 |
INTRODUCTION |
Intrinsic cortical processes
are deemed responsible for the generation of epileptiform
activity (Connors 1984
; Traub et al. 1996
; Tsau et al. 1998
). Contrary to animal
models, epileptic conditions in patients often persist for years.
Consequently, no animal model faithfully mirrors the underlying network
dysfunction, which is even speculated to be aggravated by recurrent
seizures in ways different from short-term experimental models
(Albowitz et al. 1998
; Engel et al. 1998
;
Köhling et al. 1998
). In previous studies,
synchronous sharp field potentials (SSFP) were found to occur
spontaneously in human epileptic neocortical slices
(Köhling et al. 1998
, 1999
). Such SSFP cannot be
observed in animal tissue, nor in human brain slices obtained during
tumor resections from patients without clinical signs of seizures and
may thus reflect a state of increased neuronal synchronization
characteristic of chronic epileptogenicity (Köhling et al.
1998
). Although SSFPs represent a state of neuronal
synchronization and excitation sufficiently large to generate field
potentials, on the level of single neurons, predominantly
hyperpolarizing membrane potential deflections were observed
in human epileptic tissue concomitant with SSFP (Köhling et al. 1998
). However, clear cut high-amplitude population
spikes superimposed on SSFP indicated that depolarizations
occurred in parallel (Köhling et al. 1999
). This
paper addresses the question of whether depolarizations of larger
neuronal aggregates in human epileptic tissue do actually occur during
SSFP. Further, it explores whether neuronal activity and SSFP under
epileptogenic conditions display different spatial characteristics and
whether the spontaneous initiation of prolonged epileptiform events
under such conditions can be pinpointed to distinct foci.
Voltage-sensitive dye imaging provides an ideal means to observe
neuronal activity with adequate temporal and spatial resolution
(Davila et al. 1974
). Using this method, the spread of
both stimulus-induced (Albowitz and Kuhnt 1995
;
Demir et al. 1999
) and spontaneous epileptiform activity (Tsau et al. 1998
) was explored in different animal
preparations. This study is, to our knowledge, the first such
investigation on spontaneous epileptiform activity in human tissue.
 |
METHODS |
Human neocortical tissue was a small portion of what
was excised for treatment of pharmacoresistant temporal lobe epilepsy from eight patients. Prior to the operation, the patients received carbamazepine, valproate, and one or more of the following:
lamotrigine, retigabine, phenytoin, and phenobarbital sodium.
Histopathological analysis revealed a mild degree of gliosis
(n = 4/8) and discrete focal dysplasia
(n = 4/8). The experiments were approved by the local
ethics committee, and informed consent was obtained from the patients.
Neocortical slices (n = 8) were prepared as described elsewhere (Albowitz et al. 1998
;
Köhling et al. 1998
). Briefly, from a
1-cm3 block of the inferior temporal gyrus,
450-µm coronal sections were made with a vibratome within 5 min after
resection. These were placed in a portable incubation chamber with
oxygenated (95% O2-5%
CO2) and warmed (28°C) artificial cerebrospinal
fluid (ACSF) containing (in mM) 124 NaCl, 4 KCl, 2 CaCl2, 1.24 NaH2PO4, 1.3 MgSO4, 26 NaHCO3, and 10 glucose (pH 7.35-7.45). Slices were stained with 12.5 µg/ml of the
voltage-sensitive dye RH795 1 h prior to the experiments and
washed for 1 h before transferral to a submerged-type recording
chamber (32°C) mounted on an inverse microscope. Field potentials
were recorded from layers II/III and V using conventional glass
micropipettes filled with ACSF. Slices were scanned for SSFP by
repositioning the field potential electrodes to at least six different
sites distributed roughly equally across the slice. Dye-related
fluorescence signals were detected by a 464-element honey-comb shaped
photodiode array at a rate of 785 frames/s via 10× and 20× objective
lenses, yielding total fields of view of 1500 and 750 µm diam,
respectively. Xenon lamp illumination was allowed for a maximum of
4 s per single detection period, and detection periods were
separated by at least 5 min each. Optical data were assessed as
fluorescence changes relative to resting light intensity
(dI/I) using NeuroPlex software (RedShirt Imaging, LLC,
Fairfield, CT), precluding signal variability due to inhomogeneous illumination or differences in lens properties. For trace
illustrations, optical and field potential signals were
low-pass-filtered (Butterworth filter, 30 or 50 Hz). Only data
related to field potential discharges spontaneously occurring >200 ms
after sampling start were evaluated. Unfiltered fluorescence changes
were converted to pseudocolor images scaled to the center diode. In
some cases, only the upper 10% of fluorescence decreases (equivalent
to depolarizations) were visualized (Fig.
1). The fluorescence
change dI/I for typical events was 0.05-0.3% of resting
light intensity (Figs. 1 and 2).

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Fig. 1.
Synchronous sharp field potentials (SSFP) occurring spontaneously and
concomitant neuronal depolarizations detected by voltage-sensitive dye
imaging (Opt) in chronically epileptic human neocortical slice
preparation. A: recording positions of field potential
electrode and optical recording fields (via 20× and 10× objective
lenses) are given in the diagram. WM: white matter. The fraction of
SSFP associated with extended decreases of fluorescence (neuronal
depolarizations) differed depending on the magnification.
B: spatial spread of neuronal depolarizations associated
with SSFP; single traces as in A. Optical signals at
different time points (6.4 ms interframe-interval; 64 ms before and 160 ms after SSFP peak marked by blue dot and frame) covering a field of
750 µm diam. Red dots represent relative minima of fluorescence
(upper 10% of fluorescence changes). C: single SSFP and
single optical traces (superimposed). Traces with largest (red),
substantially (<50%) smaller (black), and missing (green) optical
peaks. The location of the diodes within the array is marked by
corresponding colors. D: fraction of total recording
area (750 µm diam) with maximal fluorescence changes at different
time points as in B. Means ± SE of 13 events.
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Fig. 2.
Spontaneous ictal (>2 s duration) field potential discharge (FP) and
concomitant neuronal depolarizations detected by voltage-sensitive dye
imaging (Opt) in chronically epileptic human neocortical slice
preparation under epileptogenic conditions (low-Mg2+ ACSF).
A: recording position of field potential electrode and
optical recording fields (10× objective lens) is given in the diagram.
WM: white matter. The red line marks the photodiode line whose
fluorescence signals (Opt) are given in conjunction with the field
potential recording (FP; middle) and as pseudocolour
line-time-plot (right). Blue dot, dotted line: location
and initiiation of depolarization. B: spatial spread of
neuronal depolarizations associated with ictal discharges. Optical
signals at different time points (64 ms interframe-interval) covering a
field of 1500 µm diam. Circles: site with earliest depolarizations.
The red frame marks the initial peak of the ictal discharge (red dot in
A). C: initiation of ictal events in the
same slice the data in A and B were
obtained from. Columns represent fraction of events starting in
respective quadrants of the optical recording fields as depicted in the
diagram. Initiation was defined as initial condensation of red dots
(depolarizations) in 1 quadrant of either recording field
(n = 10 events).
|
|
 |
RESULTS |
SSFP were only detected in a fraction of slices (n = 8/21; cf. Köhling et al. 1998
, 1999
). In the
cases further investigated here, SSFP appeared at one or two of six
recording sites, located infra- or both infra- and supragranularly
(Fig. 1). When the optical recording field (20× objective lens, 750 µm diam) was centered on the field potential electrode, most (83%,
n = 18) SSFP occurred with distinct decreases of the
fluorescence signal (depolarizations) of single photodiodes (Fig.
1A). The detection of clear cut fluorescence changes became
less likely when the optical recording field was increased (10×
objective lens, 1500 µm diam). Under these circumstances, in only
64% of the events (n = 11), SSFP were correlated with optical signals clearly detectable against background levels, indicating that the neuronal clusters presumably generating the activity were small and went partially undetected with decreasing spatial resolution (Fig. 1A). The fact that doubling the
diameter of the visual field (and thus quadruplicating the area covered by a single diode) led to detection loss may indicate that activity changes were spatially inhomogeneous and in fact included "hot spots" of roughly single-diode coverage at high magnification (Fig.
1B). With gradual build-up of the SSFP, an increasing number of photodiodes detected maximal depolarizations, reaching a maximum ~14 ms after the peak of the SSFP (Fig. 1B). Individual
diodes could be distinguished at all times during the signal, creating dynamic activation patterns, and only rarely at the peak of the event
did some areas with maximal activity converge to cover larger fields.
Noise in the recordings is unlikely to account for the differences in
fluorescence changes associated with SSFP as detected by different
diodes because: 1) before the signals, all diodes showed
similar noise levels prior to the SSFP (Fig. 1C; red, black, and green traces), 2) some diodes picked up persistingly and
substantially lower activity peaks (<50%) than their immediate
neighbors (Fig. 1C, black trace), and 3) other
diodes showed a decrease of noise instead of a clear-cut
signal (Fig. 1C, green trace), possibly as a sign of
disfacilitation and revealing the noise to be biological. Although
maximal neuronal depolarizations were thus inhomogeneously distributed,
SSFP-associated depolarizations were a population phenomenon. Before
the SSFP, 2% of the total area of the optical recording field showed
maximal depolarization changes, possibly reflecting asynchronous
spontaneous action potentials of single neurons or small neuronal
clusters (Fig. 1, B and D; n = 13; 20× objective). Shortly after the peak of the SSFP, this fraction rose to 17% on average, indicating that at that point a sizeable proportion of neurons became involved. This fraction remained >4% for
>100 ms after SSFP peak. This persisting activity was not mirrored in
the field potential (Fig. 1, B and D).
Introducing epileptogenic conditions by washing nominally
Mg2+-free ACSF (Avoli et al. 1995
)
did not change the behavior of SSFP and associated optically detected
depolarizations. Specifically, the activity remained very focal and was
restricted to ~1500 µm diam. Moving the diode array from the
infragranular (cf. Fig. 1A) to a subpial position, no
fluorescence signals were observed (not shown). This reflected the
inhomogeneous distribution of areas with maximal neuronal activity,
which in the case of covering a large optical recording area (10×
objective lens, 1500 µm diam) were sparsely scattered, as already
seen with spontaneous SSFP under nonepileptogenic conditions. Again,
with the SSFP, a larger population of neurons became depolarized.
Before the event, roughly 0.5% of the total area covered show maximal
depolarizations. This rose to 8% shortly after the peak of the SSFP
was reached (n = 11).
Rarely, ictal-like activity was induced by
low-Mg2+ ACSF perfusion in slices with
(n = 2) and without (n = 1) previous
spontaneous SSFP (cf. Köhling et al. 1998
). From
the two slices displaying spontaneous SSFP, a typical example of an
ictal discharge lasting >2 s is demonstrated in Fig. 2. Also this
field potential discharge went along with distinct fluorescence
decreases, which were polyphasic like the field potential recording
(Fig. 2A, Opt). Picking one central line of diodes
perpendicular to the pial surface, the original optical signals show
that activity appeared to be initiated at a distinct diode (marked by a
blue dot in Fig. 2A). The gradual decline of fluorescence
shown here is unlikely to be related to noise since both phenomena
show different kinetics. As with spontaneous SSFP, depolarizations
were not homogeneous, as intensity differences between neighboring
diodes could amount to 20% or more. The pseudocolor line-time-plot reveals that in fact in this case, a field close to the
pial surface showed the earliest depolarizations, which subsequently reached deeper layers (Fig. 2A). Optical
recordings taken at different time points (10× objective, 1500 µm diam) confirm that earliest depolarizations were initially
clustered in an area close to the pial surface (Fig. 2B,
circles). They also show that activity peaks are distributed
inhomogeneously within the recording field and converge only with the
full-blown ictal event. This raises the question whether initiation of
activity could be also nonstereotypically located. Ten events were
recorded at both supra- and infragranular recording sites. As Fig.
2C shows, distinct initiation sites as reflected by earliest
depolarizations occurring in localized areas in different quadrants of
the hexagonal recording fields could be observed. Overall, in either
recording position, depolarizations were preferentially initiated in
quadrants directed to the pial surface of the slice, with three
possible initial "foci," which were active in parallel
(supragranular recording position) or possibly were only secondarily
invaded (infragranular recording position). Such
localized initial foci with ictal activity were not only
observed in the two slices which previously had shown spontaneous SSFP
but also in another slice which previously did not and only generated
activity with washout of Mg2+ (data not shown).
 |
DISCUSSION |
The main findings of this report are as
follows. 1) SSFP in chronically epileptic human brain tissue
are accompanied by extended cellular depolarizations. These are of
neuronal origin with at most negligible glial contribution since the
extracellular potassium concentration (the main possible source of
glial depolarizations) does not rise detectably during SSFP
(Köhling et al. 1998
). 2) Hot spots of
neuronal activity are spatially dynamic and inhomogeneous during SSFP.
3) Neuronal activity gradually builds up before SSFP, and
its decline outlasts the end of the field potential changes. 4) Ictal activity is initiated at circumscribed, variable
foci. SSFP are characteristic of chronically epileptic human brain
tissue (Köhling et al. 1998
). Our observations
suggest that they arise in a stochastic-like manner from the initial
activity of single neurons or small neuronal clusters, which start to
fire several tens of ms before field potentials actually peak, much in
the same way as in experimental epilepsy models, the firing rate of neurons increases 200 ms before EEG-spikes are observed (Elger and Speckmann 1983
). The mechanisms bringing about the spatial spread of this activity to finally constitute a small focus of ~750
µm diameter are still unclear. Synaptic processes certainly are
involved (Köhling et al. 1998
), and indeed
animal studies suggest that neocortical neurons are highly
interconnected (Markram et al. 1997
). In addition,
epileptic human brain appears to be inherently more excitable, allowing
for a wide-ranging spread of excitation at least with focal stimulation
(Albowitz et al. 1998
). Interestingly, even under
epileptogenic conditions, SSFP, and the underlying neuronal activity
patterns, can remain unchanged and rather focal, as already suggested
previously (Köhling et al. 1998
). If ictaform
activity ensues, it is initiated at distinct but variable foci (cf.
Tsau et al. 1998
). Whether these foci correspond to the
foci seen with SSFP under nonepileptogenic conditions could not yet be
verified. With such foci, ictaform activity was preferentially initiated supragranularly in a previous study, much like the initiation sites here (Köhling et al. 1999
). Coherent
activity of a local group of neurons is thought to be able to initiate
ictaform discharges (Silva et al. 1991
; Traub et
al. 1996
). In this way, we suggest that SSFP foci may start
full-blown epileptiform activity in hyperexcitable human tissue.
This work was supported by Deutsche Forschungsgemeinschaft Grant Sp
108/16-2 and Interdisziplinäres Zentrum für Klinische Forschung der Westfälischen Wilhelms-Universität
Münster Grant F2.
Address for reprint requests: R. Köhling, Institut für
Physiologie, Westfälische Wilhelms-Universität,
Robert-Koch-Str. 27a, 48149 Münster, Germany (E-mail:
kohling{at}uni-muenster.de).
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.