Optical Monitoring of Neuronal Activity During Spontaneous Sharp Waves in Chronically Epileptic Human Neocortical Tissue

Rüdiger Köhling,1 Jörg-Michael Höhling,1 Heidrun Straub,1 Dieter Kuhlmann,1 Ulrich Kuhnt,2 Ingrid Tuxhorn,3 Alois Ebner,3 Peter Wolf,3 Heinz-Wolfgang Pannek,3 Ali Gorji,1 and Erwin-Josef Speckmann1

 1Institut für Physiologie, Westfälische Wilhelms-Universität, 48149 Münster;  2Max-Planck-Institut f. Biophysikalische Chemie, 37077 Göttingen; and  3Epilepsiezentrum Bethel, 33617 Bielefeld, Germany


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INTRODUCTION
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Köhling, Rüdiger, Jörg-Michael Höhling, Heidrun Straub, Dieter Kuhlmann, Ulrich Kuhnt, Ingrid Tuxhorn, Alois Ebner, Peter Wolf, Heinz-Wolfgang Pannek, Ali Gorji, and Erwin-Josef Speckmann. Optical Monitoring of Neuronal Activity During Spontaneous Sharp Waves in Chronically Epileptic Human Neocortical Tissue. J. Neurophysiol. 84: 2161-2165, 2000. Functional changes in neuronal circuitry reflected in spontaneously occurring synchronous sharp field potentials (SSFP) have been reported to occur in human brain suffering from chronic epileptogenicity but not in primary nonepileptic tissue from peritumoral resectates. Voltage sensitive dyes and fast imaging were used to visualize spontaneously occurring rhythmic depolarizations correlated to SSFP in chronically epileptic human neocortical slices obtained during epilepsy surgery. Localized and spatially inhomogeneous neuronal depolarizations were found to underlie spontaneous SSFP, which remained unchanged and spatially restricted to foci <750 µm diam even under epileptogenic (low-Mg2+) conditions. In cases where ictaform paroxysmal activity occurred in low-Mg2+ medium, neuronal depolarizations were wide-spread but still spatially inhomogeneous, and the events were preferentially initiated at distinct foci. The findings suggest that small neuronal networks are able to establish and maintain synchronous rhythmic and epileptiform activity.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
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DISCUSSION
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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.


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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).


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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).


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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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.

Received 15 March 2000; accepted in final form 6 June 2000.


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0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society




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