1Institut de Biologie Cellulaire et de Morphologie, Université de Lausanne, 1005 Lausanne, Switzerland; 2Research Institute of Developmental Physiology, 119121 Pogodinskaya 8-2, Moscow, Russia; and 3Division of Neuroanatomy and Brain Development, Department of Neuroscience, Karolinska Institutet, S-17177 Stockholm, Sweden
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ABSTRACT |
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Kiper, D. C., M. G. Knyazeva, L. Tettoni, and G. M. Innocenti. Visual Stimulus-Dependent Changes in Interhemispheric EEG Coherence in Ferrets. J. Neurophysiol. 82: 3082-3094, 1999. In recent years, the analysis of the coherence between signals recorded from the scalp [electroencephalographic (EEG) coherence] has been used to assess the functional properties of cortico-cortical connections, both in animal models and in humans. However, the experimental validation of this technique is still scarce. Therefore we applied it to the study of the callosal connections between the visual areas of the two hemispheres, because this particular set of cortico-cortical connections can be activated in a selective way by visual stimuli. Indeed, in primary and in low-order secondary visual areas, callosal axons interconnect selectively regions, which represent a narrow portion of the visual field straddling the vertical meridian and, within these regions, neurons that prefer the same stimulus orientation. Thus only isooriented stimuli located near the vertical meridian are expected to change interhemispheric coherence by activating callosal connections. Finally, if such changes are found and are indeed mediated by callosal connections, they should disappear after transection of the corpus callosum. We perfomed experiments on seven paralyzed and anesthetized ferrets, recording their cortical activity with epidural electrodes on areas 17/18, 19, and lateral suprasylvian, during different forms of visual stimulation. As expected, we found that bilateral iso-oriented stimuli near the vertical meridian, or extending across it, caused a significant increase in interhemispheric coherence in the EEG beta-gamma band. Stimuli with different orientations, stimuli located far from the vertical meridian, as well as unilateral stimuli failed to affect interhemispheric EEG coherence. The stimulus-induced increase in coherence disappeared after surgical transection of the corpus callosum. The results suggest that the activation of cortico-cortical connections can indeed be revealed as a change in EEG coherence. The latter can therefore be validly used to investigate the functionality of cortico-cortical connections.
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INTRODUCTION |
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Noninvasive electrophysiological methods are
currently used for assessing developmental and compensatory changes in
the connectivity of the human brain. In particular, the relatively new
extension of the electroencephalographic (EEG) spectral analysis, EEG
coherence analysis, has been used to reveal and localize the underlying changes in cortical connectivity during normal and abnormal
development, while performing cognitive tasks, and in different
pathologies (Besthorn et al. 1994; Fletcher et
al. 1997
; Knyazeva and Farber 1991
; Kuks
et al. 1987
; Marosi et al. 1992
;
Merrin et al. 1989
; Terstegge et al.
1993
; Thatcher 1992
).
The application of coherence analysis to the study of cortical connectivity is based on the assumption that coherence between two EEG signals reflects functional relations between the cortical regions underlying the recording electrodes. Because coherence values depend on the stability of both power and phase relations between the signals, any factors affecting the covariance of spatially distributed EEG signals must influence the coherence values. Among them, cortical connectivity is thought to be the most important factor.
Indeed, data on the spatial distribution of EEG coherence values both
between and within the cerebral hemispheres are compatible with our
current knowledge of cortical connectivity (Knyazeva and Farber
1996; Thatcher et al. 1986
; Tucker et al.
1986
). However, the empiric justification for ascribing EEG
coherence to the activity of cortico-cortical connections in humans is
limited to a few studies in which patients with agenesis, surgical
section, or pathology of the corpus callosum showed decreased
interhemispheric coherence compared with normals (Koeda et al.
1995
; Kuks et al. 1987
; Montplaisir et
al. 1990
; Nielsen et al. 1993
; Nunez
1981
; Pinkofsky et al. 1997
; but see
Corsi-Cabrera et al. 1995
). These studies suggested that
the integrity of the interhemispheric connections influences coherence
level within the frequency range traditionally analyzed in human scalp
EEG, being most pronounced in the alpha and beta bands. For reasons
discussed below, they do not prove that coherence changes are mediated
by callosal connections.
The experimental evidence that EEG coherence reflects the activity of
cortico-cortical connections rests largely on the study of the alpha
rhythm in dogs by Lopes da Silva et al. (1973,
1980
) and Lopes da Silva and Storm van Leeuwen
(1978)
. These studies showed that coherence mediated by
cortico-cortical connections predominates over thalamocortical one
within the alpha-range, implying cortico-cortical connectivity as the
main substrate for the alpha synchronization. It should be mentioned
that these studies did not try to relate coherence levels to the
detailed structure of cortico-cortical connections, nor to any form of stimulation.
Recent studies in cats and monkeys showed stimulus-induced cortical
activity in the gamma range (Brosch et al. 1995,
1997
; Engel et al. 1990
,
1991
; Gray et al. 1989
,
1990
). This activity could be synchronized within
distributed neuronal assemblies via cortico-cortical connections
(Engel et al. 1991
; Munk et al. 1995
; Ts'o et al. 1986
). Other studies emphasized the role of
the thalamic input (or of cortico-thalamic loops) in synchronizing
cortical activity in the gamma band (Barth and MacDonald
1996
; Ribary et al. 1991
; Steriade
1997
; Steriade and Amzica 1996
).
The claim that interhemispheric cortico-cortical connections are
involved in the gamma band synchronization rests on the finding that
the synchronization of neuronal assemblies located in different hemispheres is abolished by interruption of callosal connections, or
destruction of cortical areas in one hemisphere (Engel et al. 1991; Munk et al. 1995
). Although this result is
compatible with the possibility that callosal connections mediate
interhemispheric synchronization, it does not bring conclusive evidence
that this is indeed the case. First, callosal transections were
performed several weeks before the recording sessions. Therefore the
possibility that the loss of interhemispheric synchronization is due to
local reorganizations of connections or to synaptic changes induced by
the lesion cannot be excluded. Second, callosal connections have been
shown to regulate the general level of cortical excitability (Berlucchi 1966
; Bremer et al. 1956
), and
indeed, a significant loss of multiunit responses was reported after
chronic callosotomy (Munk et al. 1995
). Because the
gamma band oscillations depend on the level of neuronal depolarization
(Steriade et al. 1996a
,b
), they might be decreased by
the loss of a tonic excitatory input to the hemisphere. Therefore
callosal transection could interfere with the stimulus-dependent
synchronization without causing it. Similar arguments weaken the
interpretation of decreased interhemispheric coherence in the patients
with callosotomy or callosal agenesis mentioned above.
The present study of interhemispheric EEG coherence during visual stimulation in ferrets has the following goals. First, we intend to determine whether stimulus-dependent changes in cortical synchronized activity can be detected using this electrophysiological technique. Second, we wish to determine whether such changes can be unequivocally ascribed to the activation of cortico-cortical connections. This is done with the purpose of using the EEG coherence analysis for assaying specific aspects of cortico-cortical connections in the normal adult or developing human brain, and in pathological conditions.
The advantage of this animal model is that it allows precise
predictions. Indeed, in ferrets as in other mammals, the corpus callosum interconnects selectively portions of the visual areas representing a narrow sector of the visual field near the vertical meridian (VM) (Colin et al. 1998; Grigonis et al.
1992
; Rockland 1985
). Thus if callosal
connections are responsible for synchronizing activity in the two
hemispheres, and, if such synchronization can be detected by studying
changes in EEG coherence, these changes should occur only with stimuli
presented near the VM. Also, the corpus callosum selectively
interconnects neurons with identical orientation specificity
(Antonini et al. 1983
; Berlucchi and Rizzolatti 1968
; Houzel et al. 1994
; Lepore and
Guillemot 1982
; Milleret et al. 1994
;
Schmidt et al. 1997
). Therefore changes in EEG coherence should occur only for stimuli that activate iso-oriented neurons in the
two hemispheres. Finally, stimulus-induced changes in interhemispheric EEG coherence should be abolished by acutely severing the part of the
corpus callosum traversed by the fibers that interconnect the visual areas.
The results of the present study met all the above conditions and thus
encourage the use of EEG coherence analysis as a way to investigate the
involvement of cortico-cortical connections in normal brain operations
(Knyazeva et al. 1999), and for assaying their function in pathological
conditions (Kiper et al. 1998a
).
Preliminary results were presented in abstract form (Kiper et
al. 1998b; Knyazeva et al. 1998
).
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METHODS |
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Subjects
Seven adult female sable ferrets were used. In six animals (all but animal FEEG.10), the same protocol of visual stimulation was performed. FEEG.10 is therefore not present in Table 1, but its results are described in the section Effects of corpus callosum transection. The animals were obtained from Marshall Europe and were maintained on a 14 h light/10 h dark cycle in the animal facility of the Institute of Cell Biology and Morphology. Animal maintenance and experimental procedures conformed to Swiss regulations, European Community directives (1997), Guidelines of the American Physiological Society (1991), and under the supervision of the Cantonal Veterinary Commission.
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Surgical preparation
Before an experiment, each animal was food deprived for 12-14
h. Initial anesthesia was induced by intramuscular injections of
ketamine hydrochloride (Ketalar, Parke-Davis, 10 mg/kg) and medetomidin
hydrochloride (Domitor, Orion, 0.08 mg/kg) and followed by an injection
of atropine sulfate (Sintectica S. A., 0.1 mg im). The femoral
vein and trachea were cannulated, and the animal was placed in a
stereotaxic apparatus. Anesthesia was continuously maintained by
inhalation of isoflurane (Forene, Abbott Laboratory) 0.5% in a
gas mixture of 50% O2-50% N2O. After
installation in the stereotaxic apparatus, the scalp was incised along
the midline, the skin and temporal muscles were retracted, and holes
were drilled through the skull to position the recording electrodes. At
this point, the animal was paralyzed with a continuous intravenous injection of gallamine triethiodide (Flaxédil,
Rhône-Poulenc Rorer, 6 mg · kg1 · h
1) diluted in lactated Ringer, and supplemented with 5%
sucrose (5.4 ml/h). The animal's temperature was monitored by a rectal probe and maintained around 37°C with a thermostatically controlled heating pad. We continuously monitored the animal's electrocardiogram (EKG) and EEG (see EEG recording). The animal was
artificially ventilated with a small-animal respirator (Harvard
Apparatus), and expired CO2 was maintained around 4% by
adjusting tidal volume or respiration rate.
Corpus callosum transection
In four animals (FEEG.4, 5,
8, and
10), the
corpus callosum (CC) was sectioned by lowering in the interhemispheric
scissure a surgical thread passed through two parallel sewing needles, spaced anteroposteriorly ~5 mm and mounted on a micromanipulator. In
the first two animals, the interhemispheric fissure was exposed by
removing the bone on one hemisphere, over the whole anteroposterior extent of the CC and by incising the dura all along. Because this procedure required coagulating venous branches terminating in the
sagittal sinus, in the last two animals two openings ~3 mm diam were
performed at anteroposterior locations corresponding to the CC splenium
and the rostrum. The surgical thread was then passed under the dura
through one of the openings, recovered through the other opening, and
then passed through the sewing needles. The needles were advanced to
the depth of ~10 mm from the surface and then retracted. The surgical
opening was then covered with agar. The location and extent of the
section was controlled histologically.
Optics and visual stimulation
Each animal was fitted with ferret contact lenses, with a
curvature of 2.6-2.9 mm, and a diameter of 4 mm, plano (Ocular Contact Lenses). The pupils were dilated by topical application of Atropine 1%
(Dispersa) and the nictitating membranes retracted by application of
Phenylephrin 5% (Lab. Chauvin). The optic disks were plotted on a
tangent screen placed in front of the animal using a reversible ophthalmoscope (Eldridge 1979), and the locations of the
foveas were estimated from these landmarks (Price and Morgan
1987
; Zahs and Stryker 1985
). To superimpose the
foveal projections on the screen, paralysis-induced misalignments of
the optical axes were corrected with the use of appropriate prisms.
The stimuli were presented on an Eizo T-560 i monitor, driven by an AT Truevision Vista graphics board, with a refresh rate of 120 Hz, interlaced. The stimuli were vertical rectangular patches of black-and-white sinusoidal gratings, with a space-averaged luminance of 32 cd/m2, presented on a uniform background of equal luminance. At the viewing distance we used (14.5 cm), the screen subtended 67 by 95° of visual angle, and the patches 31.5 by 57.5°. In all animals, the stimulus set included a reference condition consisting of a uniform gray field filling the whole screen, with a luminance of 32 cd/m2. This condition is referred to as the "background" in the remainder of the paper. In all but one animal, we used the following stimulation conditions (Fig. 1):
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1) Left hemifield stimulus. A single patch was presented to the left of the foveas' projection on the screen. The grating's orientation was horizontal, the spatial frequency 0.13 c/deg, and contrast 98%. It was drifting downward with a temporal frequency of 2 Hz. The center of the patch was located 17° from the foveas.
2) Right hemifield stimulus. Identical to 1), except that the grating was presented to the right of the foveas.
3) Bilateral identical stimuli. Gratings 1) and 2) were simultaneously presented. Therefore a 1.3° wide, vertical strip on each side of the VM was not stimulated.
4) Bilateral different stimuli. The grating to the left of the fixation point was as described above, but that on the right was vertically oriented and drifted to the right.
In addition to these four "close" stimuli, we presented the same
gratings located further away from the fixation point. In that case,
the patch centers were located 31.5° to the side of the fixation
point. Therefore the gratings did not stimulate a vertical strip of
15.8° on each side of the VM, but were still within the binocular
representation of the visual field in the known visual areas of the
ferret (Law et al. 1988; our own unpublished observations). We refer to these as the "far" conditions.
In animals FEEG.5 and FEEG.7 we also used a stimulus condition where the whole screen was filled with a downward drifting grating (spatial frequency 0.13 c/deg, contrast 98%, temporal frequency 2 Hz). In animal FEEG.10, only the whole screen stimulus was compared with the background.
EEG recording
Three active electrodes were positioned over each hemisphere.
Their locations and an EEG sample are shown in Fig.
2. The first electrode was located ~1
mm anterior to the occipital pole, over a region corresponding to the
17/18 border (Colin et al. 1998; Law et al.
1988
; Rockland 1985
). The second electrode was
positioned anterior to the first, and slightly more laterally, over a
region corresponding to area 19, as determined by other experiments
performed in our laboratory (Colin et al. 1998
). A third
electrode was placed more anteriorly and laterally, over the upper bank
of the suprasylvian sulcus. Another three active electrodes were then
stereotaxically placed at symmetrical locations on the other
hemisphere.
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The electrodes consisted of a thin (0.1 mm diam) silver wire terminated
by a small ball (0.5 mm diam). The wire was insulated by a Teflon
coating up to 5 mm from the ball. Electrodes were passed through the
holes in the skull, positioned over the dura mater, and maintained in
place with a few drops of melted bone wax. Two symmetrical reference
electrodes were screwed into the bone over the frontal sinuses. We used
separate reference electrodes to avoid artificially high
interhemispheric coherence due to shared activity under a common
reference electrode (Fein et al. 1988; Nunez
1995
). In addition, the size and remote location of the reference electrodes minimized the probability that they could be
affected by signals originating in the visual cortex. The EEG signals
derived as voltage differences between the active electrodes, and the
corresponding same-hemisphere reference were amplified, band-pass
filtered (0.1-100 Hz), and stored in a personal computer after 12-bit
A/D conversion at a sampling rate of 204.8 Hz.
Data collection
Each set of stimuli consisted of the sequence of four gratings (either far or close) and of a blank stimulus (uniform gray screen with a luminance of 32 cd/m2), each presented for 5 s, with interstimulus intervals between 2 and 3 s. The order of presentation was randomized between animals. The set of five stimuli was repeated in blocks for a minimum of 20 times. To ensure the reproducibility of our results within an experiment, blocks of visual stimuli were repeated in five of the seven animals. Before each block, the animal was presented with the blank screen to monitor the background EEG for dominant slow rhythms indicating excessively deep anesthesia, and to detect potential artifacts due to poor electrical contacts.
EEG processing
The "EEG Lab" software (Metrica, Moscow, Russia) was used for data processing. For each animal and experimental condition, a total of 1.5-2 min of artifact-free EEG in 5-s epochs was selected for analysis. The absence of artifacts resulting from pulsatory and respiratory movements of the brain, poor electrode contact, or other sources, was the only criterion for selection.
The EEGs were subjected to fast Fourier transform (FFT) (Otnes
and Enochson 1978). Primary auto- and cross-spectra estimates were averaged over the epochs (not <20), and smoothed by Parsen's window. Based on primary spectral estimates, the spectral power density
(SPD) for every channel and interhemispheric coherence (ICoh) functions
for symmetric and asymmetric electrode pairs were calculated.
Interhemispheric coherence was computed with the formula
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Because our aim is to use EEG coherence analysis to study the
cortico-cortical connectivity of individual cases (Kiper et al.
1998a), we first performed an analysis of each individual subject. For each animal, the significance of the changes induced by
different visual stimuli in either power or coherence spectra was
assessed by applying the Wilcoxon test to their mean band values for
each block of stimuli separately. Comparisons across blocks were
usually not possible, because of the EEG changes between blocks (see
further Anesthesia effects on stimulus-dependent EEG coherence).
To assess the significance of our results for the whole sample, we applied the nonparametric Friedman test for several related samples. To analyze the temporal stability of the ICoh responses, we used the Wilcoxon test for two related samples (1st vs. 2nd halves of the 5-s epochs). For pairwise comparisons (identical close vs. each of the other stimuli), we applied the Wilcoxon test. The tests were performed on the ICoh responses computed as differences in mean band ICoh values between any given stimulus and the background conditions.
Anesthesia effects on stimulus-dependent EEG coherence
The consequences of changing the levels of anesthesia on EEG
responsiveness were studied in two animals. Increasing isoflurane concentration from 0.5 to 1% or from 1 to 1.5% caused a progressive reduction of spectral power density in the gamma band, consistent with
the anesthesia effects on EEG observed in dogs (Katznelson 1981), cats (Kral et al. 1999
), and
humans (Nunez 1981
). Stimulus-induced increases in the
gamma band coherence also diminished progressively with increasing
anesthesia levels. This finding supplements clinical observations that
evoked gamma oscillations decrease their frequency and amplitude to the
point of disappearance, being indicative of suppression of sensory
information processing with increased anesthesia levels (Madler
et al. 1991
; Schwender et al. 1994
). We
therefore maintained the animals on 50% O2-50%
N2O, and 0.5% of isoflurane in all our subsequent
experiments. However, it should be mentioned that the necessary
maintenance of general anesthesia using a minimum, constant
concentration of 0.5% of isoflurane reduced the spectral power within
the beta-gamma bands gradually and clearly. It also caused a
simultaneous increase in the lower frequencies, and progressively
reduced coherence responsiveness within the gamma band.
Histological procedures
At the end of each experiment, recording locations were
marked by inserting needles coated with Procion Brown (Imperial
Chemical Industries) in the electrode holes. The animal was then killed with an overdose of pentobarbital sodium (50 mg iv), rinsed
transcardially with phosphate buffer (0.01 M, pH 7.35), and fixed with
4% paraformaldehyde in phosphate-buffered saline (0.06 M, pH 7.35).
The brain was then extracted and soaked in 4% paraformaldehyde for
8-12 h. Before cutting, the brain was allowed to equilibrate in a 30%
sucrose solution, and photographed to document the location of the
electrodes. Sections (45 µm thick) were cut with a cryostat, and
alternate sections were stained with toluidine blue,
cytochrome-oxidase, and for myelin (Gallyas 1979). On
these sections the cytoarchitectonic borders of areas 17, 18, and 19 were identified on the basis of criteria established in the literature
(Rockland 1985
) and by ourselves (Colin et al.
1998
).
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RESULTS |
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EEG spectral power changes under visual stimulation
The spectral analysis of EEG signals in the presence of the
background stimulus (reference condition) revealed a progressive decrease in power with increasing frequency, in most cases with a more
or less clear-cut hump in the beta-gamma range. Within those power
spectra, three components could be identified. Low-frequency activity
within the 1- to 6-Hz band was the most powerful. Spindles occupied
frequencies roughly from 6 to 14 Hz, were most pronounced in the
suprasylvian derivations, and were lacking from the primary visual
areas derivations in two of the animals. Higher frequency activity was
contained within a broadband in the beta/gamma range, whose limits were
set between 15 and 40 Hz. No consistent changes in SPD were induced by
visual stimulation. In particular, in the 15- to 40-Hz band, in which
coherence changes were observed (see following text), no
changes related to the stimulation condition were found in most of the
animals (FEEG.2, 3,
5, and
7). In one of the
animals (FEEG.4), all stimuli increased and, in another one (FEEG.8), decreased the SPD.
Individual analysis of EEG coherence changes induced by visual stimulation
The background interhemispheric coherence ranged between 0.80 and 90 (maximum values in the low-frequency range) and 0.13-20 (minimum values for the gamma band). Consistent stimulus-related changes in interhemispheric coherence were observed only in the 15- to 40-Hz band. Averaged ICoh within this band for the 17/18, 19, and suprasylvian electrodes are shown in Table 1. For the statistical analysis of the data, all stimulation conditions were compared with the background.
Bilateral stimulation with identical gratings located close to the VM in both hemifields systematically caused a significant coherence increase for the 17/18 and 19 electrode pairs. At each location, interhemispheric coherence increased significantly in 8 of 11 stimulation blocks. Bilateral stimulation with different gratings close to the VM resulted in an ICoh increase only in animals FEEG.3 and FEEG.4 (the 1st block).
Different results were obtained for EEG signals recorded from the suprasylvian areas, as shown in Table 1. In all conditions, the ICoh levels in the 15- to 40-Hz band were generally lower for these electrode locations than for the others. Furthermore, bilateral stimulation with identical gratings near the VM, which gave reproducible ICoh increases in the other visual areas, had no effect except in one block (animal FEEG.4). All other stimulation conditions were also ineffective.
A representative example (FEEG.2) of the ICoh spectra
obtained during these stimulation conditions is shown in Fig.
3, along with their associated power
spectra. Note that, although there were no changes in power spectra
between these stimulation conditions and the background, the identical
stimuli significantly increased ICoh for the 17/18 and 19, but not for
the suprasylvian electrode pairs. In addition, it is important to note
that in all the cases where the whole screen stimulus was presented
(FEEG.5, 7, and
8), it affected ICoh levels in the same
way as the identical stimuli located near the VM (Fig. 7).
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Figure 4 shows the results of the unilateral stimulations compared with the background for the same animal shown in Fig. 3. Unilateral stimulations did not increase ICoh levels in any of the electrode pairs (suprasylvian pair is not shown). Across all animals (see Table 1), unilateral stimulation with horizontal gratings in either the right or the left hemifield was not accompanied by a significant ICoh growth except in one stimulation block (P < 0.05).
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Similarly, we found no significant ICoh increase with any of the stimuli located far from the VM. An example of this result is shown in Fig. 5.
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To further assess the specificity of the ICoh increase produced by the identical stimuli close to the VM, we statistically analyzed the relationships between this and each of the other stimulation conditions. These comparisons showed that, as expected, coherence values were significantly higher for the bilateral stimulation with identical gratings near the VM than for any of the other stimulus conditions, both for the 17/18 and for the 19 electrodes. Indeed, in nearly all the cases where a statistically significant ICoh increase was observed for the identical close condition compared with the background, it was also higher than any of the other stimulation conditions (44 of 45 comparisons).
Group data analysis of EEG coherence changes induced by visual stimulation
The above analysis of the individual results showed that in most cases, the various stimuli affected ICoh in the way we expected. To be able to generalize these results to the whole population, we performed a statistical analysis of the whole sample. We used the nonparametric Friedman test to determine whether our ICoh responses (as defined in METHODS) were significantly different across our stimulation conditions. The responses to the close stimulation conditions varied significantly (P = 0.035 for the 17/18 pair, and P = 0.033 for the 19 pair). As expected from the examination of the individual cases, no significant changes were obtained in the suprasylvian electrode pair. Subsequent pairwise comparisons of the stimulation conditions (using the 1-tailed Wilcoxon test) showed that the identical close stimulus resulted in a significantly larger ICoh response than each of the other stimulation conditions, in both the 17/18 and 19 electrode pairs. The only exception was for the comparison between identical and different close stimuli, which failed to reach significance in the 19 electrode pair (P = 0.17). Specifically, the response to the identical stimulus was significantly higher than that to the different stimulus at the P = 0.029 level in the 17/18 pair. It was also higher than the responses to the left (P = 0.014 for the 17/18 pair, P = 0.021 for the 19 pair), and right (P = 0.023 for the 17/18 pair, P = 0.014 for the 19 pair) stimuli. There were no significant differences between ICoh responses to the far stimuli (P = 0.237 for the 17/18 pair; P = 0.647 for the 19; P = 0.675 for the suprasylvian; Friedman test).
Stability of the ICoh response
To investigate the stability of the ICoh increase for the whole sample, we compared the band ICoh values of the first versus second halves (2.5 s each) of our stimulation period. The two-tailed Wilcoxon test did not show any difference in the background or the bilateral identical condition (for 17/18 and 19 electrode locations P values ranged from 0.213 to 0.594). Analysis of individual data confirmed that, in most blocks, the response induced by visual stimulation was stable over 5 s (Fig. 6). But in two blocks (animals FEEG.4 and FEEG.5), the increase in EEG coherence was larger for epochs of analysis restricted to 2.5 s after stimulus onset, and attenuated over the following 2.5 s of stimulation. An example of this effect is also shown in Fig. 6, for animal FEEG.4 (block 3).
|
Stimulation-induced ICoh changes in heterotopic electrode pairs
Stimulation-dependent interhemispheric changes in ICoh were
not restricted to the activity recorded at homotopic points in the two
hemispheres. The Friedman test performed on the ICoh responses to
unilateral and bilateral close stimuli showed significant differences in the 17/18 and contralateral 19 (P < 0.021)
pairs, but not in the 17/18 and contralateral suprasylvian electrode
pairs. Subsequent Wilcoxon pairwise comparisons revealed that the ICoh
increase to bilateral identical stimuli was greater than to any of the other stimuli (P < 0.038). The robustness of the
responses was revealed by the individual analysis. Indeed, in all but
one animal (FEEG.7), identical stimuli presented near the VM
significantly increased the ICoh between the 17/18 and the
contralateral 19 electrodes (P < 0.05, the Wilcoxon
test). These increases are likely due to heterotopic callosal
connections that were demonstrated anatomically (Bressoud and
Innocenti 1999; Innocenti 1986
). Because we
never observed any consistent stimulus-dependent coherence changes
within a single hemisphere, the possibility that these coherence changes might instead be provided by the
intrahemispheric connectivity is not supported by our data.
Effects of CC transection
Our postmortem histological analysis showed that the CC was cut in
three of the four animals in which this had been attempted (FEEG.4, 5,
8, and
10). In two of the three animals
(FEEG.5 and FEEG.8), the transection included the
splenium and part of the body of the CC, for a total length of 2.5-3
mm (corrected for 40% shrinkage). In animal FEEG.4, the
caudal 1.5 mm of the splenium appeared intact. In FEEG.5,
the mesencephalic tegmentum under the transected portion of the CC, and
in FEEG.8, the cingulate gyrus on the left hemisphere above
the transected part of the CC were also sectioned. In
FEEG.10, the callosal transection failed, but the lesion
involved the medial part of the left hemisphere and, as in
FEEG.8, the cingulate cortex above the CC. Therefore this
animal provided a control for nonspecific EEG changes induced by the
surgical trauma involved in accessing the CC, and will be referred to
as "sham operated."
The outcomes of the CC transections were assessed by applying the Wilcoxon test to individual animals. The ICoh increase due to stimulation with the whole screen grating was abolished in both animals where the transection included the splenium (P > 0.29 for 17 and 18/19 electrode locations), but not in the sham-operated animal (the difference was still significant at P < 0.1). In the animal with the incomplete CC transection (FEEG.4), the ICoh response to the identical-close stimulus as well disappeared after the transection (P > 0.14). Examples of these results are shown in Fig. 7 (FEEG.5: complete transection of the CC; FEEG.10: sham-operated animal), which shows the ICoh spectra for the whole screen stimulus and the background conditions, both before and after the CC transection.
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DISCUSSION |
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In the present study, we intended to determine whether changes in
cortical synchronized activity that can be ascribed to the activation
of cortico-cortical connections could be detected using EEG coherence
analysis. This was done to validate the use of this technique as a way
of assaying cortico-cortical connectivity in humans (see accompanying
paper, Knyazeva et al. 1999).
We decided to use the callosal connections between visual cortical
areas as our experimental system because they have been well
characterized in several species. In particular, the sites of origin
and termination, as well as several electrophysiological properties of
the callosal fibers are known in detail in the cat (reviewed in
Innocenti 1986; see also Houzel et al.
1994
; Innocenti and Bressoud 1999
). The
organization of visual areas in ferrets is in many respects similar to
that of cats (Colin et al. 1998
; Law et al.
1988
; Redies et al. 1990
; Rockland
1985
), although their callosal connections are known in fewer
details (Colin et al. 1998
; Grigonis et al.
1992
; Rockland 1985
).
EEG coherence as a method for assaying cortico-cortical connections
Our study was based on the assumption that if EEG coherence
reflects the functional state of cortico-cortical connectivity, changes
in EEG coherence should be found when cortico-cortical connections are
activated. Among the many possible cortico-cortical connections, the
callosal connections appeared to provide several advantages for testing
this hypothesis. First, it is known that, as a rule, symmetrical points
of the two hemispheres are directly interconnected by callosal axons.
Second, in the visual areas, callosal connections are restricted to the
border between areas 17 and 18 and to parts of area 19. This reflects
the fact that in areas that are precisely retinotopically organized, it
is the representations of the VM of the visual field that are connected by callosal axons (Innocenti 1986). Third, callosal
connections originate almost exclusively from excitatory cortical
neurons, mainly from the pyramidal cells, although some of the callosal axons probably terminate on inhibitory neurons (Innocenti
1986
). Finally, anatomic and electrophysiological evidence
converge in suggesting that callosal axons interconnect mainly or
exclusively columns of cortical neurons with the same orientation
specificity (Antonini et al. 1983
; Berlucchi and
Rizzolatti 1968
; Houzel et al. 1994
;
Lepore and Guillemot 1982
; Milleret et al.
1994
; Schmidt et al. 1997
), a rule that seems
common to other cortico-cortical connections of the primary visual
areas (Gilbert and Wiesel 1989
; but see
Kisvárday and Eysel 1992
).
On this basis, we predicted that if EEG coherence reflects the activity
of cortico-cortical connectivity, an increase in EEG coherence should
be found when the two hemifields are stimulated with identically
oriented stimuli, presented close to, or crossing the VM. Indeed, in
this condition, the thalamic input to the two hemispheres can be
potentiated and time locked by the activation of callosal connections
(Engel et al. 1991; Munk et al. 1995
). We
expected that stimuli presented far from the VM would not produce changes in interhemispheric coherence because the corresponding parts
of the visual areas are not callosally connected. Finally, we also
expected that nonidentically oriented stimuli presented to the two
hemifields would fail to increase interhemispheric coherence. Our
results show that all these expectations were met. In addition, we
found that stimuli presented near the VM but restricted to one
hemifield fail to synchronize the activity of the two hemispheres.
The fact that the stimulus-dependent increase in interhemispheric coherence is abolished by an acute callosal transection supports the hypothesis that the effects are due to the activation of callosal connections, but, for reasons explained in the introduction, it does not prove it unequivocally.
In conclusion, it appears that EEG coherence increases between two
cortical sites when they are 1) interconnected and
2) simultaneously activated both through their
direct connection and their thalamic input. We presently
ignore if the second condition has always to be fulfilled, or if in
some other condition coherence can be affected solely through
activation of cortico-cortical connections. This uncertainty limits the
interpretation of EEG coherence in terms of the underlying neural
circuits as will be discussed elsewhere (Knyazeva et al.
1999).
Neural substrate and meaning of rhythmic cortical activities
Traditionally, three questions have been asked in relation with EEG activity. The first concerns the cellular correlates of EEG signals, the second, the organization of cortical and cortico-subcortical networks producing rhythmic activities, and the third, the functions of rhythms within the continuum of behavioral or mental states.
The first question was answered by Creutzfeld and collaborators, by
showing the high correlation between EEG signals and the postsynaptic
potentials recorded from nearby cortical neurons (Creutzfeldt et
al. 1966). Nevertheless, the question of the spatial origin of
the EEG signal recorded by a skull electrode in humans remains a matter
of discussion and of continuing methodological progress (see
Knyazeva et al. 1999
for discussion). The spatial origin
of the EEG signal is probably less questionable in the case of epidural
electrodes for which it can be assumed, as we did, that the signal
recorded is generated close to the electrode tip.
The question of the origin of the cortical rhythms is complex and goes
beyond the scope of the present discussion. Suffice to say that
consensus seems to emerge as to the fact that rhythms of thalamic, or
possibly also of more distal origin, play an important part in the
genesis of cortical rhythms (Ghose and Freeman 1992; Ribary et al. 1991
; Steriade and Amzica
1996
; Steriade et al. 1990
). However, cortical
neurons, even disconnected from their thalamic input can generate
periodic rhythms (Llinas et al. 1991
; Muramoto et
al. 1993
), and these can be propagated through cortico-cortical connections and, in particular, by callosally projecting neurons (Nunez et al. 1992
).
It should be mentioned that the role of callosal connections in
synchronizing the EEG activity of the two hemispheres has been a
much-debated question in the 1960s and 1970s. The interest was probably
evoked by Claes and Bremer's animal studies reporting that callosal
transection decorrelates the EEG activity of the two hemispheres
(reviewed in Bremer et al. 1956). Bremer and
collaborators introduced the concept of "dynamogenèse
réciproque" of symmetrical cortical areas, to indicate that
symmetrical cortical areas coactivate each other during sensory
stimulation as well as during resting acivity. Unfortunately, the
results of Bremer could not always be replicated (see, for example,
Batini et al. 1967
; Singer and Creutzfeldt
1969
; Susic and Kovacevic 1974
). In a review of
this literature, Berlucchi (1990)
concluded: "The
corpus callosum has long been suspected to be involved in the fine
bilateral synchronization of normal EEG activities but not in the gross
bilateral signs of the sleep-wake cycle." Indeed, it appears from the
present work that much of the disagreements may have been due to the
fact that the studies of callosal transection were focused solely on spindles and alpha waves, and were performed in nonstimulated conditions.
In recent years, the synchronization of oscillations in the gamma band
has been proposed as part of a mechanism for the binding of perceptual
features (see reviews by Engel et al. 1997;
Singer 1994
; Singer and Gray 1995
). In
that view, known as the "synchronization hypothesis," cell
populations coding different features of a given object would
synchronize their rhythmic activity, thereby linking together the
object's features and achieving a coherent perception of the world.
Although the functional significance of the synchronous oscillations
remains a matter of debate (Ghose and Freeman 1992
; Kiper et al. 1996
), the hypothesis has received
experimental support (reviewed in Engel et al. 1997
;
Roelfsema and Singer 1998
; Singer and Gray
1995
).
It should be noted that in most animal studies, synchronization was
evaluated by cross-correlation analysis of spike trains, not by
coherence analysis of EEG and local field potential signals. These two approaches have been shown to provide similar information (Guevara and Corsi-Cabrera 1996). Nevertheless, the
oscillations' frequency band observed in the cross-correlograms
between the activity of two cells or two groups of cells was usually
narrower than the band of coherence increase found in our experiments. Broadband synchronization was also observed in a study that applied coherence analysis to local field potentials in the behaving monkey (Bressler et al. 1993
). It has been shown that the
frequency of oscillations can be quite variable over short periods of
time, and across neuronal pairs (Nowak et al. 1995
;
Steriade 1997
). Thus considering that we were recording
from a large population of cells with each of our epidural electrodes,
and that we had recording periods as long as 5 s, a broadband
coherence change is not necessarily a surprise. Whether this broadband
event really represents the sum over space and time of many narrowband
oscillations, or whether it is of a different nature remains unclear.
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ACKNOWLEDGMENTS |
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We thank V. Vildavski for help with computer software and L. Grollimund for expert technical assistance.
This research was supported by PNR38 Grant 4038-043990 of the Swiss National Science Foundation.
Present address of D. C. Kiper: Institute of Neuroinformatics, University/ETH Zürich, Winterthurerstr. 190, 8057 Zurich, Switzerland.
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FOOTNOTES |
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Address for reprint requests: G. M. Innocenti, Division of Neuroanatomy and Brain Development, Dept. of Neuroscience, Karolinska Institutet, S-17177 Stockholm, Sweden.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 December 1998; accepted in final form 9 August 1999.
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REFERENCES |
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