Functional magnetic resonance imaging in awake animals

Awake animal imaging is becoming an important tool in behavioral neuroscience and preclinical drug discovery. Non-invasive ultra-high-field, functional magnetic resonance imaging (fMRI) provides a window to the mind, making it possible to image changes in brain activity across distributed, integrated neural circuits with high temporal and spatial resolution. In theory, changes in brain function, anatomy, and chemistry can be recorded in the same animal from early life into old age under stable or changing environmental conditions. This prospective capability of animal imaging to follow changes in brain neurobiology after genetic or environmental insult has great value to the fields of psychiatry and neurology and probably stands as the key advantage of MRI over other methods in the neuroscience toolbox. In addition, awake animal imaging offers the ability to record signal changes across the entire brain in seconds. When combined with the use of 3D segmented, annotated, brain atlases, and computational analysis, it is possible to reconstruct distributed, integrated neural circuits or 'fingerprints' of brain activity. These fingerprints can be used to characterize the activity and function of new psychotherapeutics in preclinical development and to study the neurobiology of integrated neural circuits controlling cognition and emotion. In this review, we describe the methods used to image awake animals and the recent advances in the radiofrequency electronics, pulse sequences, and the development of 3D segmented atlases and software for image analysis. Results from pharmacological MRI studies and from studies using provocation paradigms to elicit emotional responses are provided as a small sample of the number of different applications possible with awake animal imaging.     

A correction method for DC drift artifacts

In order to cope with the problem of drift artifacts in ERP research a new off-line correction method is described. It estimates the DC drift from all prestimulus baselines of an experiment. For this end, an amplifier reset is performed at regular intervals and the DC off-set preceding any reset is stored. A regression is calculated between the prestimulus baseline amplitudes of consecutive trials and the time that has passed by. The amplitude trend which can be explained by either a linear or non-linear regression model is then subtracted from all data points.The power of the method is illustrated by two examples. The first shows that detrending increases the signal-to-noise ratio in ANOVA designs. The second shows that amplitude differences of event-related slow potentials, which appeared between the first and the second half of an experiment, could be explained by a non-linear drift component.     

A microelectrode for depth recording in awake animals

The manufacture is described of a metal, glass-coated microelectrode for single-cell recording from deep structures of the brain during behavioral testing. It is thin, long, and straight, and has excellent electrical properties.     

Different neural systems for recognizing plants, animals, and artifacts

The purpose of this study was to investigate functional organization in the human brain involved in the representation of knowledge regarding plants. We measured the brain activity of eight male volunteers during the recognition of visual stimuli representing plants, animals and artifacts, using positron emission tomography. The participants were presented with and were required to name silently two different images each of 15 entities belonging to three ontological categories, and 30 series of four to six digits. Marked increases in regional cerebral blood flow were found in the hippocampus and the parahippocampal areas bilaterally and the right lateral occipital cortex during the silent naming of all three categories, compared with that during the silent reading of digits. The right lateral occipital cortex was specifically activated in association with the naming of plants, and the right fusiform cortex was specifically activated in association with the naming of animals. In addition, the right temporo-occipital cortex was activated only during animals and plants, not artifacts. Our results indicate that there were a few characteristic activations for the different categories, and that entities belonging to the different categories are not necessarily represented in different locations of the brain.     

Air-driven eye shutter system for vision experiments using awake behaving animals

When conducting physiological and behavioral experiments for vision research, we often need to present stimuli monocularly. In this report, we describe a new eye shutter system that was developed to occlude individual eyes during experiments with awake behaving animals. The eye of choice is occluded by an air pressure-driven opaque mask, the opening and closing of which is controlled by computer. This new eye shutter system has several advantages over other systems: (1) it can completely shield the eye from light using a physically opaque eye mask; (2) contamination of electrophysiological recordings by electrical noise is avoided because the actuator is not electrical; (3) the left and right eyes can be randomly occluded in each trial; (4) space for apparatus in front of the animal's face is not required. We used this eye shutter system to record neural activity from the primary visual cortex of awake monkeys and confirmed that this system is suitable for the monocular presentation of visual stimuli during electrophysiological recordings from awake animals.     

Technique for applying neurotropic substances onto single units in awake animals

Methods are shown for the stereotaxic placement of twin cannulae, one for recording single unit activity and the other for microinjecting test substances directly on the recording site. The device is inexpensive, occupies a small space on the calvarium, and remains operational in the same animal for several months. This technique is being used to study the effects of various neurotransmitters and neuromodulators on the activity of single units in the hypothalamus of unanesthetized rabbits.     

Automated detection of EEG artifacts during sleep: preprocessing for all-night spectral analysis.

This paper describes a simple artifact detection algorithm which can be used when large amounts of EEG data are to be automatically processed via spectral analysis techniques in a general purpose digital computer, and visual inspection of each EEG epoch becomes an impossible task. The technique is based on a chi-square (chi(2)) goodness-of-fit test to a Gaussian distribution (CSQ), and it was applied to EEG epochs each 30 sec long. This test proved to be very sensitive to non-stationarities in the EEG amplitude distribution for a particular epoch, and it produced a large value for the chi(2) coefficient when an artifact was present. EEG epochs that gave rise to chi(2) coefficients of value larger than a heuristically determined minimum were discarded from further analysis. The above technique enabled efficient data reduction and reliable automatic off-line processing of 50 nights of sleep EEG via spectral techniques.     

Retrospective motion correction protocol for high-resolution anatomical MRI

Modern computational brain morphology methods require that anatomical images be acquired at high resolution and with a high signal-to-noise ratio. This often translates into long acquisition times (>20 minutes) and images susceptible to head motion. In this study we tested retrospective motion correction (RMC), common for functional MRI (fMRI) and PET image motion correction, as a means to improve the quality of high-resolution 3-D anatomical MR images. RMC methods are known to be effective for correcting interscan motion; therefore, a single high-resolution 3-D MRI brain study was divided into six shorter acquisition segments to help shift intrascan motion into interscan motion. To help reduce intrascan head motion, each segment image was reviewed for motion artifacts and repeated if necessary. Interscan motion correction was done by spatially registering images to the third image and forming a single average motion-corrected image. RMC was tested on 35 subjects who were considered at high risk for head motion. Our results show that RMC provided better contrast-to-noise ratio and boundary detail when compared to nonmotion-corrected averaged images.     

A multiple source approach to the correction of eye artifacts

Previously published methods correct eye artifacts by subtracting proportions of the EOG from EEG electrodes. The implicit assumption made by these methods is that the EOG signals are a good measure of eye activity and contain no EEG. In this paper a new multiple source eye correction (MSEC) method of eye artifact treatment based on multiple source analysis is presented, which incorporates a model of brain activity. An accurate, head model-independent estimate of the spatial distribution of eye activity can be obtained empirically from calibration data containing systematic eye movements and blinks. Using the resulting spatial vectors together with the brain model, eye activity in EEG and event-related response data can be estimated in the presence of overlapping brain activity and corrected. A consequence of the MSEC approach is that data at EOG electrodes can be included in analyses of brain activity. In addition, by suitable selection of the spatial vectors, the eye activity can be split into signals which identify vertical and horizontal movements and eyeblinks.Using auditory ERP data sets with and without large eye artifacts, the MSEC method is compared with a “traditional” method in which brain activity is not modelled, particularly with respect to the spatial distribution of the corrected EEG. Traditional eye correction methods are shown to alter the spatial distribution of the EEG, resulting, for example, in changes in location and orientation of modelled equivalent sources. Such distortion is much reduced in the MSEC method, thus enhancing the precision of topographical EEG analyses.     

The correction of ocular artifacts: a topographic perspective.

OBJECTIVE: To evaluate the scalp topography of the potentials related to saccades and blinks. METHODS: The scalp topographies of the potentials associated with saccades and blinks were recorded in 60 subjects. The topographies were analyzed using both source components and attenuation factors, with each factor representing the fraction of the potential recorded in peri-ocular electrodes that contributes to the EEG recorded from a particular scalp location. RESULTS: Blinks and upward saccades generated potentials with very different topographies. Left and right saccades and up and down saccades generated equal but inverted fields except at peri-ocular locations where subtle inequalities occurred. The potentials associated with lateral saccades were consistently larger in female subjects than in male subjects. CONCLUSIONS: The differences in the scalp topographies between blinks and vertical saccades can be explained by the different ways in which they are generated. Blink potentials are caused by the eyelids sliding down over the positively charged cornea, whereas saccade potentials are caused by changes in the orientation of the corneoretinal dipole. Any compensation procedure for ocular artifacts must take into account the topographic differences between blinks and upward saccades.     

A split microdrive for simultaneous multi-electrode recordings from two brain areas in awake small animals

Complex cognitive operations such as memory formation and decision-making are thought to be mediated not by single, isolated brain structures but by multiple, connected brain areas. To facilitate studies on the neural communication between connected brain structures, we developed a multi-electrode microdrive for chronically recording ensembles of neurons in two different brain areas simultaneously. The "split drive" contains 14 independently movable microdrivers that were designed to hold tetrodes and to permit day-to-day adjustment of dorsoventral position in the brain. The limited weight of the drive allowed rats to adjust well to the headstage after recovering from surgery and permitted stable recording sessions across at least several weeks. In addition to describing the design and assembly of the split drive, we also discuss some important individual parts of microdrives used for tetrode recordings in general. Furthermore, the split drive was applied to two widely separated and connected brain structures, the hippocampus and ventral striatum. From these two areas, stable ensemble recordings were conducted in rats performing a reward-searching task on a triangular track, yielding group sizes of about 15 and 25 units in the dorsal hippocampus and ventral striatum, respectively.     

A fully automated correction method of EOG artifacts in EEG recordings.

OBJECTIVE: A fully automated method for reducing EOG artifacts is presented and validated. METHODS: The correction method is based on regression analysis and was applied to 18 recordings with 22 channels and approx. 6 min each. Two independent experts scored the original and corrected EEG in a blinded evaluation. RESULTS: The expert scorers identified in 5.9% of the raw data some EOG artifacts; 4.7% were corrected. After applying the EOG correction, the expert scorers identified in another 1.9% of the data some EOG artifacts, which were not recognized in the uncorrected data. CONCLUSIONS: The advantage of a fully automated reduction of EOG artifacts justifies the small additional effort of the proposed method and is a viable option for reducing EOG artifacts. The method has been implemented for offline and online analysis and is available through BioSig, an open source software library for biomedical signal processing. SIGNIFICANCE: Visual identification and rejection of EOG-contaminated EEG segments can miss many EOG artifacts, and is therefore not sufficient for removing EOG artifacts. The proposed method was able to reduce EOG artifacts by 80%.     

The design and application of three speaker-based stimulating devices for cutaneous stimulation in anesthetized and awake animals

Those wishing to study neuronal plasticity in sensory systems are confronted by the need to deliver equivalent stimuli to the organism at time intervals separated by hours, days or months. This problem is particularly acute in the somatosensory system where delivering an equivalent stimulus generally requires a second physical contact with the same point on a geometrically complex surface. This requirement is difficult to fulfill. We have designed two stimulators that avoid or minimize the importance of this requirement by obviating the need for the stimulator to be at a fixed distance from the skin. As well, we have redesigned a system for whisker stimulation originally proposed by Simons. The first stimulator is appropriate for experiments in anesthetized animals; the surface to be stimulated is immersed in water warmed to body temperature and the tactile stimulus is generated as an hydraulic pulse. The second uses a high velocity pulse of air shaped so that it can be transmitted significant distances without attenuation. The redesign of the Simons’ vibrissa stimulator provides larger amplitude displacements and lower controlling voltages more readily generated by equipment normally found in laboratories. We also described the design of a chamber for restricting the awake rat during chronic study and the electrodes used for recording and for delivery of drugs in awake animals held in such a chamber.     

Application of multiline two-photon microscopy to functional in vivo imaging

High spatial resolution and low risks of photodamage make two-photon laser-scanning microscopy (TPLSM) the method of choice for biological imaging. However, the study of functional dynamics such as neuronal calcium regulation often also requires a high temporal resolution. Hitherto, acquisition speed is usually increased by line scanning, which restricts spatial resolution to structures along a single axis. To overcome this gap between high spatial and high temporal resolution we performed TPLSM with a beam multiplexer to generate multiple laser foci inside the sample. By detecting the fluorescence emitted from these laser foci with an electron-multiplying camera, it was possible to perform multiple simultaneous linescans. In addition to multiline scanning, the array of up to 64 laser beams could also be used in x-y scan mode to collect entire images at high frame rates. To evaluate the applicability of multiline TPLSM to functional in vivo imaging, calcium signals were monitored in visual motion-sensitive neurons in the brain of flies. The capacity of our method to simultaneously acquire signals at different cellular locations is exemplified by measurements at branched neurites and 'spine'-like structures. Calcium dynamics depended on branch size, but 'spines' did not systematically differ from their 'parent neurites'. The spatial resolution of our setup was critically evaluated by comparing it to confocal microscopy and the negative effect of scattering of emission light during image detection was assessed directly by running the setup in both imaging and point-scanning mode.     

Setup and data analysis for functional magnetic resonance imaging of awake cat visual cortex

Functional magnetic resonance imaging（fMRI）is one of the most commonly used methods in cognitive neuroscience on humans.In recent decades,fMRI has also been used in the awake monkey experiments to localize functional brain areas and to compare the functional differences between human and monkey brains.Several procedures and paradigms have been developed to maintain proper head fixation and to perform motion control training.In this study,we extended the application of fMRI to awake cats without training,receiving a flickering checkerboard visual stimulus projected to a screen in front of them in a block-design paradigm.We found that body movement-induced non-rigid motion introduced artifacts into the functional scans,especially those around the eye and neck.To correct for these artifacts,we developed two methods：one for general experimental design,and the other for studies of whether a checkerboard task could be used as a localizer to optimize the motioncorrection parameters.The results demonstrated that,with proper animal fixation and motion correction procedures,it is possible to perform fMRI experiments with untrained awake cats.     

Principles of acoustic motion detection in animals and man

Motion provides one of the most important cues for survival, because it helps to break the camouflage of a predator or a prey and because it allows predictions about the future path of an object. Recent data on the processing of acoustic motion have yielded some astonishing findings, suggesting that the psychophysical, neurological and neurophysiological mechanisms underlying the detection and representation of acoustic motion are quite similar to those underlying the detection and representation in other modalities, especially in vision.  A further comparison of these similarities and differences with respect to the different environmental constraints posed for the different modalities may help in understanding general problems associated with motion computations.     

A container for transporting small laboratory animals for magnetic resonance imaging

We have constructed a simple container, consisting of a propylene tube, a High-Efficiency Particulate Aerosol (HEPA) filter and a rubber glove, for transporting small animals to magnetic resonance imaging (MRI) facilities that are located outside a pathogen-free environment. Results of pathogens analysis indicate that the container is able to prevent infection by several microorganisms. The quality of the MR images of mice and rats transported in, and imaged while in the container was satisfactory. This container can be useful for examinations that required the use of instruments located outside clean animal units, ensuring safety for both humans and animals.     

Functional coupling shows stronger stimulus dependency for fast oscillations than for low-frequency components in striate cortex of awake monkey

It has been argued that coupling among the neural signals activated by a visual object supports binding of local features into a coherent object perception. During visual stimulation by a grating texture we studied functional coupling by calculating spectral coherence among pairs of signals recorded in the striate cortex of awake monkeys. Multiple unit activity (MUA) and local field potentials (LFP, 1-140 Hz) were extracted from seven parallel broad band recordings. Spectral coherence was dominated by high-frequency oscillations in the range 35-50 Hz and often by additional low-frequency components (0-12 Hz). Functional coupling among separate cortical sites was more stimulus specific for MUA than for LFP: MUA coherence at high and low frequencies depended highly significantly on: (i) the similarity of the preferred orientations at the two sites - the more similar the higher the coherence; (ii) the orientation of the stimulus grating - with highest coherence at half angle between the preferred orientations at the two sites; (iii) cortical distance - coherence decreases to noise levels at approximately 3 mm (MUA) and 6 mm (LFP). Coherence of fast oscillations did not depend on the degree of coaxiality of the orientation-sensitive receptive fields, whereas low frequencies showed significant dependency. This indicates that different frequency components can engage different coupling networks in the striate cortex which probably support different coding tasks. Changes in average oscillation frequency with stimulus orientation were highly significant for fast oscillations while there was no dependency for low frequencies. Finally, stimulus-related spectral power and coherence of fast oscillations were considerably higher than of low frequency components. Fast oscillations may therefore contribute more to feature binding and coding of object continuity than low-frequency components, at least for texture surfaces as analysed here.