior to the imaging session, animals are lightly
anaesthetised with isoflurane gas. A topical anaesthetic is applied to the skin and
soft tissue around the ear canals and over the bridge of the nose. The head is secured
into a stereotaxic-like support system with ear bars and nose clamp. The body of the
animal is placed into a body restrainer that is mechanically independent from the
head holder. This design isolates all of the body movements from the head restrainer
and minimises motion artifact. After the animal is set up, the isoflurane gas is removed
and the restraining system is positioned in the magnet. Animals are fully conscious
within 10–15 min and functional imaging can proceed. Central nervous system activity
may, of course, be influenced by residual effects of anaesthesia required for setting
up animals in restrainers, however, by using anaesthetics with rapid elimination from
the body (such as isoflurane), such issues are minimised. With conscious imaging,
at least any effects of the anaesthetic are minor and are temporally removed as far
as possible from functional scans, by protocols in which anatomical images are collected
first. Thus, in general, in our laboratory, functional scans are carried out at least
30 min after animal subjects have regained consciousness, and are likely to be minimally
affected by prior anaesthesia. Determining with certainty whether the use of anaesthesia
for setting up restraint affects functional MRI data is difficult because it would
be impossible to set up an animal without use of sedation for control comparisons.
Following an imaging session there are several simple ways to assess motion artifact:
(i) subtraction of anatomical data across the imaging session; (ii) qualitative analysis
of time series movies looking for voxel displacement; and (iii) analysis of raw data
time series for course spikes. Movement of the head by a single voxel or more over
the course of an imaging session will appear as a ‘ghost’ image following subtraction
of anatomical data sets (Fig. 9). Time series movies show ‘blips’ or distortions that
correlate with course spike activity in the raw data. More elaborate software programs
have been written to evaluate and correct for motion artifacts (29).
FIG. 9
Shown are anatomical images collected over the time from the same brain slice. Voxel
by voxel subtraction of these images show no evidence that there was any movement
of the head during the imaging study (35).
Controlling for stress
The stress caused by immobilisation and noise from the MR scanner during functional
imaging in fully conscious animals is a major concern. To address this problem, animals
are routinely acclimated to the imaging procedure prior to their first scanning session.
The acclimation procedure is essentially a simulated scanning session. Animals are
anaesthetised with isoflurane as described above for securing the animal into the
restrainer. When fully conscious, the animal is exposed to simulated experiment by
placing the restraining unit into a black opaque tube ‘mock scanner’ with a tape-recording
of an MRI pulse sequence. This procedure is repeated every day for 4 days. Following
acclimation, rats show a significant decline in body temperature, motor movements,
heart rate and plasma corticosterone levels compared to their first day of restraint
(30). Another advantage of acclimating animals for imaging is an increase in CNR.
The reduction in motor movement decreases the baseline level of noise resulting in
better signal resolution (30). Interestingly, basal blood flow to the brain is not
affected by acclimation. There is no difference in basal CBF in animals exposed to
their first restraint and imaging session compared to the same animals after several
days of acclimation. This finding is particularly important because as noted above,
changes in baseline perfusion affect the magnitude of the BOLD response.
The common marmoset monkey (Callithrix jacchus) is an excellent subject for fMRI studies.
Marmosets acclimate in only 2–3 days and can be imaged while fully conscious without
the need for surgery or paralysis to minimise motion artifact (31). Their small size
(300–500 g) allows them to be imaged in ultra high field, small bore scanners. Rhesus
macaques also prove to be adequate subjects for fMRI because they can be acclimated
and trained to relax quietly in a sphinx like position in a horizontal bore (25, 26)
or sit up-right in a vertical bore (32). Indeed, Gash et al. (33), at the College
of Medicine, University of Kentucky, resolved many of the issues associated with imaging
fully conscious rhesus monkeys and published a comprehensive description of their
equipment and procedures. Frederick et al. (34), at McLean Hospital, Brain Imaging
Center, recently acclimated and imaged cynomolgus monkeys for up to 2 h using a totally
noninvasive restraint device developed by Insight Neuroimaging Systems (Worcester,
MA). Monkeys were exposed to both photic and noxious thermal stimulations. Areas of
activation corresponded to those activated by the same stimuli in unanaesthetised
human volunteers. Motion during these procedures did not exceed < 0.3 mm (translational)
and < 0.15° (rotational) during scans. This level of motion is below that typically
observed in human studies and was adequately dealt with by motion correction.
Eliciting behavioural responses in the magnet with natural stimuli
What behaviours can be studied in the magnet and how can they be triggered during
an imaging session? Obviously, any behaviour that requires a consummatory act would
be very difficult to study with fMRI because the immobilisation alone most likely
would prevent the motor response that defines the behaviour. However, fear, anger
and hunger are examples of internal states of arousal and motivation. These types
of behavioural conditions are fertile areas of investigation using fMRI. An investigator
can collect a library of smells and visual images that have proven ethological significance
in the animal's natural habitat and in the seminatural environment of the laboratory
setting. These smells and images can be used to communicate with the animal in the
magnet. For example, we have used odours of novel receptive female marmosets to elicit
sexual arousal in male marmosets during an imaging session (35, 36). Vocalisations,
while a key form of communication in many animals, are hard to use in imaging studies
because of the noise created by the scanner. To date, no method has been developed
for using vocalisations to elicit changes in brain activity in conscious animals during
an imaging session.
Another way of communicating with animals in the magnet is to bring the natural activating
stimulus directly into the bore of the magnet. In a recent study, we imaged changes
in BOLD signal in dams in response to pup suckling (37). This was accomplished by
attaching a cradle with pups to the restrainer beneath the ventrum of the mother.
A shield separates the pups from the mother's teats. When the shield is pulled away,
the pups immediately begin to suckle (Fig. 10) resulting in activation of the mesolimbic
dopaminergic pathway (Fig. 11). In another example, we built a perforated Plexiglas
vivarium that can be fitted into the magnet bore directly in front of the subject
being imaged. The vivarium is scented with the bedding of the subject's home cage.
Robust changes in brain activity can be elicited by placing different ‘intruders’
into the vivarium (e.g. male competitors or sexually receptive females).
FIG. 10
Photographs showing a conscious dam restrained in the device used for functional magnetic
resonance imaging. A cradle of the dam's pups is positioned to allow access to the
teats for suckling. © 2005 The Society for Neuroscience.
FIG. 11
Three-dimensional activational map showing that pup suckling activates the reward
system in lactating dams. Upper left picture is a translucent shell of the brain viewed
from a dorsal perspective showing colour coded volumes of interest (VOIs) corresponding
to anatomical geometries of the mesocorticolimbic and nigro-striatal dopamine systems.
Colours have been melded into a single functional VOI (yellow) showing localisation
of positive (red) and negative (blue) BOLD signal changes with pup stimulation. The
top left brain (below the colour coded shell) includes both dopamine systems while
the bottom left brain has masked the caudate/putamen and substantia nigra comprising
the nigro-striatal dopamine system revealing the mesocorticolimbic or reward system.
Pictures on the right hand side are the corresponding negative BOLD images for each
functional volume, respectively. The middle columns show traditional activation maps
of contiguous brain sections with labelled regions of interest (37).
Identifying discrete behavioural responses
How do we tease apart the elements of a complex behaviour and correlate these changes
with brain activity using fMRI? With careful attention to the temporal pattern of
behavioural activation, it may be possible to identify and correlate nuances in a
complex behaviour with changes in BOLD signal. For example, the presentation of olfactory
and visual stimuli of a predator to elicit a fear response in an animal would be expected
to activate olfactory and visual pathways, limbic and cortical structures mixed with
motor pathways involved in initiating freezing or escape behaviour. Outside the magnet,
this stimulus-evoked behaviour could be videotaped, time coded and analysed to yield
a record of the sequence and chronology of the behavioural response in the form of
a time-event table. With lag sequence analysis, the contingency and timing between
subtle behavioural events can be predicted. The fMRI acquisition parameters can be
set to provide the temporal resolution necessary to match this time-event table. When
combined with well-designed control studies, it may be possible to correlate the spatial
and temporal pattern of brain activity with discrete events in the complex behaviour.
Indeed, it is routine in our laboratory to run our studies on the bench top before
an actual imaging session. For example, in a just completed study, we imaged the neural
circuitry contributing to the genesis of generalised tonic/clonic seizure by collecting
continuous, high resolution, multislice images at subsecond intervals following administration
of an epileptogenic agent (38). Coordinating the timing of the image acquisitions
with the predicted onset of seizure was based on electroencephalogram recordings and
muscle contractions, performed on the bench top.
Data analysis
Although there are many programs available for fMRI data analysis, many of these are
aimed at analysing human imaging data. Some programs, including Stimulate (39) and
AFNI (29), are readily available and suitable for analysing animal imaging data. Additionally,
we have developed our own registration, segmentation, and cumulative analyses tools,
now commercially available as MIVA (40). MIVA employs a quantitative analysis strategy,
successfully developed and optimised for the rat brain. Each subject is registered
or aligned to a fully segmented rat brain atlas that has the potential to delineate
and analyse more than 1200 distinct anatomical volumes within the brain. These detailed
regions are collected into 96 subvolumes (e.g. dentate gyrus, insular cortex, anterior
dorsal thalamus, accumbens) that are grouped into 12 major regions of the brain (e.g.
amygdaloid complex, cerebrum, cerebellum, hypothalamus). In addition, the atlas is
designed to represent functional neuroanatomical systems, such as the primary and
vomeronasal olfactory systems (Fig. 12).
FIG. 12
Shown in the background is a graphical interface with both image and geometry data
displayed. The left displays are magnetic resonance (MR) imaging image data from a
rat brain. The lower right display is geometry-based two-dimensional display of a
segmented rat brain corresponding to the registered rat brain image in the lower left
corner. The upper right display is a three-dimensional view of the geometry-based
rat brain shell. Superimposed within the shell are the MR image on the left and the
segmented slice from the bottom of the display. Shown in the lower right is an MR
image of a rat brain slice registered to a cross section of the fully segmented atlas.
The matrices that transformed the subject's anatomy shells to the atlas space are
used to embed each slice within the atlas. All transformed pixel locations of the
anatomy images are tagged with the segmented atlas major and minor regions creating
a fully segmented representation of each subject. The inverse transformation matrix
[T
i]−1 for each subject (i) is also calculated. An interactive graphic user interface
facilitates these shell alignments (41). Approximately 15–20 min per subject are required
to create the slice perimeters, run the marching cube, align the geometries and create
the final segmented anatomy.
Statistical t-tests are performed on each subject within their original coordinate
system. The control window is a defined set of image acquisitions over the first 3–5
min. The stimulation window is a defined set of image acquisitions lasting from 5
to 30 min. The imaging is continuous without interruption. The t-test statistics use
a 95% confidence level, two-tailed distributions and heteroscedastic variance assumptions.
Due to the multiple t-test analyses performed, a false-positive detection controlling
mechanism is introduced (42). This subsequent filter guarantees that, on average,
the false-positive detection rate is below our cutoff of 0.05. These analysis settings
provide conservative estimates for significance. Those pixels deemed statistically
significant, retain their percent change values (stimulation mean minus control mean)
relative to control mean. All other pixel values are set to zero.
Applications in behavioural neuroscience research
Brain/environment interactions during development
There are myriad examples in animal studies showing early emotional or environmental
insult can affect brain development with long-term neurobiological and behavioural
consequences. Insights into the aetiology of mental illness and drug addiction may
be gleaned by longitudinal studies examining the interaction between a vulnerable
gene pool, a stressful environment, and/or exposure to drugs of abuse. Because fMRI
is noninvasive, it can be used to study the same animal over the course of its life.
Presently, studies are underway in the laboratory examining the psychosocial and cognitive
effects of methylenedioxymethamphetamine (MDMA) given orally to adolescent marmoset
monkeys in doses that mimic recreational drug use in human adolescents. Functional
imaging is used to follow the changes in brain activity in response to acute and chronic
MDMA exposure as monkeys grow from adolescence into adulthood (43). Functional imaging
can also be used to study animal models of aspects of drug addiction, such as progressive
changes in response to repeated doses [whether increased responding (i.e. sensitisation)
or decreased responding (i.e. tolerance)]. Repeated administration of drugs of abuse
modifies behavioural responses in rats under many experimental conditions. Alterations
in drug-induced behaviour over time are usually measured as increases in locomotor
activity or stereotypic behaviour, preference for a drug-conditioned environment,
or reinforcement of drug self-administration. For example, daily administration of
a single cocaine dose progressively enhances locomotor behaviour in rats termed behavioural
sensitisation. Although the initial actions of cocaine occur primarily within the
mesolimbic dopamine system, there is a growing body of studies showing that other
brain regions and many other neurotransmitter systems are involved in the long-term
response to this drug. Imaging the progressive changes in brain activity following
repeated administration of cocaine or other drugs of abuse permits an integrated view
of the same animal over time during its transition to a sensitised state.
We recently assessed the effects of acute and chronic cocaine administration on brain
activity in awake, acclimated male rats (44–46). Our data showed that an intracerebroventricular
dose of 20 µg of cocaine produces increases in BOLD signal in areas corresponding
to the brain mesocorticolimbic or reward pathway. This dose of cocaine also elicits
increases in locomotor activity and dopamine metabolism in animals studied outside
the magnet. Rats administered cocaine repeatedly for one week before imaging showed
only modest increases in brain BOLD activity when given a challenge dose during functional
imaging. It appears that with chronic administration brain activity in response to
cocaine is reduced as compared to the first exposure, an effect akin to pharmacological
tolerance. To assess whether these changes in neural responsiveness to cocaine with
chronic exposure corresponded to altered cerebrovascular reactivity, animals were
given a very brief hypercapnic challenge in order to stimulate a ‘mock’ BOLD response
in the absence of neuronal activity. We found no differences in haemodynamic-related
BOLD responses between acute and chronically treated animals, suggesting altered brain
responsiveness to cocaine instead. Results from these studies showed remarkable spatial
concordance with previous studies looking at cocaine stimulation of metabolic activity
using the 2-deoxyglucose autoradiography technique (47).
Imaging the neural circuitry of emotion
Three areas of behaviour that carry high emotional valence are fear, sexual arousal,
and aggression. Much has been learned about the neural circuitry involved in these
emotions from imaging human volunteers (48). However, there are limitations in the
types of experiments that can be performed on humans. Moreover, many human studies
are confounded by the heterogeneity of the population sample and the psychosocial
history of the volunteers. Consequently, this is an exciting area of study using animals.
However, the biggest problem in imaging emotions in animals is data interpretation.
Although humans can provided a semiquantitative measure of their emotions using visual
analogue scales and other assessment methods, this level of communication is absent
in animal imaging.
To evaluate brain activity associated with sexual arousal, we imaged male marmoset
monkeys during presentation of vaginal odours of receptive females versus odours from
ovariectomised females (35, 36). Again, prior to the imaging session, we studied the
stimulus response pattern of male marmosets during exposure to periovulatory odours
of novel females. Measures of sexual arousal were collected and correlated with changes
in physiology and endocrinology (49). The anticipated changes in autonomic physiology
were corroborated during presentation of periovulatory odours during an imaging session.
Interestingly, a common neural circuit comprising the temporal and cingulate cortices,
putamen, hippocampus, medial preoptic area and cerebellum shared showed positive BOLD
response to peri-ovulatory odours but negative BOLD response to odours of ovariectomised
females. The negative BOLD data were interpreted as a reduction in brain activity
(for more on interpreting negative BOLD data, see response to question 4 in ‘Questions
for the bench’, below). These data suggest the odour driven enhancement and suppression
of sexual arousal affect neuronal activity in many of the same general brain areas.
These areas included not only those associated with sexual activity, but also areas
involved in emotional processing and reward.
Nursing has reciprocal benefits for both mother and infant helping to promote maternal
behaviour and bonding. To test the ‘rewarding’ nature of nursing, fMRI was used to
map brain activity in lactating dams exposed to their suckling pups versus cocaine
(37). Suckling stimulation in lactating dams and cocaine exposure in virgin females
activated the dopamine reward system. By contrast, lactating dams exposed to cocaine
instead of pups showed a suppression of brain activity in the reward system. These
data support the notion that pup stimulation is more reinforcing than cocaine, underscoring
the importance of pup-seeking over other rewarding stimuli during lactation.
Following these studies, we hypothesised that oxytocin, released in the maternal brain
during breastfeeding, may be involved mechanistically. To follow-up the above studies,
we exposed postpartum dams to pup suckling before and after administration of an oxytocin
receptor antagonist; we also administered oxytocin alone to a control group of dams.
Dams exposed to oxytocin showed brain activation that was extremely similar to that
activation seen when dams were suckling pups. The brain activation seen in suckling
dams was almost completely attenuated by the oxytocin antagonist. Overall, data suggest
that oxytocin strengthens mother-infant bonding through acting on key brain areas
for reward, emotion, and olfactory discrimination; namely, the nucleus accumbens,
prefrontal cortex, ventral tegmental area, amygdala, insular cortex, several cortical
and hypothalamic nuclei and the olfactory system (50).
Investigating effects of psychotherapeutics
Functional MRI shows incredible potential for probing the effects of pharmacological
agents on brain activity. Not only does fMRI allow us to understand the fundamental
mechanisms of brain function, but it also allows the testing and in vivo investigation
of the neural actions of potential brain medications. Both acute and long-term progressive
effects of drug treatment on brain function can be evaluated using fMRI. The study
by Hagino et al. (24), examining changes in brain activity following acute and prolonged
exposure to dopamine receptor agonists and antagonists, is an obvious application
of fMRI in animal studies. For example, serotonin reuptake inhibitors cause a prompt
increase in brain levels of serotonin and changes in behaviour (51). Nonetheless,
patients treated for depression or obsessive compulsive disorder with serotonin reuptake
blockers require weeks of treatment before reporting an improvement in their condition.
This would suggest drug efficacy for the treatment of mental illness is due to secondary
changes in the serotonin system or other interrelated neurochemical signals and pathways
that are slowly affected by the continuous exposure to elevated serotonin with chronic
drug treatment. With fMRI, the same animal can be imaged over long periods of time
following progressive changes in brain activity associated with continuous drug treatment.
Currently, our group is using the ability of fMRI to follow progressive changes within
the same animal to investigate the therapeutic mechanisms of antipsychotic drugs.
Similar to selective serotonin reuptake inhibitors (SSRIs), antipsychotic medications
require several weeks of administration to exert a therapeutic effect; unlike SSRIs,
multiple drugs with radically different pharmacological profiles have demonstrated
an ability to abate the positive symptoms of psychotic disorders such as schizophrenia
with sustained administration. By using fMRI of pharmacological challenges to specific
neurotransmitter systems, we are investigating the changes in neural signalling that
occur within the same animal following several weeks of treatment with neuroleptic
medications. Correlated with changes in behaviour, the localisation of changes in
neural activation resulting from a dopaminergic challenge that appear after sustained
treatment with neuroleptic medications may provide a marker of therapeutic action,
and may facilitate the future development of novel medications with reduced side-effects
or enhanced efficacy.
With respect to the above cited studies, knowledge on the systemic effects of drugs
is paramount for fMRI. Intravenous or intraperitoneal administration of psychostimulant
drugs such as cocaine and amphetamine can produce physiological disturbances that
hamper fMRI data. Also, some drugs have direct effects on the vascular endothelium
in the brain, possibly affecting haemodynamic responses to brain activity which provide
the basis for the BOLD signal. These physiological perturbations can be overcome by
prior benchtop work to determine dose-effects on systemic physiology of animals. Testing
different doses and routes of administration that can eliminate or delay the onset
of systemic effects (i.e. cerebroventricular, oral, subcutaneous) can be used in animals
and can help improve the resulting fMRI data.
Such studies of drugs of abuse and psychotherapeutics are of relevance for neuroendocrinologists
as they illustrate the methodological possibility of administering drugs interacting
with neuroendocrine systems (via numerous routes of administration) and following
brain activational responses.
Testing cognitive performance
Deficits in learning and memory are recognised as components of the symptomology of
several mental disorders such as attention-deficit disorder (52) and schizophrenia
(53). Many learning paradigms do not require any signs of overt behaviour, making
them amenable to testing with fMRI. Because animals will readily respond to peripheral
stimulation when in the magnet for fMRI, they may be used in studies of classical
conditioning. For example, foot shock can be used as an unconditioned response in
associative learning paradigms. When coupled with a conditioned stimulus such as light,
it can be used in learning studies examining discrimination and perception. Operant
conditioning would be more difficult because a behavioural action (e.g. bar pressing
eliciting rewarding or punishing stimuli) would be necessary. However, a study by
Logothetis et al. (32) demonstrated that awake rhesus monkeys can be trained to press
buttons during MRI protocols. These advances in the use of conscious animals open
the area of cognitive neuroscience to investigation with fMRI. Because many hormones
are involved in cognitive change resulting from ageing and/or disease processes, the
development of the ability to image cognitive function in model animals whose neuroendocrine
status can be manipulated is an important development.
Limitations and advantages of functional MRI
Functional MRI is a new method available to behavioural neuroscientists to help study
the brain. Although there are different fMRI methods, they all involve a change in
blood flow to achieve a change in signal contrast. The change in blood flow is coupled
to an increase or decrease in brain metabolism. Consequently, from the onset of a
stimulus, there is a temporal delay of 2–3 s (54) for BOLD and CBF contrast and even
longer for CBV contrast. To achieve a statistically reliable change in signal following
a stimulus, it is necessary to average multiple data acquisitions collected over 1–2
min. Imaging contrast that depends on haemodynamic changes will never achieve the
temporal resolution of electrophysiology. Therefore, it is not possible to image the
initial activation of a behavioural neural circuit in real time. Instead, you are
left with a ‘haemodynamic finger print’ of the stimulus response a few minutes after
its onset.
Spatial resolution in fMRI is a function of field strength and the radiofrequency
electronics. A majority of the data thus far reported in animal studies ranging from
mice to monkeys, employed a 64 × 64 matrix with an in-plane resolution of 400–500
µm2. When overlaid into a segmented atlas with a resolution of 50–100 µm2, it is possible
to see patterns of activation associated with many nuclear areas. However, a clear
delineation of discrete functional neuroanatomical subgroups, such as those described
in the amygdaloid complex (> 20 discrete areas), has not been realised in either animals
or humans. Indeed, fMRI is not capable of identifying functional changes in single
neurones in vivo. However, this spatial limitation is not perceived as a problem because
many neurones in discrete locations and of a similar phenotype behave as an ensemble
with a coordinated pattern of activation. With greater magnetic field strengths and
improved electronics becoming available in the future, it may be possible to parcel
out the functional activity of the many discrete nuclei that subdivide the major areas
of the brain.
Functional MRI has two features that distinguish it from other methods in behavioural
neuroscience research. First, it can be entirely noninvasive. No surgery is required
in preparation for imaging, and it is not necessary to euthanise the animal after
imaging. Animals can be acclimated to imaging procedures, minimising the stress. A
single animal can be imaged multiple times over the course of its natural life. Because
neuroadaptation is a developmental process critical to understanding behaviour, fMRI
functions as a window to the brain, enabling scientists to follow changes in brain
activity in response to factors such as age, environment, hormones, and drugs of abuse.
Second, fMRI has the spatial and temporal resolution to resolve patterns of neuronal
activity across the entire brain in less than a minute. Synchronised changes in neuronal
activity across multiple brain areas can be viewed as functional neuroanatomical circuits
coordinating the thoughts, memories and emotions for particular behaviours.