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Optical Imaging of Epileptiform Events in
Visual Cortex in Response to Patterned
Photic Stimulation
Theodore H. Schwartz1
In a subset of patients with epilepsy, patterned visual stimuli can
trigger clinical seizures. The etiology of this phenomenon, and the
complex interaction between functional architecture and epilepsy,
were investigated in ferret visual cortex. Optical imaging of intrinsic
signals was used to visualize maps of orientation, ocular dominance
and spatial frequency. Acute interictal spike foci were then induced
within V1 using focal iontophoresis of bicuculline methiodide and
optically mapped during presentation of patterned visual stimuli. We
found that specific orientations and spatial frequencies could preferentially trigger epileptiform events, depending on the location of the
epicenter of the epileptic focus within the columnar architecture of
visual cortex. These data support a cortical etiology of the clinical
phenomenon of pattern-sensitive epilepsy. We were not able to
demonstrate a spatial correlation between the functional architecture maps and the topography of the epileptic focus. These findings
implicate short-range rather than long-range horizontal excitatory
connections in the lateral spread of interictal spikes, which may be
specific to the epilepsy model of acute focal disinhibition. Orientation
and spatial frequency maps were severely disturbed in the region of
the focus but were unaltered in the surrounding cortex. Thus, optical
imaging of intrinsic signals can be used to simultaneously map
epilepsy and normal functional anatomy with high spatial resolution.
One of the best clinical examples of the interaction between
functional brain architecture and epilepsy is photosensitive
epilepsy. This phenomenon occurs in up to 15% of patients with
epilepsy, of which as many as 70% patients will demonstrate a
phenomenon called ‘pattern-sensitive epilepsy’, in which
epileptic events are triggered by patterned visual stimuli (Binnie
and Wilkins, 1998; Dorothée and Trenité, 1998). Clinical studies
have demonstrated that the most effective stimuli consist of
alternating black and white linear gratings oscillating in a direction orthogonal to their orientation, and the most important
parameters are spatial frequency, binocular presentation and
luminance (Binnie and Wilkins, 1998). In rare cases of astigmatism, one particular orientation is more likely to trigger an event
than all other orientations (Chartrian et al., 1970; Wilkins et al.,
1979). The pathophysiology of pattern-sensitive epilepsy is
unknown, but the etiology is thought to arise from dysfunction
in an inhibitory network surrounding a trigger zone comprising
a population of orientation selective neurons in visual cortex
(Wilkins et al., 1979). Laboratory studies of experimentally
induced interictal spike (IIS) foci in mammalian visual cortex
using focal disinhibition, however, have only succeeded in triggering IISs following changes in luminance but not orientationspecific stimulation (Ebersole and Levine, 1975; Gabor and
Scobey, 1975, 1980; Gabor et al., 1979; Ebersole and Chatt,
1981). Although the etiology of this phenomenon remains
unclear, the ability of physiological stimulation to trigger
paroxysmal discharges implies a direct functional interaction
between the normal cortical architecture and the genesis of
epileptiform events.
One of the fundamental organizing principals of the
neocortex is that neurons with similar response properties are
organized into columnar modules. These functional columns
have anatomic correlates found in the arborization of thalamocortical afferents, as well as the interconnectedness of isoorientation domains via long-range horizontal excitatory
connections (Mountcastle, 1957, 1982; Hubel and Wiesel, 1962,
1963; Gilbert and Wiesel, 1989; Malach et al., 1993; Weliky et
al., 1995; Bosking et al., 1997; Dalva et al., 1997). For many
years, investigators have postulated that the cortical architecture, in particular the functional column, may play an important
role in the initiation and propagation of epileptic events
(Ebersole and Levine, 1975; Gabor et al., 1979; Reichenthal and
Hocherman, 1979; Gabor and Scobey, 1980; Ebersole and Chatt,
1981). By focally applying small doses of epileptogenic agents
and performing cortical transections to isolate slabs of cortex in
vivo, attempts have been made to define the minimal volume of
cortex necessary to generate an IIS (Morrell and Hambery, 1969;
Reichenthal and Hocherman, 1977; Lueders et al., 1980; Bashir
and Holmes, 1993). Since the cortical column might possess
sufficient recurrent excitatory inter-connections to facilitate
epileptogenesis (Ayala et al., 1973), several investigators have
theorized that the cortical column may be the basic epileptic
unit (Goldensohn et al., 1977; Reichenthal and Hocherman,
1977, 1979; Gabor et al., 1979). Data from rodent neocortical
slices, however, have shown that the minimal volume of cortex
necessary to elicit an IIS is actually much smaller than a cortical
column, and there appears to be no relationship between the
two structures (Gutnick et al., 1982; Silva et al., 1991; Chesi and
Stone, 1998). These findings have not been confirmed in vivo.
In addition to influencing the initiation of epileptiform events,
the columnar organization of the cortex has also been suggested
as an important factor in the lateral spread of neocortical
epilepsy. Simultaneous electrophysiological recordings from
variable distances around the IIS trigger-zone, in both in vitro
and in vivo preparations, have demonstrated a non-uniform
horizontal propagation velocity (Tharp, 1971; Goldensohn et
al., 1977; Chervin et al., 1988; Wong and Prince, 1990; Wadman
and Gutnick, 1993). This spatial periodicity is thought to reflect
variations in the density in long-range horizontal excitatory
connections, which have been shown to link populations of
neurons with similar receptive field properties (Gilbert and
Wiesel, 1989; Malach et al., 1993; Weliky et al., 1995; Bosking et
al., 1997; Dalva et al., 1997).
Our understanding of the cortical column has evolved in
recent years with the introduction of optical recording techniques that permit the simultaneous sampling of large areas of
Cerebral Cortex V 13 N 12 © Oxford University Press 2003; all rights reserved
Cerebral Cortex December 2003;13:1287–1298; DOI: 10.1093/cercor/bhg076
Intoduction
Max-Planck-Institute für Neurobiologie, 82152 Martinsried,
Germany
1Present address: Department of Neurological Surgery, Weill
Medical College of Cornell University, 525 East 68th Street,
Box 99, New York, NY 10021, USA
cortex (Grinvald et al., 1988; Cohen, 1989; Ebner and Chen,
1995; Bonhoeffer and Grinvald, 1996). These studies have
demonstrated that the distribution of orientation preference
across visual cortex does not occur in a box-like, discrete
fashion, as proposed by earlier models based on electrophysiological data (Hubel and Wiesel, 1963). Rather, the range of
orientations is smoothly distributed around a central point in a
circular, pinwheel-like pattern (Bonhoeffer and Grinvald, 1991,
1993). Rapid transitions, or fractures, only occur at pinwheel
centers, or ‘singularities’ (Blasdel and Salama, 1986; Bonhoeffer
and Grinvald, 1991). This finding calls into question the relationship between the epileptic generator and the cortical column. In
addition, the existence of additional overlying maps in visual
cortex, which have been demonstrated with optical recording
techniques, such as spatio-temporal frequency and directionality
maps, further confuses the spatial limits of the alleged epileptic
generator (Shmuel and Grinvald, 1996; Weliky et al., 1996;
Hübener et al., 1997; Shoham et al., 1997).
The purpose of this study was to examine the interaction
between an acute pharmacologically-induced IIS focus and the
surrounding functional architecture in ferret visual cortex with
optical imaging of intrinsic signals. In vivo optical mapping of
an IIS focus has been recently demonstrated by Schwartz and
Bonhoeffer (2001). In this study, we generated an interictal
focus in ferret visual cortex and mapped both the columnar
organization of V1 and V2 as well as the epileptic focus in order
to visualize directly how the two phenomena interact. Although
we were able to preferentially trigger an IIS with oriented visual
stimuli, we found little evidence that either the minimal volume
of cortical tissue required for an IIS or the lateral margins of horizontal spread has any relationship with the functional architecture. We cannot exclude that these findings may be specific to
this experimental model based on the acute injection of a
disinhibiting epileptogenic agent.
Materials and Methods
Animal care, surgery and optical imaging were done as previously
described (Bonhoeffer and Grinvald, 1996; Chapman and Bonhoeffer,
1998; Schwartz and Bonhoeffer, 2001). All procedures were approved by
regional government authorities.
Surgery
Nine adult (6 months to 5 years) ferrets (Marshall Farms, New Rose, NY)
were used in this study. Anesthesia was induced with a mixture of ketamine (15–30 mg/kg i.m.) and xylazine (1.5–2.0 mg/kg i.m.), supplemented with atropine (0.15 mg/kg i.m.). Animals received a tracheotomy
and were ventilated with 60–70% N2O, 30–40% O2 and 1.4–1.6% halothane (halothane was reduced to 0.8–0.9% for the imaging). End-tidal
CO2 was maintained at 3.2–3.8% and animals were hydrated with 2 cc/
kg/h dextrose/Ringer’s solution and paralyzed with intravenous gallamine triethiodide (10 mg/kg/h). EKG, EEG and rectal temperature were
continually monitored. The scalp was incised, then a craniotomy was
performed over the dorsal occipital lobe and the dura was retracted.
Withdrawal of cerebrospinal fluid from the cisterna magna was
performed when necessary to reduce cerebral pulsations. Animals were
fitted with contact lenses to focus the eyes onto the screen, keep the
corneas moist and atropine (0.5%) was used to dilate the pupils. Agar
(2%) and a glass coverslip were placed over the cortex for imaging.
Visual Stimulus
For visual cortex mapping, animals were presented with a 20-condition
random sequence of moving sine-wave gratings projected onto a frosted
glass screen. The sequence consisted of four different orientations at two
spatial frequencies (0.08 and 0.2 cycles per degree) interleaved with four
blanks, iso-luminant with the oriented stimuli. Each non-blank stimulus
was presented using computer controlled eye shutters to randomly alter-
1288 Epileptic Focus and Functional Architecture • Schwartz
nate monocular stimulation, for a total of 20 behavioral conditions.
Stimuli appeared on the screen and then began moving in a direction
perpendicular to their orientation after 1800 ms. Visual stimuli were
generated with a VSG Series Three stimulator (Cambridge Research
System, Rochester, UK). During epilepsy mapping, animals were
presented with six different visual stimuli, each for 1 min, in a
nonrandom order: blank, 0°, 45°, 90°, 135°, blank.
Optical Imaging
The brain was illuminated (707 ± 10 nm) with two fiber-optic light
guides connected to a halogen lamp. The signal was recorded with a
cooled CCD camera (Theta system—ORA 2001, Germantown, NY)
equipped with a tandem lens (Ratzlaff and Grinvald, 1991) focused
∼500 µm beneath the cortical surface. Orientation, spatial frequency and
ocular dominance maps were first generated in normal cortex prior to
epileptogenesis. Five frames of 600 ms duration were recorded at the
start of stimulus movement, exactly 1800 ms after stimulus onset, for
each of 20 visual stimuli (conditions). These were then averaged over 24
repetitions. To map epileptic foci, 120 frames of 500 ms duration were
recorded for each of six visual stimuli (conditions). Each of these 6 min
blocks was separated by a 95 s inter-block interval, during which images
were digitized at 12-bit resolution and stored on the hard drive.
Electrophysiology and Iontophoresis
Epidural electrocorticography (ECoG) was monitored with two AgCl
electrodes on either side of the craniotomy, ∼5 mm from the epileptic
focus. Field potential recording (f.p.) and iontophoresis were performed
through two glass micropipettes (o.d. = 1.0 mm, i.d. = 0.58 mm) pulled
and broken to a tip diameter of 5 µm and separated by 500 µm. F.p.
micropipettes were filled with 3 M NaCl. ECoG and f.p. signals were
amplified, band-pass filtered between 1 and 100 Hz, and digitized at
200 Hz. Single-units were recorded during visual stimulation and quantitative orientation and direction tuning curves were calculated after spikesorting based on spike shapes (Brainware, Oxford, UK). Interictal foci
were induced with iontophoresis of 5 mM bicuculline methiodide [BMI
(Sigma, St Louis, MO)] diluted in 165 mM NaCl, pH 3.0. The micropipette
was advanced 800 µm with an approach angle of 30–40° in order to place
the tip in cortical layers II–III of area V1. The currents used were –15 to
–20 nA for retention and + 50 to 500 nA for epileptogenesis. Positive
currents were maintained until stereotypical biphasic IIS appeared
(∼5 min) and then titrated to the minimal dose to maintain a constant
amplitude and frequency with no afterdischarges.
Single-unit Analysis
Responses to drifting gratings of 16 different directions from the optically
determined ‘focus’ and ‘surround’ regions were bandpass filtered
between 0.3 and 3 kHz and digitized using Brainware. Orientation tuning
averaged over five trials was quantified by fitting smooth curves to the
data with a fast Fourier transform using the zero-, first- and second-order
components (Wörgotter and Eysel, 1987). Spontaneous activity was
assessed by the response to a blank stimulus. Bursting was defined by the
number of inter-spike intervals < 5 ms using the Friedman test (Wyler and
Ward, 1980). In addition, inter-spike intervals from 4 to 8 ms were
analyzed.
Imaging Analysis
Visual Cortex Maps
Orientation, ocular dominance and spatial frequency maps were
computed in a standard fashion (Bonhoeffer and Grinvald, 1996) using
commercially available analysis programs (MAPS, Germantown, NY). In
brief, differential orientation maps were generated by dividing images
obtained while animals viewed one orientation by images obtained
during presentation of the perpendicular orientation. Single condition
maps are created by dividing images obtained during presentation of an
orientation by images obtained during presentation of the blank stimulus.
Vectorial summation of the single-condition maps yields an angle map
of orientation preference that can then be color-coded. Fracture
maps, which demonstrate rapid transitions orientation preference, are
produced from the two-dimensional derivative of the angle map. Spatial
frequency maps result from dividing the sum of images obtained while
the animal views all orientations presented at one spatial frequency by
the sum of all orientations presented at a second spatial frequency; ocular
dominance maps were computed accordingly. All images were clipped
to set the upper and lower 1.5% of pixel values to gray levels of 255 and
0 respectively, and high-pass filtered to cut-off components larger than
the field of view of the camera. Orientation tuning was quantified for
each pixel within a region of interest by fitting smooth curves to the data
with a fast Fourier transform in a fashion similar to that used for the single
units (Wörgotter and Eysel, 1987). The area of the column was determined by thresholding the top 15% pixel values in the differential maps.
The determination of this value was based on prior reports (Chen et al.,
2001) and our own data, which, when varied, did not significantly
change the results.
Epilepsy Maps
Blank-divided (BD) epilepsy maps were produced by dividing each individual frame acquired during interictal spiking from an average of 30
control images obtained with a negative holding current when no
interictal spiking occurred. Spike-triggered (ST) epilepsy maps were
obtained by dividing a single frame following the IIS by a single frame
preceding the IIS (Schwartz and Bonhoeffer, 2001). The optical area of
the IIS was derived from the BD maps by thresholding to a pixel value
1 SD above the pixel values from the same location of the focus during
control conditions. This threshold was chosen since it most reliably
reproduced the appearance of the raw data. This area was manually
outlined in NIH image (NIH) and calculated from the known area of the
field of view of the camera. Control images were generated by triggering
divisions from frames in which spikes did not occur. Both BD maps and
ST maps could be visualized as single frame divisions or averages of
multiple frames to increase the signal to noise ratio. BD maps were
spatially-filtered with a high-pass to cut off components larger than the
field of view of the camera. ST maps were unfiltered. As a control, maps
were generated in a similar fashion with a large negative iontophoretic
current.
Correlating the Epilepsy and Visual Cortex Maps
Contour plots of a region of interest surrounding the focus were superimposed over the angle, spatial frequency and ocular dominance maps
using IDL (Research Systems, Inc.). Four evenly spaced contours were
chosen to represent the range of the optical amplitude of the focus
starting at a level equal to one standard deviation above the mean pixel
value in the image. A warping algorithm, based on alignment of the blood
vessel pattern, was used in cases in which the camera was moved
between mapping trials. Two-dimensional cross correlations were
performed between a region of interest (ROI) surrounding the IIS focus
and the same region in the functional map with bin size equal to the size
of a pixel. To obtain statistical significance, the ROI containing the focus
was randomly shifted to 1000 locations over the functional maps. The
correlation coefficient (r) of the actual data was then compared with the
distribution of randomly generated r-values. Since multiple statistical
tests were performed, which increase the risk of false positive finding,
we chose P < 0.01 as the cut-off for significance. For these statistical
comparisons, maps were smoothed with a 120 µm low-pass filter to eliminate both false negative correlations from single pixel deviations and
false positive correlations from noise. Images were not thresholded for
this comparison to avoid the loss of any subtle structure to the signal,
which might be of significance.
Figure 1. Optical imaging of intrinsic signals reveals the functional architecture in ferret visual cortex. (A) Blood vessel pattern of the surface of the visual cortex. (B) Differential
map produced by dividing images obtained with 0° and 90° stimulation. (C) Differential map produced by dividing images obtained with 45° and 135° stimulation. The size of the
45° and 135° iso-orientation domains is smaller than that of the 0° and 90° domains. (D) The angle map is generated by color-coded vectorial summation of each single condition
map on a pixel-by-pixel basis. (E) Fracture map generated from the two-dimensional derivative of the angle map demonstrates rapid changes in orientation preference at pinwheel
centers. (F) Ocular dominance map in the ferret has a seemingly disorganized structure and column diameters vary widely both between and within animals. The V1/V2 border
correlates with the margin between the caudal contralateral and rostral ipsilateral eye bands. (G) Spatial frequency maps demonstrate high spatial frequency preference caudally
and low-spatial frequency preference rostrally. The spatial frequency border is roughly parallel to the ocular dominance border but shifted caudally. R, rostral; C, caudal; L, lateral;
M, medial. Scale bar = 1 mm.
Cerebral Cortex December 2003, V 13 N 12 1289
Results
Functional Architecture of Ferret Visual Cortex
Orientation, ocular dominance and spatial frequency maps were
produced from ferret visual cortex (n = 6) using optical
recording of intrinsic signals (Bonhoeffer and Grinvald, 1996).
Orientation preference in the ferret is organized in a similar
manner as in the cat, tree-shrew or primate (Fig. 1D) (Blasdel
and Salama, 1986; Grinvald et al., 1986; Bonhoeffer and Grinvald, 1991; Chapman et al., 1996; Fitzpatrick, 1996; Weliky et
al., 1996; Rao et al., 1997). Vertical columns consisting of populations of neurons preferring the same orientation are distributed in a smooth circular pattern into ‘pinwheel’ shapes
surrounding a central point (Bonhoeffer and Grinvald, 1991).
These ‘singularites’, or ‘fractures’, are characterized by rapid
changes in orientation preference between neighboring
neurons (Maldonado et al., 1997). By calculating the two-dimensional derivative of the angle map, we generated fracture maps,
which revealed that, similar to the findings of Rao et al. (1997,
pinwheel centers had a density of 4.8 ± 0.8/mm2 and more
discontinuities than in the cat (Fig. 1E). By thresholding the
images and quantifying the pixels preferentially responding to
each orientation, we determined that the area of cortex devoted
to horizontal and vertical orientations was on average 26 ± 14%
larger than the area devoted to oblique orientations (Fig. 1B,C).
This anisotropy was found in all animals. A similar, although
less dramatic, contrast was reported in the visual cortex of the
developing and adult ferret in earlier reports (Chapman and
Bonhoeffer, 1998; Coppola et al., 1998). The average width of
the orientation columns derived from differential maps was 340
± 60 µm (Fig. 1B,C), which is equivalent to an average area of
0.12 mm2. However, columns were variable in length, some
being circular and others oblong.
Ocular dominance maps have a bizarre, irregular structure in
the ferret with a large degree of inter-animal variation (Law et
al., 1988; Redies et al., 1990; Issa et al., 1999; White et al.,
1999). Although three separate regions with differing ocular
dominance patterns have been identified, our maps were limited
to the most rostral map on the surface of the occipital cortex
which is most easily imaged in the in vivo anesthetized preparation (Law et al., 1988; White et al., 1999). In this region, a caudal
contralateral eye band sits adjacent to a more rostral ipsilateral
eye band (Law et al., 1988; White et al., 1999). Single-unit
recordings have shown that these ocular dominance areas are
almost completely monocular and their interface represents the
V1/V2 border (White et al., 1999) [but see Issa et al. (1999) for
contrasting data on binocularity of neurons in V1]. As in
previous reports, we found a complex inter-digitation of V1 into
V2, consisting of large irregularly shaped blobs which fragments
V2 into isolated cortical territories (Fig. 1F) (Issa et al., 1999;
White et al., 1999). Although widely variable, the average area of
the OD columns was 0.94 ± 0.78 mm2.
Optical imaging and 2-DG data have recently demonstrated that
spatial frequency is also organized in a columnar fashion in
visual cortex of both the cat and primate (Tootell et al., 1981;
Bonhoeffer et al., 1995; Hübener et al., 1997; Shoham et al.,
1997; Issa et al., 2000). Spatial frequency columns have not been
examined in ferret visual cortex. Although a wide range of
spatial frequencies have been shown in the cat to be represented in a continuous fashion (Issa et al., 2000), for the
purposes of these epilepsy experiments we only mapped high
and low spatial frequency. We found a clear preference for high
1290 Epileptic Focus and Functional Architecture • Schwartz
spatial frequency stimuli caudally and low spatial frequency
rostrally in the region of occipital cortex available for optical
imaging (Fig. 1G). Within these larger areas, smaller regions of
spatial frequency preference were identified that had a mean
area of 0.61 ± 0.57 mm2. It is recognized in the cat that neurons
in area V1 are tuned preferentially for high spatial frequency
while those in area V2 are tuned for low spatial frequency
(Movshon et al., 1978; Bonhoeffer et al., 1995; Issa et al., 2000).
Although the border between regions with high and low spatial
frequency preference in the ferret was almost parallel to the V1/
V2 border as defined by the ocular dominance bands, they were
clearly separated by as much as 1 mm.
Interictal Spikes are Triggered by Specific Orientations
and Spatial Frequencies
After visualizing the functional architecture, an acute IIS focus
was created using iontophoresis of BMI into cortical layers II–III.
Within 3–5 min of starting a +500 nA ejection current, IIS could
be recorded from the local f.p. As demonstrated previously
(Schwartz and Bonhoeffer, 2001), these events began as small
negative deflections (0.2–0.4 mV) in the local field potential
(f.p.) and rapidly developed into well-formed biphasic spikes
(2–6 mV), occurring at regular intervals (0.26–0.76 Hz) and
lasting for several hours (Fig. 2A,B). Optical recording of
intrinsic signals was used to generate spike-triggered epilepsy
maps (Schwartz and Bonhoeffer, 2001). These maps provide an
image of the location and topography of the population of
neurons participating in each IIS with a spatial resolution of
Figure 2. Optical epilepsy maps reveal the area of cortex involved in each IIS. (A)
Cortical surface map with the location of the BMI iontophoresis and f.p. recording
electrode. (B) Periodic biphasic interictal spike recorded from the field potential
electrode reach a stable amplitude within a few minutes after starting iontophoresis.
(C) Epilepsy maps from four different animals generated with spike-triggered image
division. The first map in the upper left-hand corner corresponds with the cortex and
spikes presented in (A) and (B). Each map is the an average of the spike-triggered
image division generated from all spikes occurring in a 6 min period, which varied from
70 to 160 spikes, depending on the frequency. Scale bar = 1 mm.
<100 µm. During the early small amplitude events, the optical
epilepsy maps demonstrated a focal change in reflectance in
light that increased in size in parallel with the amplitude of the
IIS. After several minutes, well-developed epileptic foci were
generally circular, centered on the location of the micropipette
tip with an area of 2.84 ± 1.59 mm2 and a sharp border (Fig. 2C).
Control injections using a negative current did not produce any
changes in reflectance. In order to evaluate whether visual stimulation with a particular orientation was sufficient to trigger an
IIS, blank and grating stimuli were shown in sequence and the
number of IIS occurring in the first 500 ms after initiating
stimulus movement were quantified. As is demonstrated in
Figure 3A, the probability of an IIS was greater during a window
from 40 to 300 ms following presentation of oriented stimuli
than in a similar window after presentation of the blank stimuli.
This was seen in all cases (n = 6) (Fig. 3A). In many cases, one or
two particular orientations were more likely to trigger an IIS
(Fig. 3B). In one animal, presentation of 0°-oriented stimuli
consistently precipitated an afterdischarge, a small, self-limited,
seizure-like event, which was documented in both the electrical
and optical recordings (Fig. 3C). Afterdischarges were not seen
following presentation of the other orientations.
The preferred orientation for triggering the IIS was determined by the quantity of IISs that appeared within a 500 ms
window after the onset of presentation of each orientation. The
most common orientations that triggered an IIS were 0° and
135° (Table 1). In order to determine the explanation for this
phenomenon, we hypothesized that the preferred orientation
for triggering the IIS would correlate with the columnar location
of the iontophoresis pipette and the epicenter of the IIS optical
map. By superimposing the epilepsy maps derived from the
earliest small amplitude spikes over the angle maps of the orientation columns derived from the same area of cortex at an earlier
time, prior to iontophoresis of BMI (Fig. 4), we could show that
the location of the epileptic focus was strongly correlated with
the orientation of the visual stimulus that preferentially triggered
Figure 3. Preferential triggering of IIS and afterdischarges by oriented stimuli. (A) The timing of each IIS following initiation of movement of the grating stimuli pooled over all
animals. Each diamond represents the occurrence of an IIS in response to the stimuli displayed on the X-axis. Time 0 corresponds to the onset of the movement of the orientated
stimulus and the corresponding time point following the blank stimulus. The Y-axis demonstrates the latency of the spike. Grating stimuli are more likely to elicit an IIS in a window
from 40 to 300 ms following stimulus movement than are blank stimuli. (B) Examples of the number of spikes that occur in a window from 40 to 300 ms following stimulus
movement in four different animals averaged over all trials. In these examples, the orientations most likely to trigger an IIS were 135° > 0° > 90° > 45° (upper left), 135° = 0°
> 90° > 45° (upper right), 0° > 45° > 135° (lower left) and 0° (lower right). Error bar = SEM. Stimuli which always triggered a spike have no error bars. (C) Optical (top trace)
and f.p. (bottom trace) recordings from one animal show that 0° stimuli trigger an afterdischarge in two separate trials. Timescale of f.p. recording is expanded to facilitate visual
inspection.
Cerebral Cortex December 2003, V 13 N 12 1291
the IIS (Table 1). The columnar location was derived from the
number of pixels in the angle map preferring each orientation
underneath the area of the superimposed focus.
Another way to determine the preferred orientation for triggering an IIS is to examine the angle map, resulting from visual
stimulation with an IIS focus in visual cortex. Angle maps are
created by color-coding the vectorial summation of responses to
each orientation. The length of each vector is the magnitude of
the response to that orientation divided by a genuine blank stimulus (single-condition map). If one orientation were more likely
to elicit an IIS than the blank, the epileptic focus would appear
in the single-condition map, otherwise it would be divided out.
Thus, in the resulting angle map, the dominant color in the focus
reveals the orientation most likely to trigger the IIS. Figure 5A
(middle) reveals the resulting angle maps from an IIS focus triggered preferentially by 0° > 45° stimuli.
We also found that spatial frequency, in addition to orientation, was a powerful trigger for epileptiform events. In Figure 5B
(middle), a sum of the optical responses to high spatial
frequency stimuli is divided by the optical response to low
spatial frequency stimuli. If IISs were equally likely to be triggered by stimuli of both spatial frequencies, the optical signal
from the focus would be divided out of the resulting image. In
the example shown in Figure 5B, IISs are clearly more likely to
Table 1
Relationship between the location of the imaged focus with respect to the underlying orientation
columns and the preferred orientation of the visual stimulus for triggering the interictal spikes (IIS)
Preferred orientation triggering IIS
Location of imaged focus
1
135° = 0° > 90° > 45°
135° > 0°=90°
2
0°
0° > 45°
3
135° > 0°
135° > 0°
4
135° > 0° > 90° > 45°
135° > 0°
5
0° > 45° > 135°
0° > 45°
6
0° = 45° = 135° > 90°
0° = 45° = 90° = 135°
occur in response to low spatial frequency stimuli since the
optical signal from the focus appears to have a uniform shading
in the divided image. Similar results were found in all six
animals.
It is not clear from examining the angle maps in Figure 5A,
however, if the orientation preference of the neurons within the
focus has also been shifted, since the signal from the IIS is so
large. In order to distinguish between these two possibilities, we
performed extracellular single-unit recordings from the same
neuron before, during and after induction of an IIS focus. Singleunits (n = 7 units in four animals) within the focus demonstrated
a significant increase in overall firing frequency and bursting
simultaneous with the IIS (P < 0.05). Concomitantly, there was a
marked widening of the tuning curve from control values. The
orientation selectivity index (Wörgotter and Eysel, 1987)
decreased from a mean of 1.15 ± 0.48 before epileptogenesis to
0.85 ± 0.42 during the period of IIS, then returned to 0.95 ± 0.64
after reversal of the iontophoresis. There was also a mean shift in
orientation preference by 23.4 ± 35° (Fig. 6A,B). Such a shift in
orientation preference has also been reported following iontophoresis of sub-epileptic doses of BMI (Toth et al., 1997).
Hence, although the number of single units examined is limited,
the data support the idea that the optical signal recorded from
the focus represents both the preferred orientation for eliciting
IIS and a shift in orientation preference of the involved neurons.
The Functional Architecture in the ‘Surround’ is not
Affected by the Focus
Epilepsy maps generated with optical imaging of intrinsic signals
demonstrate a sharp border between the focus and surrounding
cortex (Schwartz and Bonhoeffer, 2001). Neurons in the cortex
surrounding an interictal focus are known to exhibit inhibitory
post-synaptic potentials (IPSPs) concurrently with each IIS,
presumably triggered by recurrent long-range inhibitory connections from within the focus (Prince and Wilder, 1967; Dichter
and Spencer, 1969a,b). In order to assess how this decrease in
neuronal activity in the ‘inhibitory surround’ affects the functional architecture, we imaged orientation preference in the
cortex surrounding an IIS focus and compared it with the
functional architecture maps obtained before and after epilepto-
Figure 4. Optical maps of the earliest, small amplitude IIS, superimposed over the orientation maps. (A) Example of the f.p. recording from the first low-amplitude spikes at the
beginning of the development of the IIS focus. (B) Blank-divided maps obtained by thresholding to the pixel value 1 SD above the mean prior to the appearance of the first spikes
(seen in A) superimposed over the angle maps from that same area of cortex. In these examples, epileptic foci are located over 0° > 45° (left) and 135° > 0° (right). The area of
the focus is indicated below the map. The irregular contour of the spike foci is likely caused by vascular artifacts since the blank divided maps highlight the vasodilation that
accompanies the IIS focus.
1292 Epileptic Focus and Functional Architecture • Schwartz
Figure 5. Orientation- and spatial-frequency triggering of IISs can be seen in the angle and differential maps. Optical recording from the surrounding cortex reveals preserved
cortical architecture and intact cerebral autoregulation. (A) Angle map generated in the presence of an IIS focus. The intrinsic signal within the focus is distorted by the occurrence
of the spikes. The intrinsic signal from the surrounding cortex is unaffected. The dominant color in the focus indicates that 0° stimuli were more likely to trigger spikes than other
orientations. Sample of the f.p. recording simultaneous with the imaging. Scale bar = 1 mm. (B) Spatial frequency maps from the same animal demonstrate that low-spatial
frequency stimuli are more likely to trigger IIS than high-spatial frequency stimuli.
genesis. It was not clear if the alterations in cerebral hemodynamics induced by the metabolic demands of the IIS focus
would affect the optical signals from adjacent cortex.
As can be seen in Figure 5, the layout of the orientation
columns outside the area of the epileptic focus is not significantly affected by the adjacent IIS. Extracellular single-unit
recordings (n = 7 units in four animals) from neurons in the’
surround’ confirm the optical data (Fig. 6C,D). Although these
neurons represent only a small fraction of the entire neuronal
pool, unlike those recorded from the focus, they did not manifest bursting simultaneous with the IIS and showed a mean
decrease in their firing rate by 36% (P < 0.05). Mean change in
orientation preference was –1.01 ± 6.83°.
The Shape of the Interictal Focus Does not Correlate with
the Functional Architecture
In order to determine if the spatial distribution of the IIS
conforms to the underlying functional architecture, we superimposed the epilepsy maps over the cortical architecture maps.
Epilepsy maps generated with spike-triggered image division
were used for this analysis since blank-divided (BD) maps have
larger vascular artifacts that could induce false positive correlations. Contour plots of the IIS superimposed over the functional
maps did not reveal any obvious relationship between the focus
and the underlying architecture (Fig. 7). For example, the
margins of the IIS did not spread preferentially into any one
orientation or avoid crossing either the borders between ocular
dominance and spatial frequency columns or the fractures in the
orientation maps. Neither was there a region outside the border
of the focus, within a particular functional ‘column’, in which
the intrinsic signal rose >1 SD from the mean of the entire image.
We investigated any subtle relationship between the topography
of the focus and the underlying functional architecture using
two-dimensional cross correlations. A region of interest
surrounding the interictal focus was correlated with the same
region in the functional architecture maps and the resulting rvalue was compared with the distribution of r-values obtained
by randomly shifting the focus to 1000 different locations within
the functional map. There were no correlations at P < 0.01 for
either the orientation, ocular dominance or spatial frequency
maps. To explore the possibility that a rapid change in contour
might occur in the region of a fracture, a similar comparison was
performed between the two-dimensional derivative map of the
focus and the fracture map. No significant correlations were
found.
The size and shape of the minimal epileptic aggregate
has no relationship to the functional architecture
To determine the minimal epileptic aggregate capable of
sustaining an IIS, we used BD maps acquired during the first,
low-amplitude spikes, which appear early in the development of
the interictal focus. BD maps, in contrast to ST maps, represent
the full optical signal since the residual signal from the prior
spike is not subtracted out of the final image (Schwartz and
Bonhoeffer, 2001). Although BD maps have more artifacts, they
are also more sensitive to small signal changes. Since we could
not reliably produce optical maps from a single spike at the
earliest stages of the evolution of the focus, we averaged the
maps from the first 10 spikes, which were of similar amplitude.
The first ten spikes in seven different foci had a mean amplitude and width of 0.43 ± 0.07 mV and 25.9 ± 6.3 ms respectively.
Simultaneously acquired BD maps had a mean area of 0.12 ±
Cerebral Cortex December 2003, V 13 N 12 1293
Wilder, 1967; Dichter and Spencer, 1969a,b; Bragin et al.,
2002a). The acute application of disinhibiting pharmacological
agents such as penicillin (PCN) or bicuculline (BMI), which act
via blockade of GABAA receptors, is one of the oldest and most
frequently utilized models of the IIS focus (Fisher, 1989).
Although this model is not a perfect replication of the chronic
epileptic focus, intracellular recordings from neurons within the
disinhibited focus reveal paroxysmal depolarizing shifts similar
to recordings from neurons in more chronic foci, as well as facilitation of ‘fast ripple’ activity (Matsumoto and Ajimone-Marsan,
1964; Prince, 1968; Bragin et al., 2002a).
Figure 6. Radial plots of orientation preference from extracellular single unit recordings
from the focus and surround. (A) Two examples of units in the focus from two separate
animals demonstrate a shift in orientation preference, a decrease in selectivity and an
increase in spike frequency in the presence of the IISs. (B) Two units from the surround
recorded from two separate animals show minimal change in orientation preference
and selectivity although there is a clear decrease in firing frequency. This electrophysiological data confirm the optical data in Figure 5 and indicate that the orientation
preference of the neurons in the focus does shift.
0.02 mm2 with a range of 0.08–0.14 mm2 (Fig. 4). Hence, the
size of the minimal epileptic aggregate is similar to the size of
the iso-orientation domains but much smaller than the ocular
dominance and spatial frequency columns. In addition, there is
much less variance in the size of the minimal epileptogenic
aggregate than in the functional columns, which vary widely
from column to column and animal to animal. Since BD maps are
more affected by surface vasculature, their contour is not
symmetrical. To test whether any anisotropy was caused by
correlations with the underlying functional architecture, these
first-spike BD maps were cross-correlated with the functional
architecture maps and compared with the distribution of rvalues obtained by randomly shifting the position of the focus to
1000 different locations. No significant (P < 0.01) correlations
were found.
Discussion
The IIS is the electrophysiological hallmark of epileptic cortex,
which consists of the synchronous paroxysmal depolarization of
a population of pathologically interconnected neurons, typically
lasting less than 100 milliseconds (Dichter and Ayala, 1987).
These neurons are also capable of producing high frequency
oscillations termed ‘fast ripples’ (Ylanen et al., 1995; Bragin et
al., 2002a). Surrounding the epileptic focus, neurons exhibit
varying degrees of inhibition, felt to be important in controlling
the spread of hypersynchronous excitatory activity (Prince and
1294 Epileptic Focus and Functional Architecture • Schwartz
How Does the Functional Architecture Inf luence the
Initiation of the Epileptic Event?
By presenting an oriented stimulus that was iso-luminant with
the blank stimulus, we were able to preferentially trigger IISs
with oriented stumuli. These results provide direct evidence
that the neurophysiologic basis of photosensitive and patternsensitive epilepsy resides in primary visual cortex. Earlier
attempts to trigger IIS foci in visual cortex with orientationspecific stimuli were not successful (Ebersole and Levine, 1975;
Scobey and Gabor, 1977). We attribute our success to the fact
that the initial presentation of the oriented stimulus did not
cause a change in luminance and spikes were triggered with
stimulus movement rather than stimulus presentation. Since
change in luminance is a known trigger of visually evoked
epileptic events (Scobey and Gabor, 1977; Wilkins et al., 1979),
it is probable that the orientation-specific response in prior
studies was masked by the large luminance change at stimulus
onset.
We also show that certain orientations are more likely to
trigger epileptiform events than other orientations and we can
predict this preference based on the orientation column into
which the BMI is iontophoresed. Hence, the initiation of epileptiform events is directly related to the underlying columnar
architecture and can be explained based solely on focal alterations in neocortical physiology.
The ‘Minimal Epileptogenic Unit’
The minimal number of neurons required to support paroxysmal population activity has been investigated both in vivo and
in vitro. In vivo cortical transections can isolate small areas of
cortex to define the minimal amount of tissue required to
support an IIS, generally reported between 0.5 and 0.8 mm2
(Morrell and Hambery, 1969; Reichenthal and Hocherman,
1977; Lueders et al., 1980; Bashir and Holmes, 1993). In vitro
techniques, on the other hand, have shown that subdivided
neocortical slices consisting of only one or two lamina are
capable of sustaining paroxysmal events (Gutnick et al., 1982;
Silva et al., 1991; Chesi and Stone, 1998).
The area of cortex from which intrinsic signals were recorded
during the earliest IISs in our study was on the order of 0.1 mm2.
One reason the size of the minimal epileptogenic aggregate we
report is smaller than reported in previous in vivo studies
involving cortical transections is that we are recording the
amount of tissue participating in each event, rather than the
minimal amount required to sustain the event. Surgical transections undoubtedly damage a considerable number of neurons
adjacent to the incision, which decreases the number of functional neurons in the remaining slab of tissue. Our technique
probably overestimates the size of the epileptogenic aggregate
for two additional reasons. First, we summed the maps from the
Figure 7. Lack of correlation between the IIS focus and the functional architecture. (A) Epilepsy map generated with spike-triggered image division. Contour plot of the focus
superimposed over the (B) 0/90° differential map, (C) 45/135° differential map, (D) spatial frequency map and (E) ocular dominance map. (F) The two-dimensional derivative of the
epilepsy map highlights the rapid change in optical signal at the edge of the focus. (G) Superimposition of the contour plot on the two-dimensional epilepsy map over the fracture
map indicates no correlation between rapid changes in orientation preference and the edge of the focus. Scale bar = 1 mm.
first 10 spikes in an evolving focus to increase our signal to noise
and the tenth spike was larger than the first. Also, the intrinsic
signal represents subthreshold activity in addition to action
potentials and thus includes spatial information about EPSPs in
surrounding neurons outside of the bursting focus (Das and
Gilbert, 1995). Our findings are further supported by optical
recordings of spontaneous events from slices stained with
voltage-sensitive dyes (VSDs), which report that events arise
from a population of neurons with a volume of <0.04 mm3 or an
area of 0.09 mm2 (the spatial resolution of the technique) (Tsau
et al., 1998, 1999). In addition, Chesi and Stone (1998) recently
compared the minimal epileptogenic unit in vitro to the rodent
barrel fields and found that the epileptic generator was clearly
smaller than the volume of an individual barrel and that there
was no apparent relationship between the two structures.
Both primary and secondary visual cortices are organized into
multiple overlying maps, including direction, spatial frequency
and ocular dominance in addition to orientation, which are
distributed relatively smoothly across the cortical surface
(Bonhoeffer and Grinvald, 1991, 1993; Shmuel and Grinvald,
1996; Weliky et al., 1996; Hübener et al., 1997; Shoham et al.,
1997; Issa et al., 2000). The size of these columns is extremely
variable, even within a single animal. Hence, it is not clear how
there could be a single epileptogenic unit that could conform to,
or be determined by, such a structurally diverse and complex
cortical organization.
How Does the Functional Architecture Inf luence the
Propagation of the Epileptic Event?
In vivo and in vitro field recordings from disinhibited epileptic
foci have reported that horizontal propagation occurs in ‘fits and
starts’ over preferred pathways across the cortex, possibly
related to columnar architecture (Tharp, 1971; Petsche et al.,
1974; Chervin et al., 1988; Wong and Prince, 1990; Wadman
and Gutnick, 1993). The rate of horizontal propagation in these
studies (0.04–0.08 m/s) implicates synaptic transmission medi-
ated by axon collaterals of excitatory pyramidal cells (Chervin et
al., 1988; Wong and Prince, 1990; Wadman and Gutnick,
1993;). Both excitatory and inhibitory short-range horizontal
excitatory connections (<500 µm) are spatially symmetric,
targeting neurons of all orientations in visual cortex, whereas
long-range excitatory connections (>1 mm) are patchy and
anisoptropic, connecting domains with similar receptive fields
(Weliky et al., 1995; Bosking et al., 1997; Dalva et al., 1997; Toth
et al., 1997). Hence, the reports of inhomogeneous horizontal
epileptic spread implicate long-range rather than short-range
connections.
In vitro optical recordings of epileptic spread with voltage
sensitive dyes (VSD), however, have not shown a similar nonuniform horizontal spread but rather smooth propagation (Sutor
et al., 1994; Tanifuji et al., 1994; Albowitz and Kuhnt, 1995),
perhaps caused by higher doses of pharmacological disinhibition used in the VSD experiments (Telfian and Connors, 1998).
Experiments in ferret neocortical slices have shown that high
doses of BMI can alter horizontal propagation velocities and
eliminate the influence of long-range horizontal projections
(Nelson and Katz, 1995). Hence, a model of epilepsy based on
disinhibition may underestimate the role of long-range projections.
Our in vivo optical recordings were made with the minimal
dose of BMI that could sustain constant amplitude IISs and we
found a homogeneous, symmetrical spike topography that did
not appear to correlate with the underlying columnar architecture. Our negative results may be attributable to the temporal
resolution of the intrinsic signal which is on the order of several
hundred milliseconds (Frostig et al., 1990; Bonhoeffer and Grinvald, 1996). In support of our data, London et al. (1989) optically recorded BMI-induced IIS in vivo using VSDs and a
photodetector with a temporal resolution of <5 ms and reported
no spatial inhomogeneities in the lateral spread of the optical
signal.
Cerebral Cortex December 2003, V 13 N 12 1295
How Does the IIS Focus Inf luence the Functional
Architecture?
Although a small percentage of patients with chronic epilepsy
show an overall decline in cognitive abilities, it is thought to
result more from long-term anticonvulsant therapy or an underlying structural abnormality rather than from the epileptic
events themselves (Cassidy and Corbett, 1997). Nevertheless,
there is a convincing evidence that the constant barrage of electrical events can induce changes in the nervous system that may
underlie some of the chronic cognitive/behavioral decline found
in epileptic patients (Engel et al., 1991; Lothman, 1997).
Our results indicate that focal epileptogenic disinhibition not
only induced a large decrease in orientation selectivity but also a
small, but significant shift in the orientation preference of
neurons in the focus, compared with neurons recorded from
the inhibitory ‘surround’. Studies of orientation preference
following focal application of subepileptic disinhibiting as well
as inhibiting pharmacological agents also show significant
changes in orientation preference several hundred microns
away from the site of iontophoresis (Crook and Eysel, 1992;
Sillito et al., 1980; Toth et al., 1997). Toth et al. (1997) demonstrated that these shifts occur in the direction of the orientation
preference of the cortical column into which the disinhibiting
agent was applied. Hence, it is likely that the changes in orientation preference found in our study may be more attributable to
pharmacological disinhibition than the epileptic events.
Optical and electrophysiological data from the inhibitory
‘surround’, however, is a more reliable reflection of the influence of the IIS on the surrounding architecture since the recordings are performed 4–6 mm from the application site, which is
beyond most estimates of diffusion distance following iontophoretic application (Fox et al., 1989). We found that the architecture of the iso-orientation domains did not change in the
‘surround’ despite the overall decrease in neuronal activity and
the proximity to the epileptic focus. However, an acute model
of epileptogenesis may not be sufficient to induce alterations in
synaptic plasticity found in chronic epilepsy.
Our ability to optically map functional architecture in close
proximity to the epileptic focus parallels the fMRI literature.
Cerebral hemodynamics appear tightly regulated around pathological lesions such as epileptic foci and vascular malformations
which might interfere with blood flow and subsequently influence the optical signals (Morris et al., 1994; Latchaw et al.,
1995; Maldjian et al., 1996; Schwartz et al., 1998). The ability to
optically map functional architecture adjacent to pathological
lesions is critical for intraoperative applications of intrinsic
signal imaging to guide neurosurgical resections (Schwartz and
Bonhoeffer, 2001).
Notes
Special thanks to Tobias Bonhoeffer, in whose laboratory these experiments were performed. Also thanks to Sven Schütt, who contributed
substantially to the data analysis, Frank Sengpiel and Mark Hübener, for
expert advice throughout the study and finally, Volker Staiger, Iris
Kehrer and Frank Brinkmann, for providing outstanding technical assistance. This work was supported by the Max-Planck Gesellschaft, as well
as by grants to T.H.S. from the Epilepsy Foundation of America, the van
Wagenan Fellowship of the American Association of Neurological
Surgeons and the Alexander von Humboldt Stiftung.
Address correspondence to Theodore H. Schwartz, M.D., Department
of Neurological Surgery, Weill Medical College of Cornell University,
525 East 68th Street, Box 99, New York, NY 10021, USA. Email:
[email protected].
1296 Epileptic Focus and Functional Architecture • Schwartz
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