Brain Research, 508 (1990) 105-117

1(15

Elsevier BRES 15161

Laminar interactions in rat motor cortex during cyclical excitability changes of the penicillin focus Daniel S. Barth t'2, Christoph B a u m g a r t n e r L'4 and Shi Di ~ 3 Departments of 1Neurology and 2psychology, University of California, Los Angeles, Los Angeles, CA 90024 (U.S.A.) and ¢Mental Health Institute, Beijing Medical University, Beijing (People's Republic of China) and 4Neurological University Clinic. Vienna (Austria) (Accepted 3 July 1989)

Key words: Focal epilepsy: Current source-density; Principal component analysis; Source modeling; Penicillin spike: Laminar analysis

Laminar interactions between neurons in rat motor cortex during cyclical seizure episodes in the penicillin focus were studied using a combination of current source-density (CSD) and principal component analysis (PCA), combined with computer-based physical modeling. These data suggest that all phases of cyclical seizure phenomena are produced by interactions between two distinct populations of neurons, the same neuronal circuits previously reported to give rise to the direct cortical response (DCR) and electrically evoked interictal penicillin spikes (EIIS). The first population consists of small pyramidal cells in the supragranular layer, and the second population consists of larger pyramidal cells in the infragranular layers with apical dendrites extending to the cortical surface. The supragranular cells serve as a trigger zone for initiating both spontaneous interictal spikes (IIS) and polyspike bursts (PSB) during seizures. Fast activity in the supragranular cells is typically followed by a hyperpolarizing slow wave that may be the result of Ca2~-activated K * currents. This slow wave increases during seizures, possibly reflecting changes in extracellular Ca2+ associated with seizure onset and termination. The monophasic response of infragranular cells is similar for both IIS and PSB and consists of a large depolarizing shift followed by a rapid but p,artial repolarization period and a subsequent gradual repolarization period lasting several hundred milliseconds. The infragranular response is similar in polarity and morphology to the intracellularly recorded paroxysmal depolarization shift (PDS) and may indicate that these deeper neurons are mainly responsible for this phenomena in neocortex. Finally, there is a marked posticta[ slow oscillation between the supra- and infragranular layers. This oscillation appears first and largest in the supragranular cells and may reflect a disturbance in excitatory feedback in these cells produced by the disinhibitory effect of penicillin, a disturbance capable of pathologically synchronizing the epileptic neuronal aggregate sufficiently for activation of the spike-generating mechanism and subsequent seizures.

INTRODUCTION W h e n penicillin is directly applied to the surface of neocortex, a series of epileptiform electrographic events evolve that typically range in time course from 10-50 ms ictal and interictal spikes (IIS) to slower 100-1000 ms waves following IIS and bursts of seizure discharge ~5' 25,26,32,35. Yet, there have also been infrequent reports of uitraslow changes in activity of the penicillin epileptic focus that occur over minutes and appear as a slow cycling of epileptic excitability ~7~941"44"45. Excitability cycles in the penicillin focus are of interest because they may provide clues to electrophysiological mechanisms underlying the onset and termination of seizures. Ultraslow changes in epileptic excitability were first recorded in experiments using low frequency stimulation to evoke interictal spikes (EllS) in strychninized cortex ~' 47. In these studies it was noted that while individual EIIS could be successfully evoked by low frequency stimulation (approx. l/s), they were limited to trains of 10-30 s

duration before the focus became unresponsive. Continued spike driving could only be achieved by resuming stimulation after some delay. Using continuous low frequency afferent and electrocortical stimulation, Prince later reported that trains of driven E l l s would alternate with trains of evoked responses, a p h e n o m e n a termed 'cyclical spike driving '3~. Cyclical spike driving has been recorded in both isolated 37 as well as intact neocortex 3s _~9,4~ and therefore probably reflects mechanisms of excitability control contained within cortical neuronal circuitry. With larger concentrations of epicortical penicillin, spontaneous seizures often alternate with postictal suppression and trains of IIS in regular cycles that are similar in periodicity to that found in cyclical spike driving but without the r e q u i r e m e n t of exogenous stimulation 44. The cycling of seizures may continue with regularity for periods up to several hours. Although the occurrence of cyclical seizure episodes in the penicillin focus is highly artificial, it serves as a useful model for studying

Correspondence: D.S. Barth, 3155 Reed Neurological Research ()enter, Department of Neurology, University of California, Los Angeles. Los Angeles, CA 90024, U.S.A.

0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

106 e l e c t r o p h y s i o l o g i c a l c h a n g e s t h r o u g h o u t ictal, postictal, and interictal periods. O u r i n t e r e s t in cyclical seizure p h e n o m e n a lies in h o w l a m i n a r i n t e r a c t i o n s of cortical circuitry m a y be a l t e r e d during

seizures,

and

how

this

differs

from

laminar

i n t e r a c t i o n s d u r i n g the IIS c o m p l e x . R e c e n t studies of IIS h a v e s u g g e s t e d a u n i q u e i n v o l v e m e n t of cortical n e u r o n s at c e r t a i n l a m i n a in t h e g e n e r a t i o n o f discharges b e t w e e n seizures3,12,31,34 36. By using l a m i n a r e l e c t r o d e s to r e c o r d e x t r a c e l l u l a r field p o t e n t i a l s and a p p l y i n g c u r r e n t sourced e n s i t y ( C S D ) analysis to r e v e a l l a m i n a r locations of

Fig. 1. Microelectrode array used for laminar recording. Sixteen 10 × 10/~m Ag/AgC1 contacts are spaced by 150 ~um, covering a total laminar depth of 2.25 mm when inserted perpendicular to the cortical surface.

e x t r a c e i l u l a r sinks and sources, it has b e e n possible to m e a s u r e spatial and t e m p o r a l a c t i v a t i o n p a t t e r n s of cells w i t h i n the cortical l a m i n a d u r i n g the e l a b o r a t i o n of the IIS c o m p l e x . T h e s e studies h a v e i n d i c a t e d the possible r o l e o f cells n e a r the b o r d e r of layers II1 and IV in initiating IIS 7'1s, and the s u b s e q u e n t p a r t i c i p a t i o n of n e u r o n s in d e e p e r cortical layers giving rise to p o t e n t i a l s t h a t are similar in latency and d u r a t i o n to the intracellularly r e c o r d e d p a r o x y s m a l d e p o l a r i z a t i o n shift ( P D S ) 25

26,40

W e h a v e r e c e n t l y a p p l i e d the m u l t i v a r i a t e statistical

m e t h o d of p r i n c i p a l c o m p o n e n t analysis ( P C A ) c o m b i n e d with c o m p u t e r - b a s e d physical m o d e l i n g to s e p a r a t e spatially and t e m p o r a l l y o v e r l a p p i n g C S D p a t t e r n s d u r i n g direct cortical r e s p o n s e s ( D C R s ) and E l l S in the s a m e a n i m a l s 3. T h e results of this w o r k s u g g e s t e d that similar n e u r o n a l circuits w e r e a c t i v a t e d in a similar s e q u e n c e d u r i n g the D C R and E l l S , and only the timing of the circuits was a l t e r e d by the epileptic process. T h e o b j e c t of the p r e s e n t study was to e x t e n d this m e t h o d to the analysis

of d y n a m i c

changes

in l a m i n a r

interactions

a s s o c i a t e d with cyclical seizure episodes. MATERIALS AND METHODS Three adult Sprague-Dawley rats (280-300 g) were studied. Following an injection of atropine sulphate, general anesthesia was administered and maintained using a combination of ketamine HCI (100 mg/ml) and xylazine (20 mg/ml), Normal body temperature was maintained throughout the experiments using a regulated heating pad. A unilateral craniectomy exposed a region over the right hemisphere extending from 3 mm rostral to bregma as far caudally as lambda, and laterally from the midline to a point 7 mm ventral to the temporal ridge. The dura mater was resected and exposed cortex moistened with saline throughout the experiment. A 1 mm 2 piece of filter paper soaked in penicillin G potassium (2000 IU//A) was placed on the surface of the right motor cortex. The filter paper was removed after approximately 5 min, when spontaneous penicillin spikes appeared. After approximately 1 h, the animals displayed clear ictal events recorded at the site of penicillin application. Seizures were characterized by transformation of IISs into repetitive 50-100 ms polyspike bursts (PSBs) with an interburst interval of 0.5-1 s. Unlike the IIS, each PSB was accompanied by twitching of the contralateral forepaw. In no animals did the seizures progress to higher frequency discharges classically associated with tonic and clonic phases. Seizure episodes occurred in regular cycles with an average seizure duration of approximately 30 s, followed by a 5-20 s postictal period, and resumed interictal spiking for approximately 30-100 s before another seizure. This

cycle of ictal excitability was regular for up to 3 hr of recording time. The laminar profile of extracellular field potentials associated with both interictal and ictal discharges was measured from a linear micro-array of 16 Ag/AgCI chambered electrodes (Fig. 1; OttoSensors Corp., Cleveland, OH; 10 × 10ktm 2 sensing area per electrode: 150/~m electrode spacing) covering a total distance extending from the cortical surface to a depth of 2.25 mm. Recordings were referenced to an Ag/AgCI ball electrode on the contralateral nasal bone. The array was stereotaxically inserted perpendicular to the cortex into the center of the penicillin focus with the upper electrode positioned just beneath the surface under microscope control. Potentials were digitally sampled in 10 s blocks (200 Hz. 12 bitsl and stored on disk for further analysis. One-dimensional CSD was computed from the 16 channels of laminar potentials to identify discrete regions of extraceUular current sinks and sources associated with membrane depolarization and hyperpolarization along the axis of the electrode array 34'272943. The averaged CSD was then computed separately for IIS and PSB from at least 100 events. Averaging was achieved by visually identifying and marking a particular temporal component of the IIS or PSB complex using a manually controlled cursor and high resolution computer graphics monitor. The crest of the initial current sink (associated with the first large surface negative spike of the IIS and PSB) in the fourth channel (450/~m below the cortical surface) was consistently used for all animals because of its large and easily identified morphology. However, the averaged results showed little sensitivity to the particular channel or temporal component used for time marking. The averaged CSD waveforms of the IIS and PSB (using an averaging window of 0.25 s before and 1 s after time zero) were further analyzed with PEA 3'1°'17'20'2~. The use of PCA in laminar analysis, and the strengths and weaknesses of such multivariate statistical procedures in this application have been described in detail previously3. The basic assumption of PCA in the present context was that the CSD waveforms were generated by only a few synchronously active neuronal populations, which were fixed in distinct cortical lamina and had transmembrane currents that varied uniquely over time (neuronal populations that produce spatially and temporally distinct CSD patterns will be called neuronal elements). The goal of PCA was to determine whether variance of the 16 laminar CSD waveforms (system variance) associated with the averaged IIS or PSB, could actually be explained by a linear combination of a significantly smaller number of fundamental waveforms (principal components). Each principal component was associated with a temporal pattern of component scores (representing the morphology of the fundamental waveform of that component) and a spatial pattern of 16 component loadings (indicating the weighted contribution of the component's fundamental waveform at each of the laminar electrode sites). To reconstruct the spatiotemporal pattern of a given principal component, its scores were multiplied by its loadings. The complete laminar CSD profile of the averaged IIS and PSB was reconstructed by adding the spatiotemporal patterns of the principal components. Each significant principal component was assumed to represent a

107 unique neuronal element participating in the discharge complex. The spatial loading patterns were used to estimate the laminar distribution of extracellular sinks and sources along the vertical axis of the element, and the component scores, the temporal pattern of transmembrane currents unique to the element. Finally, a physical model was used to physiologically interpret the P C A results -~. The model assumed that CSD patterns measured along the axis of the electrode array, perpendicular to the neocortical surface, were primarily produced by elongated apical dendritic processes of pyramidal cells 27. To approximate these apical dendritic generators of the CSD, a simple computer-based model of a vertically oriented current dipole was used. The length of the dipole, and the location, extent, and strength of sinks and sources along its length were variable parameters of the model, with the constraint that the sinks balance the sources. The component loadings, the hypothetical CSD patterns of the individual underlying neuronal elements, were used to estimate the optimal parameters of the physical model by a least squares method. The optimal model parameters were then used to estimate the regions of transmembrane currents given rise to the CSD of each neuronal element.

RESULTS

Fig. 2 shows a typical epileptic excitability cycle recorded from the most superficial electrode in the array. The trace begins with frequent PSB; however, when plotted at this low temporal resolution, the polyspikes during each burst cannot be discerned. The seizure abruptly terminates with a 20 s postictai depression, followed by 100 s of IIS before the onset of another seizure. The morphology of the potential and CSD profiles recorded from the 16 channel electrode array are shown in higher temporal resolution for single events in Fig. 3. The potential profile of the IIS was similar to that previously reported for EIIS 3 and consisted primarily of a positive-negative spike and low amplitude slow wave at the cortical surface that reversed polarity at a depth of 1.2 mm. The potential profile of the PSB was close to that of the IIS, with a similar polarity reversal in the depth. The PSB was characterized by a long duration potential shift with multiple spikes superimposed. CSD profiles of the IIS and the PSB complex showed extracellular current sinks in lamina where large negative potentials were present and sources in the locations of positive potentials. Because CSD is computed as a smoothed second derivative of potential, regions of sinks and sources were more PSB

spatially confined than their potential counterparts and the gradient between regions of opposite polarity was steeper. The remaining analysis was based on CSD wave forms. Both the IIS and PSB morphologies were stable over successive events within animals as shown in the superimposed traces of Fig. 4. For this reason, averaged CSD profiles were accurate and used for subsequent analysis. However, because of the short and variable interburst interval of the PSB, only a 200 ms window around the time marker (arrow; Fig. 4) was undistorted by successive bursts. The increased spatial resolution of CSD highlighted a feature that was present but less apparent in single event potential profiles, that the appearance of a symmetrical pattern of opposite polarity between the cortical surface and depth was only approximate. In fact, the morphology of the IIS and PSB was not uniform across the lamina, suggesting that both complexes were produced by differently timed neuronal elements in different cortical layers. This averaged CSD profile for the IIS (Fig. 5A) and PSB (Fig. 8A) was quite consistent across the 3 animals studied. Turning first to the IIS complex, reconstructions of averaged CSD profiles from only the first two principal

Potential A)

CSD c)

IIS

F

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2:7)', B)

/i

+

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l0 sec Fig. 2. Typical cyclical seizure episode recorded at the cortical surface. The trace begins with approximately 2/s polyspike bursts (PSB) associated with seizures, a postictal period with no spikes for 20 s, and 100 s of interictal spikes (IIS) before the next seizure. Cycles similar to this were regular over periods up to 3 hrs.

250 msec

I

sink

Fig. 3. Laminar recordings of individual IIS and PSB. A: the potential profile of the IIS consisted primarily of a positive-negative spike and low amplitude slow wave at the cortical surface that reversed polarity at a depth of 1.2 mm. B: the potential profile of the PSB was close to that of the IIS, with a similar polarity reversal in the depth. The PSB was characterized by a long duration potential shift with multiple spikes superimposed. C and D: CSD profiles of the single trial IIS and PSB were used for subsequent analysis. Vertical calibration: 1 mV potential, 50 /~A/mm 2 CSD.

1 (t~

components accounted for over 99% of the total variance in all animals (Fig. 5B). The first (Fig. 5C) and second (Fig. 5D) components accounted for 86 and 13% of the variance, respectively. Reconstruction of each principal component was performed by multiplying the spatial patterns of component loadings (shown in the vertical boxes of Fig. 5) with the temporal patterns of component scores (horizontal boxes in Fig. 5). Both the component loadings and scores were also consistent across animals, as shown in the superimposed traces of Fig. 5. The loading patterns of both components were simple, with two regions of opposite polarity in the cortical depth, indicating dipolar laminar distributions of extracellular sinks and sources. Physical modeling indicated a dipolar source for the first component extending over most of the cortical thickness and reversing polarity deep in layer V. The second component was produced by a more superficial dipole that reversed polarity in layers II and III. These loading patterns for the two principal components of the spontaneous IIS were quite similar to those obtained in our previous analysis of the DCR and EI1S complex (Fig. 6). As in these previous experiments, it appeared that the spontaneous IIS was produced by two distinct neuronal elements, one in the infragranular (component 1) and the other in the supragranular layer (component 2). For simplicity, principal components 1 and 2 will be referred to as the infra- and supragranular cells for the remainder of the paper. Fig. 7 shows an enlargement of reconstructed data (top laminar profiles) and scores (bottom traces) for the two principal components, reflecting temporal patterns of transmembrane currents in the infra- and supragranular neuronal elements over the time course of the spontaneous IIS complex. Based on the similarity of PCA results between animals, the data shown here are grand A)

CSD

B)

PCA (components1+2)

A)

Single Traces

C)

Averaged Traces

IIS

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Lamina ~¢r.z . . . . . . . . . .

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Fig. 4. Single trial and averaged CSD patterns. Both the IIS (A)and PSB (B) morphologies were stable over successive events as shown in these superimposed traces. For this reason, averaged CSD profiles (C,D) were considered accurate representations of the single events. Time zero for averaging is indicated with an arrow. Vertical calibration: 50/~A/mm 2.

averages computed across animals. Four latencies of the complex are labeled at peaks of the spike and slow wave sequence (Fig. 7a-d). Prior to any activity in the infragranular cells, the complex began with activation of the supragranular cells consisting of a source in layers I-II and a complimentary sink in layers V-VI (Fig. 7a). This pattern soon reversed in the supragranular cells along with activation of the infragranular cells consisting C)

PCA (component l)

D)

PCA (component2)

Lamina 1

II

+ m V VI

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T -----T I I/,/

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Fig. 5. PCA results for the averaged IIS, superimposed for all animals. Reconstructions (B) of averaged CSD profiles of the IIS complex (A) computed from only the first two principal components accounted for over 99% of the total variance in all animals. The first (C) and second (D) components accounted for 86 and 13% of the variance respectively. Reconstruction of each principal component was performed by multiplying the spatial patterns of component loadings (shown in the vertical boxes) times the temporal patterns of component scores (horizontal boxes). Vertical calibration: 50 t~A/mm 2.

109 of large sink in layers I-V and source in layers V-VI (Fig. 7b). At the beginning of the slow wave (Fig. 7c), the pattern of transmembrane currents reversed again at the supragranular layer but remained of constant polarity but decreasing amplitude in the infragranular layer. The slow wave sequence terminated with a return to baseline in the infragranular cels and a slower decay of currents in the supragranular layers (Fig. 7d). The PSB complex was also closely fit by only two principal components (accounting for 99% of the variance; Fig. 8B). The first and second components accounted for 74 and 25% of the variance, respectively. Physical modeling of the spatial loading patterns indicated that the first component was produced by a dipolar source that reversed in polarity slightly deeper than the first component of the IIS, near the border of layers V and VI. The second component was produced by a more superficial dipole very close in location to that of the second component of the IIS complex, reversing in polarity in layers II and III. Due to the similarity of these component loadings to those of the IIS, components 1 and 2 of the PSB complex will still be referred to as the infra- and supragranular cells, respectively. Although the morphology of component scores differed between the IIS (Fig. 7) and PSB (Fig. 9), the sequence and polarity of activation between the supragranular and infragranular layers were similar. The first spike of the PSB was produced by activity in the supragranular cells consisting

of a source in layers I-II and sink in layers V-VI (Fig. 9a). This pattern quickly reversed polarity in the supragranular cells, accompanied by an extended activation of the infragranular cells consisting of a large sink in layers I-V and a source in layer VI (Fig. 9b). Unlike the IIS, the PSB then displayed one or more small spikes consisting of a rapid polarity reversal in both the supraand infragranular cells (Fig. 9c, 9c'). In the infragranular cells, polyspikes were superimposed on the large steady currents. The PSB complex terminated in a manner similar to the IIS, with a gradual return to baseline of transmembrane currents in the infragranular cells, and a layer l-II source/layer V-VI sink in the supragranular cells that took longer to return to baseline. However, observations of averaged activity at latencies greater than 200 ms must be considered inaccurate because of temporal overlap of successive PSB. Although PCA results indicate that the averaged llS and PSB complexes were produced by similar neuronal elements in the supra- and infragranular layers, it was unclear whether information was being lost in the averaging process. Specifically, we wished to determine if the component loadings determined from averaged data, representing the infra- and supragranular CSD distribu-

I

II + III IV v

Infragranular Reconstruction DCR

EIIS

IIS VI

I

11 + 111 IV

Component 1 Supragranular Reconstruction

V

Lamina I

III IV Component 2

V VI

Fig. 6. Loading patterns for the two principal c o m p o n e n t s of the s p o n t a n e o u s IIS compared to those obtained previously for the D C R and EIIS complex. As in these previous experiments, it appears that the s p o n t a n e o u s IIS was produced by two distinct neuronal elements, one in the infragranular (component 1) and the other in the supragranular layer (component 2).

Inffagranular Scores

J

~

Supragranular Scores a~ b c

d

250 msec

fig. 7. Enlarged data reconstructions (laminar profiles at top) and scores (lower 2 traces) for the two principal c o m p o n e n t s of the IIS. Based on the similarity of P C A results between animals, the data shown here are grand averages c o m p u t e d across animals. Four latencies of the complex are labeled at peaks of the spike and slow wave sequence (a-d), Vertical calibrations is omitted to emphasize the fact that these are statistical quantities and not measured currents.

1 IU A)

CSD

B)

PCA (components 1.2)

C)

PCA

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PCA {component 2)

( c o m p o n e n t 1)

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250 msec Fig. 8. PCA results for the averaged PSB, superimposed for all animals. 99% of the variance was accounted for by only two principal components. The first and second components accounted for 74 and 25% of the variance respectively. The first component was produced by a dipolar source that reversed polarity slightly deeper than the first component of the IIS, near the border of layer V and VI. The second component was produced by a more superficial dipole very close in location to that of the second component of the IIS complex, reversing polarity in layers If and III. Note that the polarity reversal of loadings for one of the animals is shifted approximately 100 #m higher than the other two, probably indicating a slight change in electrode position during this recording of the PSB. Vertical calibration: 50/~A/mm 2.

tions in a g i v e n animal, could be applied to reconstruc-

results of the averaged IIS and PSB c o m p l e x e s . Succes-

tion of large s e g m e n t s of raw ictal, postictal, and interictal data in the s a m e animal. For each anmal, a representative loading pattern was c o m p u t e d for the first

sive 10 sec blocks of raw C S D data were a n a l y z e d using

and s e c o n d principal c o m p o n e n t s from the c o m b i n e d

I II

+

III IV Infragranular Reconstruction

these loading patterns to c o m p u t e scores for thwe two c o m p o n e n t s that a c c o u n t e d for a m a x i m u m a m o u n t of the variance. In all blocks, it was possible to account for 9 0 - 9 5 % of the raw C S D variance using the representative loadings and c o m p u r t e d scores. Fig. 10 shows the score patterns c o m p u t e d for 90 s of raw data, covering o n e c o m p l e t e seizure cycle. T h e plot begins just at the termination of a prior seizure, a period that was charac-

V VI

2.

(Infra) I

n + llI IV Supragranular Reconstruction

4)

5)

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Supragranular Scores

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250 msec

Fig. 9. Enlarged data reconstructions and scores for the two principal components of the PSB. Five latencies of the complex are labeled at peaks of the polyspike and slow wave sequence (a-d). Observations of averaged activity at latencies greater than 200 ms must be considered inaccurate because of temporal overlap of successive polyspike bursts. Vertical calibration is omitted to emphasize the fact that these are statistical quantities and not measured currents.

Fig. 10. Score patterns computed for 90 s of raw data, covering one complete seizure cycle in 10-s blocks. The plot begins just at the termination of a prior seizure, with rhythmic oscillation at approximately 1.6 Hz (block 1). This progressed to larval (blocks 2 and 3) and fully developed (block 4) IfSs. PSBs accompanied seizure onset (blocks 5-8) with the appearance of slow interburst afterwaves in the supragranular scores. Seizures terminated abruptly with postictal suppression followed by resumption of slow oscillations (block 9). Vertical calibration is omitted to emphasize the: fact that these are statistical quantities and not measured currents.

111

c)

c

IIS

D:n

EIIS b

"- . . . . .

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lnfragranular PSB

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Fig. 11. Phase plots applied to PCA scores of the averaged IIS and PSB complex, as well as to the EIIS complex from a previous study. Infragranular scores are plotted on the horizontal axis and supragranular scores are plotted on the vertical axis. The coordinates are shifted gradually along a diagonal from the upper left to lower right with time, Previously identified latencies of the IIS (a-d) and PSB (a-d) complexes are labelled on the phase diagrams to assist in understanding their relationship to more familiar time series plots. Similar latencies have been identified for the EIIS.

J

f terized by a rhythmic osicllation at a p p r o x i m a t e l y 1.6 Hz that was largest in the scores for the supragranular cells but was also present in the scores of the infragranular cells (Fig. 10, block 1). The oscillation in the infragranular cells increased in amplitude and began to show small spikes that a p p e a r e d to be incompletely d e v e l o p e d IIS, occurring at a regular phase of the oscillation (Fig. 10, block 2). W h e n completely d e v e l o p e d IIS a p p e a r e d later (Fig. 10, block 3), they occurred at the same phase of the oscillation. The a p p e a r a n c e of the scores for the raw IIS (Fig. 10, block 3 and 4) was quite similar to that o b t a i n e d for the averaged IIS complex (Fig. 7), and consisted of an e x t e n d e d m o n o p h a s i c spike in the infragranular cells and a brief biphasic spike with subsequent slow wave in the supragranular cells. This similarity also held for the raw scores c o m p u t e d during PSB (Fig. 10, blocks 6, 7 and 8). Seizures typically terminated suddenly (Fig. 10, block 9), and were immediately followed by the onset of the rhythmic oscillation, apparent first in the supragranular cells and later in the infragranular cells. Fig. 11 shows a different m e t h o d of plotting the scores for the first and second principal c o m p o n e n t s that emphasizes their phase relationships. In this figure, the phase plots are applied to scores of the averaged IIS and PSB complex, as well as to the E I I S complex from a

Infragranular

f Fig. 12. Phase diagrams computed from PCA scores of one complete seizure episode in each animal. Diagrams for the 3 animals (C-D) begin with a postictal period (marked with arrows) where there was no apparent epileptic spiking, followcd by a series of IIS that increase in frequency as they lead into a seizure and subsequent postictal period. Phase plots of the averaged IIS (A) and PSB (B) complex are also shown for comparison to those computed for the raw component scores of the interictal and ictal periods. Of particular interest in these plots are the postictal periods. Here, the phase plots in all animals appear as a slow spiral, indicating that the slow postictal oscillation noted in time series plots of raw scores is systematically out of phase between the supragranular and infragranular layers.

previous study 3. Scores for the first c o m p o n e n t , representing activity of the infragranular cells, are plotted on the horizontal axis while the second c o m p o n e n t scores, or activity of the supragranular cells, are plotted on the vertical axis. The coordinates are shifted gradually along a diagonal from the u p p e r left to lower right with time, Previously identified latencies of the iIS (Fig. 7 a - d ) and

112 PSB (Fig. 9a-d) complexes have been labelled on the phase diagrams to assist in understanding their relationship to more familiar time series plots. The IIS complex began with an upward deflection along the vertical axis representing initial activation of the supragranular cells (Fig. lla), and was quickly followed by a left deflection along the horizontal axis representing the rapid activation of infragranular cells. Both components peaked (Fig. 1lb) and returned to baseline, forming a long loop with two humps (Fig. llc,d) representing the slow waves of the supragranular cells. The phase diagram for the I1S was similar to that of the EllS derived from previous experiments 3, differing mainly by the presence of a brief stimulus artifact in the EllS. Fig. 11 also depicts the characteristic phase plot of the PBS. This plot reflects the more rapid initial activation of the supragranular cells (Fig. l l a ) but shows a looping pattern to the lower left similar to the IIS as both components peak (Fig. llb). The return to baseline is interrupted by a second smaller loop (Fig. llc,c') representing a large secondary spike. Phase diagrams of one complete seizure episode in each animal are depicted in Fig. 12. Each digram begins with a postictal period (marked with arrows) where there was-no apparent epileptic spiking, followed by a series of IIS that increase in frequency as they lead into a seizure and subsequent postictal period. Phase plots of the averaged IIS (Fig. 12A) and PSB (Fig. 12B) complex were similar to those computed for the raw component scores of the interictal and ictal periods in all animals. Of particular interest in these plots are the postictal periods. Here, the phase plots in all animals appear as a slow spiral, indicating that the slow postictal oscillation noted in time series plots of raw scores is systematically out of phase between the supragranular and infragranular layers. DISCUSSION These data suggest a simple electrophysiological model in which cyclical seizure episodes are produced by similar laminar interactions between two basic populations of pyramidal neurons, one in the infragranular layer with apical dendrites extending near the surface of the cortex, and another shorter population in the supragranular layers. The sequence and pattern of interactions recorded here are similar to those previously recorded for the EIIS and DCR complex, suggesting that the same neuronal circuits subserve activity ranging from normal evoked responses to focal seizures. However, some of the simplicity in our model may have resulted from a loss of detail attributed to the methods. These methodological limitations are fully described in a previous paper 3 and will only be reviewed

here. Extracellular potentials were recorded with relatively large diameter electrodes compared to microelectrodes used in single cell recording. The ,esulting potentials are therefore the sum of many thousands of neurons differing in shape, location, and laminer extent. Much of the fine spatial and temporal activity in subpopulations of neurons is lost in the population sum. Only highly synchronized postsynaptic potentials produced by dendritic process extending along the axial length of the recording array can contribute substantially to the summed potentials and subsequent CSD analysis. Other problems are associated specifically with CSD analysis. Because CSD is computed as a spatial derivative of potential, it has a tendency to emphasize noise in the data and must therefore be spatially filtered 3'27. Spatial smoothing limits the spatial resolution of the measure to potentials that extend along much of the cortical thickness. Potentials from very short cells with steeper spatial gradients would be greatly attenuated or lost. CSD is also far less sensitive to postsynaptic potentials of cell somas compared to extended dendritic processed 27. Because most inhibitory synapses are at or near pyramidal cell soma, this has the effect of making CSD selectively more sensitive to excitation over inhibition 27. However, this does not rule out large inhibitory potentials at the soma or hyperpolarizing potentials produced synapticall or nonsynaptically on the dendrites. Other methodological limitations exist with the use of PCA to interpret CSD waveforms. The basic assumption underlying PCA in this context is that there be temporally distihct neuronal elements of fixed laminar location, with only transmembrane currents that vary over time. While this requirement is physiologically realistic, propagating potentials would not be well represented by PCA. Furthermore, PCA in this application cannot distinguish between subpopulations of cells that have similar spatial distributions of transmembrane currents but are active at different phases of the epileptic process. These are subsumed under a single principal component and regarded as a single neuronal element. Finaly, PCA is a purely statistical procedure that has no basis in physiological reality 3'~7"46. This basis must be established with physical modeling. Our physical model was that of a simple, vertically oriented current dipole, fit to the spatial patterns of component loadings. This was sufficient for approximating the location and extent of underlying neuronal elements. More detailed information about the gross electrophysiological anatomy of contributing cells might have been obtainable from a model that accurately represented soma and dendritic membranes. These methodological limitations are therefore expected to yield an approximate picture of the neuronal

113 complexity underlying epileptiform discharge. Yet, the simple structure of the P C A loadings (varimax rotation 23 had almost no effect on the loading patterns), the constancy of P C A results between successive epileptic events within a given animal and between animals, and the similarity of these results to previous analyses of the D C R and EIIS, suggest that these data reflect fundamental laminar circuits active during all phases of cyclical seizure episodes.

The spontaneous IIS complex Fig. 13 depicts the sequential activation of supra- and infragranular neuronal elements during the spontaneous IIS complex. The neuronal elements are shown in cartoon form as a short pyramidal cell in the supragranular layer and a longer pyramidal cell in the infragranular layer with apical dendrites extending to the cortical surface. The complex begins with an extracellular sink proximal to the soma of the supragranular cells (Fig. 13a) followed by a sink on the distal apical dendrites of these and the infragranular cells (Fig. 13b). This initial discharge sequence is the same as that previously recorded for the D C R and EIIS 3 and has been identified by others as the most characteristic feature of IIS T M 13,3o,33.35. Intracellular and unit recordings have revealed strong depolarizing currents during this early phase of the IIS complex, suggesting that the sinks recorded here repre-

a

b

c

d

/ ,,a

.At..,

j

= sink

~ = source

Fig. 13. Sequential activation of supra- and infragranular neuronal elements during the spontaneous IIS complex. The neuronal elements are shown in cartoon form as a short pyramidal cell in the supragranular layer and a longer pyramidal cell in the infragranular layer with apical dendrites extending to the cortical surface. The complex begins with an extracellular sink proximal to the soma of the supragranular cells (a) followed by a sink on the distal apical dendrites of these and the infragranular cells (b), and ends with a source at the distal apical dendrites of the supragranular cells and proximal source on the infragranular cells (c,d).

sent excitatory postsynaptic potentials (EPSP) ~A2"25. In both the EIIS and IIS, the proximal dendritic region of the supragranular cells is the first to depolarize and appears to act as a trigger zone for initiating IIS. It is important that this finding holds for spontaneous IIS, where no cortical electrical stimulus was used. The trigger zone is therefore not simply a direct result of the evoking stimulus. However, the absence of an evoking stimulus may explain the smaller amplitude and longer duration of the initial depolarizing wave of the IIS when compared to the EIIS 3 (Fig. I1B). Through studying the developing penicillin focus in visual cortex, Ebersole and Chatt ~ have proposed that stellate cells in layer 1V may serve as the initial excitatory trigger for IIS, followed by amplifying activation of pyramidal cells near the border of layers lit/IV, subsequent hypersynchronized depolarization of other supragranular pyramidal cells, and finally, activation of large pyramidal cells in the infragranular layer. Our findings are in essential agreement with this proposed sequence of laminar interaction with the exception of a trigger zone in layer IV. The initiating discharge of IIS in the present study had a spatial CSD pattern that was well fit by a physical model of a vertically oriented current dipole. This is representative of pyramidal cells with extended apical dendrites, and not stellate cells with their approximately radially symmetric dendritic processes and closed field electrical properties 24. In the same light, these data do not rule out the contribution of stellate cells since their closed field configuration should make them difficult to resolve in one dimensional CSD analysis 27. Our study suggests that laminar interactions recorded previously, during the developing penicillin focus, may also be discriminated and studied in the fully developed I1S complex if PCA is used to separate spatially and temporally overlapping activation of discrete laminar neuronal populations. The later phases of the IIS have typically been associated with hyperpolarizing intracellular currents ~' ~4,1~,2~,~2. These appear in the supragranular cells as a source at the distal apical dendrites (Fig. 13c). Due to the distal dendritic location, the source might reflect nonsynaptic hyperpolarizing currents, possibly Ca2+-activated K + currents 2'~6 similar to those hypothesized for the E l l s 3. With the CSD method, it is impossible to determine whether the [ate configuration of distal sink and proximal source in the infragranular cells is due to continued depolarization of the apical dendrites or beginning hyperpolarization proximal to the soma, since both events would create the same spatial pattern of transmembrane currents (Fig. 13c). Whereas a mixture of hyperpolarizing and depolarizing potentials is possible, active inhibition at the soma should be difficult to resolve

114 in the C S D 27 and is alo unlikely given the GABA blocking properties of penicillin 9"22. For this reason, the slow wave measured here probably reflects a gradual and prolonged depolarization of distal apical dendrites of the infragranular cells. Thus, in contrast to the supragranular cells, activation of the infragranular neurons is monophasic, beginning with a sharp depolarization lasting 50-100 ms, a fast repolarization phase ending at a depolarized level, followed by a subsequent slow phase of repolarization lasting several hundred milliseconds before returning to baseline. This temporal pattern is remarkably similar to paroxysmal depolarization shifts (PDS) recorded intracellularly from in vitro neocortical slices perfused with penicillin 1~ and extracellularly in cat neocortex during penicillin spiking eS. The present data suggest that neurons primarily responsible for prolonged PDS in vivo may be pyramidal cells in the infragranular layer. This conclusion is supported by recent intracellular recordings of normal rodent somatosensory cortex, revealing a population of large pyramidal cells in layer V~ displaying readily evoked bursts of spikes 1"7"1s. Cells capable of intrinsic bursting are thought to be primarily responsible for PDSs in epileptic cortex and prerequisite for the development of IIS 42. Another feature of the slow wave complex in the infragranular cells is the lack of any late hyperpolarizing currents when compared to previous recorded responses in these cells during the EIIS 3. A similar loss of hyperpolarizing currents in cells displaying prominent PDSs has been recorded intracellularly in neocortical neurons during the transition from IIS to tonic seizure discharge 26. In the present data, the loss of hyperpolarizing currents may reflect increased excitability of the infragranular neurons even during interictal phases of cyclic seizure episodes, compared to the EIIS in cortex with smaller concentrations of intracortical penicillin.

The spontaneous PSB complex Polyspike bursts during seizures began in the same way as the IIS, with a proximal sink in the supragranular cells (Fig. 14a) followed by distal sinks in these cells and in the infragranular cells (Fig. 14b). As in the IIS, this sequence of sinks probably also reflects initial depolarizing currents in both cortical layers. Because of the repetitive nature of PSB and the short interburst interval, the initiating event is ambiguous. However, if it is taken as the first sharp spike after a prolonged slow wave interval, then the proximal dendritic region of the supragranular cells once again may be considered the trigger zone of each paroxysm. This relationship between the supra- and infragranular layers holds for subsequent spikes in the burst, starting with proximal depolarization of the supragranular cells (Fig. 14c) followed by distal depolarization

of these cells and the infragranular cells (Fig. 14c'). Aside from the presence of polyspikes, the PSB complex is quite similar to the IIS. Fast depolarization sequences in the supragranular cells associated with polyspikes are followed by subsequent prolonged hyperpolarization at the distal dendrites (Fig. 14d). The infragranular cells remain depolarized for the duration of the PSB complex, once again going through a rapid then slow repolarization. The onset of repolarization is delayed by the presence of polyspikes. The polyspikes themselves appear to be superimposed on a large continuous depolarization in the infragranular cells. These data suggest that the PSB is produced by neuronal circuitry very similar to that underlying the IIS. During both the IIS and PSB, infragranular cells appear to be primarily responsible for prolonged depolarization. Once again, it is the activity of the infragranular cells that is in best agreement with intracellular recordings of PDS. With the increase in excitability associated with transition to seizures, depolarization associated with PDS increases in duration and frequently displays a superimposed rhythmic discharge 26. This morphology is similar to that recorded from the infragranular cells in the present study. However, it is unclear from the grand averaged data what changes have taken place to cause the transformation from IIS to PSB. The amplitude

a

b

c'

c

!I! ~ = sink

J{ _J k,

j = source

Fig. 14. Sequential activation of supra- and infragranular neuronal elements during the spontaneous PSB complex, The PSB began in the same way as the IIS, with a proximal sink in the supragranular cells (a) followed by distal sinks in these cells and the infragranular cells (b). Subsequent spikes in the burst start with proximal depolarization of the supragranular cells (c') followed by distal depolarization of these cells and the infragranular ceils (c). Fast depolarization sequences in the supragranular cells associated with polyspikes are followed by subsequent prolonged hyperpolarization at the distal dendrites (d). The infragranular cells remain depolarized for the duration of the PSB complex, going through a rapid then slow repolarization.

115 of depolarizing and hyperpolarizing components appear quite similar between the complexes. The fundamental sequence of laminar interactions remains constant between the cortical layers. This would suggest that another influence is involved in the transition to seizure that was not recorded here. However, there is a clue in the raw data that is not apparent in the averaged data. Close examination of unaveraged activity in the infragranular cells during the transition to seizure (Fig. 10, blocks 4-8, upper trace) reveals no remarkable change in the basic monophasic pattern of currents between the interictal and ictal state. Yet, this is not true for activity in the supragranular cells (Fig. 10, blocks 4-8, lower trace). The most noticeable change from the interictal to ictal state is seen in the stow, hyperpolarizing wave following each IIS or PSB. The slow wave is relatively small and variable during IIS (fig. 10, block 4) and grows in amplitude with stereotyped morphology during PSB. Indeed, during seizures, the slow wave appears to pace the interburst interval. We have suggested that the supragranular slow wave is probably produced by Ca 2+activated K + currents. This possibility is interesting in light of the present observation because it suggests that K + conductance increases during the ictal period and that this increase may be due to changes in extracellular C a 2 + . Support for this hypothesis is found in studies of the slow time course of extracellular Ca 2+ during cyclical spike driving of the penicillin focus in neocortex 21 and in hippocampus 19. During trains of evoked IIS, levels of Ca 2+ fall steadily to a minimum where the train abruptly ends. In the quiet period following each train of spikes, Ca 2+ rises rapidly and then slowly falls again, heralding the onset of the next train. It is possible that the slow changes in Ca 2÷ associated with excitability cycles of driven IIS are similar to those associated with PSB onset in the present experiment and the appearance of hyperpolarizing slow waves in the supragranular cells. Postictal oscillations At almost no time during the 3-4 h that each animal was studied did activity in the epileptic focus appear random. Reconstructions of hypothetical activity of the supra- and infragranular cells using raw data extending over many seconds and representative loading patterns computed from averaged events, highlighted systematic laminar interactions during all phases of cyclical seizure episodes. Laminar interactions during IIS and PSB in the raw records were similar to those computed for the average, with some variability (Figs. 10, 12). Yet, even during the postictal period when no paroxysmal spikes were present, the supra- and infragranular layers appeared to oscillate out of phase at a frequency of approximately 1.6 Hz in all animals (Fig. 10, blocks 1 and

9; and Fig. 12A-C, arrows). This pattern was similar to but lower in frequency than oscillations reported in rabbit neocortex 3~'35. The postictal oscillation was closely connected to the later generation of interictai spiking. Spiking first appeared as 'larval I1S' and occurred at a regular phase of the background oscillation (Fig. 10A, blocks 2 and 3). When full IlS appeared, they occurred at the same phase of the slow oscillation wave (Fig. 10, block 3). This is similar to previously reported postictal oscillations recorded intracellularly that are capable of occasionally, but not always, triggering PDS 2~'. These observations suggest that slow oscillating interactions between the supra- and infragranular layers may be a primary electrophysiological event underlying the cyclical seizure phenomena. The mechanism of postictal oscillations is unclear. They are consistently much larger in the supragranular cells and occur later in the infragranular cells primarily as a synchronized larval spike complex. The fact that they appear many seconds before noticeable I1S in either the supra- or infragranular cells suggests that their presence, amplitude, and timing is independent of the spike generating mechanism. However, the spike generating mechanism is not necessarily independent of the slow oscillations since both larval and fully developed IIS occur at a select phase, at least in the early reappearance of I1S. It is conceivable that the oscillations reflect a synchronizing influence within the epileptic population. As more neurons are recruited into synchrony, a spike generating threshold is reached in certain subpopulations, resulting in larval IIS. As the process of recruitment continues, the threshold for fully developed IIS is eventually reached. Therefore, slow oscillations may indicate a second, more fundamental, involvement of supragranular cells in pathological synchronization of the epileptic aggregate. It was indicated earlier that supragranular cells serve as a trigger zone, initiating both IIS and PSB complexes. Slow oscillations that appear first and largest in this same lamina suggest that the trigger may be initially paced by yet another slow membrane process. The fact that slow oscillations appear in the virtual absence of activity in the infragranular projection cells may indicate that the underlying neural network responsible for these waves does not require feedback from the infragranular layers or from thalamocortical interactions and is instead self-contained within cells of the supragranular layer. One possible explanation is that the slow oscillation is produced by excitatory feedback within the supragranular neural network, feedback which is usually damped by the presence of strong inhibition that has been compromised by the action of penicillin. Whatever the underlying mechanism, the slow oscillations observed

11(~ here must reflect a fundamental frequency at which the epileptic n e u r o n a l aggregate can be synchronously excited during the interictal period. This frequency is very close to the highest frequency at which I1Ss are typically observed, and is also quite close to the optimum frequency for reliably evoking IISs in the penicillin Of particular interest is that this slow

fOCUS37"38'41

stimulus frequency has been determined to be ideal for the generation of cyclical spike driving. Although no exogenous stimulation was used in the present studies,

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the slow 1.6 Hz oscillations observed here may represent a form of e n d o g e n o u s stimulation of the penicillin focus capable of establishing cyclical seizure episodes in this preparation.

Acknowledgements. This research was supported by USPHS Grants 1-R01-NS22575, NSF Grant BNS-86-57764, Whitaker Foundation Grant $880620, and in p.art by the Fonds zur F6rderung der wissenschaftlichen Forschung Osterreichs (Erwin Schr6dinger Stipendium J246M and J334MED).

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