Laminar interactions during neocortical epileptogenesis

Brain Research, 298 (1984) 253-271 Elsevier

253

Laminar Interactions During Neocortical Epileptogenesis JOHN S. EBERSOLE and ALLEN B. CHAqT

Epilepsy Center, Neurology Service, Veterans Administration Medical Center, West Haven CT06516 an.dDepartment of Neurology, Yale University School of Medicine, New Haven CT06510 (U.S.A.) (Accepted September 6th, 1983)

Key words: epilepsy - - experimental - - neocortex - - focal - - laminar sensitivity - - penicillin - - cat

Interactions among laminar subpopulations of cat striate cortical neurons were assessed during the evolution of discrete and temporary epileptic foci, which were induced by selective microinjection of penicillin into different cortical layers. Field potentials and multiunit cellular discharges, evoked by selective visual field stimulation, were recorded simultaneously from 3 layers by multibarreled glass microelectrodes. Laminar response profiles at distinct stages of epileptogenesis were characterized for loci induced in superficial pyramidal, middle stellate, and deep pyramidal layers. Layer 4 was verified to be the most susceptible to epileptogenesis. Penicillin's action within this stellate layer appeared to be sufficient for epileptogenesis and was supportative of, if not necessary for, the development of foci originating in pyramidal cell layers. These findings could not be fully appreciated by monitoring only spontaneous interictal spike potentials. Of the two types of neuronal discharge routinely observed, early latency bursting was principally a characteristic of layer 4 stellate populations, whereas longer-latency bursts comparable to paroxysmal depolarization shifts were recorded equally well from both stellate and pyramidal layers. Epileptiform alterations in both field potential and unit responses were quickly evident in cortical laminae having known anatomic connections with the layer where the focus was induced: e.g. in layers 2-3 with layer 4 loci, in layers 5-6 with layers 2-3 loci, and in layer 4 with layers 5-6 loci. The spread of epileptogenesis was slower between laminae where pathways are purported to be less well developed, and appeared to be principally dependent upon the diffusion of penicillin. INTRODUCTION Many advances in characterizing the cellular and field potential correlates of epilepsy have come from the in vivo study of acute focal epilepsy in animal models (for review, see refs. 1, 13, 42, 48, 53). Much of this e x p e r i m e n t a t i o n utilized the topical application of convulsants, the principal one being penicillin. All these studies d e p e n d e d upon the gradual diffusion of drug into the cortex, and most awaited the resultant spontaneous interictal spiking to begin analysis. U n d e r s t a n d a b l y , these relatively large and fully-evolved foci yielded recordings showing a rather uniform involvement of neurons in the pathologic, paroxysmal depolarization shift (PDS) process 34. Perhaps due in part to this assumed h o m o g e n e i t y within the epileptic aggregate, most of the past investigation has been given to characterizing the PDS and postulating its origin. P r o p o r t i o n a t e l y little attention

has been directed at determining where penicillin acts within the cortex. A r e its effects uniform across cortical layers or is there some hierarchy in epileptogenic susceptibility among neuronal subpopulations? D o certain neurons or groups of neurons have specific roles in epileptogenesis and do these result in standard patterns of recruitment within and between cortical columns? Spatial, as well as functional, analyses of cortical epileptic loci are few in number. T o p o g r a p h i c a l examination of spontaneous interictal potentials from the cortical surface has characterized systematic changes in the amplitude and polarity of epileptic potentials when recording from regions progressively m o r e distant from the center of the focus 23. L a m i n a r analyses of interictal spike potentials and D C shifts during tonic seizure discharges have provided the same information for varying depths within the epileptic focus19. 24. The finding of synchronous hyperpo-

Correspondence: J. S. Ebersole, Department of Neurology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, U.S.A. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.

254 larizations in cells outside of the focus, rather than the massive depolarization of the PDS, has led additionally to the theory of an 'inhibitory surround'S~ 46,47. Ectopic spike generation was shown to delineate the central, interictally active portion of a focus from the ictally recruitable surround 20. In nearly all these endeavors fully-evolved foci, which were employed for their stability, may have obscured differences in responsivity of constituent neuronal populations that existed at earlier stages of development. An appreciation for the fact that the various cortical layers have a differential susceptibility to penicillin-induced epileptogenesis has recently been gained. Initial studies reported that the superficial layers were the most sensitive and probably contained elements essential for development of focal epilepsy 39. Such experiments using topical convulsant necessarily biased any effects in favor of these upper layers, however, since they were affected first and with a higher drug concentration. Recent independent observations using intracortical microinjection 3,4,12 or microiontophoresis 27.30,31 of penicillin have concluded that the middle cortical layers are the most susceptible to epileptogenesis. More specifically, in cat visual cortex we have found the responses from layer 4 to be most effectively and rapidly altered by penicillin 3,4,12. These studies have set the stage for a systematic investigation of the functional relationships among laminar neuronal subpopulations during epileptogenesis. In an attempt at this clarification, we have recorded the evolution of temporary epileptic loci induced by microinjection of penicillin into the different cortical layers. Multiple simultaneous perspectives were gained by recording local field potentials and multi-unit extracellular activity from muitibarreled microelectrodes with longitudinal tip separations. Although such recordings cannot offer detailed information on membrane physiology, they do provide useful information about the aggregate activity of neuronal populations, including those of smaller stellate neurons that are difficult to record intracellularly. The present investigation will demonstrate that epileptiform responses recorded from different layers within a cortical column evolve in stereotyped patterns depending upon the level at which penicillin is introduced. By correlating these spatiotemporal pat-

terns of epileptogenesis with known cortical morphology, we will speculate as to the roles of laminar neuronal subpopulations in the onset and spread of epileptiform abnormalities within the cortex. Preliminary results of this work have been presented elsewhere14-16. MATERIALS AND METHODS Data were obtained from 18 adult cats. Anesthesia, surgical preparation, ophthalmologic corrections, and animal maintainance procedures were the same as those previously detailed 13. Three- or 4-barrel micropipet arrays with fixed tip separations were fabricated from single- and double-barrel glass capillary tubing. After being drawn and beveled to an outside tip diameter of 5-10 pm, single-barrel pipets were angled approximately 30° at the tip with a microforge. A double-barrel pipet of twice the diameter formed the straight core to which one or two singlebarrel pipets were separately cemented under microscopic control with longitudinal tip separations of 400-800 pm. 4 M sodium chloride solution was placed into pipets at each level for extracellular multiunit and field potential recording; sodium penicillin solution (35 mM) was placed into the second barrel of the twin pipet for microinjection. Electrode configurations with the penicillin pipet deep, middle, or superficial in the array were employed (see Fig. 1). As small as picoliter volumes of penicillin were ejected from the micropipet by an automated pressure injection system 35. Quantification procedures for approximating injection volumes have been presented previously 13. To minimize tissue distortion, small injections (100-150 pl) were made recurrently with each stimulus cycle until a target volume was reached or until a certain level of response alteration was achieved. Multi-barrel micropipets were positioned under microscopic visual control to enter area 17 of the visual cortex. Penetration just medial to the area 17-18 border along the topographic representation of the visual vertical meridian (stereotaxic coordinates: posterior 0-5 ram, lateral 2 ram) afforded the best opportunity for penetration perpendicularly into a single cortical column. Mineral oil was poured into the plastic chamber over the exposed cortex and warm dental wax was layered over this to produce a hydraulically closed recording system, which mini-

255

Normal 0_

i

Epileptiform

-

V~

--

\

! 700

2OO0 ;s

i

ON

_j-Visual SC ~OFF Assoc. Cx Fig. 1. A schematic representation of the cytoarchitectonics of cat striate cortex using information developed by Lund et al. 32and Gilbert and Wiesel2L Predominant cell types together with their principal afferent and efferent connections are illustrated. The configuration of a typical 4-barrel drug injection/recording electrode spanning the layers is shown on the left. To the right are representative field-potentialswhich were recorded every 200 ~m during the withdrawal of an electrode array from striate cortex. These were evoked by punctate photic stimuli before (Normal) and after (Epileptiform) the microinjection of approximately 1 nl of 35 mM penicillin into layer 4. Note that the largest primary potentials are seen in layer 4 before penicllin injection and the largest enhanced primary (EPRs) and late responses (LRs) are also seen in layer 4 after penicillin injection (arrowheads). A reversal of polarity of the LR potential in the deeper cortical layers (positive; upward-going) and an absence of EPRs in both superficial and deep layers are shown. Calibrations: 50 /~V, 110 ms sweep time. LGN

LGN

mized cortical pulsations. A 1° square visual stimulus, one log unit in brightness above a b a c k g r o u n d illumination of approximately 10 Cd/m 2 was used to e v o k e regional field potentials. Stimuli were p r e s e n t e d recurrently - - a 2-s ON followed by a 2-s O F F . A n interspaced 10-s period without stimulation was e m p l o y e d when monitoring spontaneous activity. The center of the c o m b i n e d receptive fields of the local neuronal p o p u l a t i o n was d e t e r m i n e d by searching with each eye for the visual field location at which the O N - O F F stimulus e v o k e d the largest p r i m a r y potential for a given recording level. The less p r e f e r r e d eye was occluded and control responses were o b t a i n e d by stimulating the central area of the receptive field. Recording sites were d e t e r m i n e d by a combination of depth m e a s u r e m e n t and response criteria that have proven accurate when c o m p a r e d with iontopho-

retic and autoradiographic marking techniques in c o m p a n i o n studies3. 4. The latter were not e m p l o y e d in the present investigation because the i m m e d i a t e removal of tissue, necessary to minimize diffusion and provide accurate localization, would preclude recording the full evolution of the focus and its concommittant laminar interactions. Physiologic verification of electrode depth was important in adjusting for variable cortical compression (dimpling) which can occur during electrode penetration. L a y e r 4 can be accurately defined as the region where p r i m a r y latencye v o k e d potentials are of greatest amplitude 13,17. With the center of this tissue band located, the laminar positions of the other recording electrodes in the fixed array could be d e t e r m i n e d based u p o n their physical separations (see Fig. 1). Regional activity from each recording site was amplified and divided into two frequency bands by se-

256 lective filtering. Laminar field potentials were processed by low pass filtering (< 100 Hz); multi-cellular unit spikes were isolated by high pass filtering (> 200 Hz). Raw data were recorded on magnetic tape for later off-line analysis where they could be transcribed onto real time hard copy with a Mingograf jet ink writer, or digitized in 250 ms sweeps and averaged in sums of 4-8 by a PDP/8 computer. Laminar data will be presented and discussed in terms of 3 levels: pyramidal layers 2-3, stellate layer 4, and pyramidal layers 5-6, which correspond in area 17 to cortical depths of approximately 200-700, 700-1300 a n d 1300-2000 #m, respectively. The rational for this laminar division into cortical bands of 500-700 #m is based upon differing predominant cell types, neuronal receptive field characteristics, and stages of input processing. These bands are well within the depth resolution of the techniques used. Furthermore, laminar analysis of field-potentials recorded every 100 um during electrode withdrawal revealed no additional potentials either before or during focus evolution that were not monitored by our standard recording configuration (see Fig. 1, every 200#m shown). RESULTS

Epileptic foci induced in layer 4 Field potentials Microinjection of approximately 1 nl of sodium penicillin solution (0.02 units (I.U.)) was accompanied by a rapid and consistent sequence of local response alterations in all 12 experiments where it was introduced into layer 4. As illustrated in Fig. 2, the earliest change was an enhancement of the primary evoked response (EPR), which could be appreciated within 10-20 s of starting the injection. This amplitude increase was limited to 50-200%, and there was no other significant change in response configuration or latency. Reliably within 30 s a new, longer-latency negative potential developed (LR, the 'late response'). With time this potential customarily showed a great increase in amplitude and a gradual shortening of latency until a distinct EPR component was obliterated. The rather stereotyped, large negative potential that remained was the equivalent of a classic interictal spike potential (IIS). By this stage, similar potentials were being generated sponta-

neously as well. Under these experimental conditions, the time required for full development within the cortical layer was approximately 2-3 min. Separate EPR and LR components were again discernible during the decline of the focus, during which time the LR waned in amplitude, increased in latency, and finally disappeared, leaving behind the EPR. Separation of these two components of an evoked IIS was possible even at the height of a focus by increasing the stimulus rate or decreasing the stimulus intensity. Typical of an epileptiform potential with a longer refractory period, the late response was also particularly sensitive to such experimental manipulations, as has been shown previously ]3,1s,34. The entire evolution took 14-30 min to achieve a return to control levels. This response sequence has been reviewed in detail in previous reports from our laboratory 1~,13,1s. In the present experiments, simultaneous recordings were also made from both supra- and infragranular layers during this evolution. It was the response from superficial pyramidal layers that most closely followed the sequence of alterations induced in layer 4. Immediately following penicillin injection, the early EPR of layer 4 was associated with some increase in the normally small and somewhat longer-latency response of layers 2-3. In a dramatic fashion, however, any LR in layer 4 was associated with a large layer 2-3 potential (e.g. see 30 s response in Fig. 2). During early or late stages in the evolution of a layer 4 focus, when the LR latencies were longer, it could be appreciated that the large amplitude response of layers 2-3 occurred in synchrony with the layer 4 LR, rather than with the EPR. Notable as well was the degree of increase in this superficial pyramidal layer response in relation to its control. As a comparison, the EPR of layer 4 was routinely 2-3 times the amplitude of the control primary response. Its LR could be 10-15 times this size, while in layers 2-3 the LR was often 20 + times the amplitude of its control response. The entire evolution of the LR in layer 4 was closely paralleled by potentials from the superficial pyramidal layers. There appeared to be little change in layer 4 responsivity that was not reflected immediately in the laminae above. Moreover, layer 2-3 LRs never appeared independently of a layer 4 LR in this situation. As we have demonstrated elsewhere [4], LRs can be recorded from both levels before penicillin has spread beyond layer 4 boundaries. Additional effects

257

20

Pre-NaPen

30

70

170

480

950

1200 sec

-C'--

Layer 2 L

k LR

~/

250~v

i!

50 ms

Layer 4

~

~

Layer 5

~,S'~-.~

~ •

~

~

v%,

lnL NaPen

vV~.s ~

~

,'~

~ ~ _

-~/~-

LR

Fig. 2. Simultaneous 3 layer recording during the development of an epileptic focus originating in layer 4 induced by the microinjection of 1 nl of penicillin. Note the rapid onset of response alterations (EPR and LR) from layer 4, the prominent concurrent LR potentials from layer 2, and the slower spread of epileptiform activity into layer 5. Separate EPR and LR components from layer 4 are only obliterated at the height of the focus (interictal spike potential, IIS). The layer 2 LR response faithfully follows the layer 4 LR throughout the evolution. Layer 5 LR potentials deteriorate more rapidly, leaving behind only a positive potential at the same latency, which was also apparent during early focus development. Recording depths were approximately 300,900 and 1450/xm, respectively. Elapsed time following the onset of penicillin is given on the top line of this and subsequent figures.

beyond these rapid, apparently projected influences from layer 4, which might be related to the gradual accumulation of penicillin in layers 2-3 due to diffusion, could not be differentiated. In contrast, there was much less of a close association between a layer 4 focus and the response alterations of deep pyramidal layers 5-6. Often there was little or no change in the normal layer 5-6 response until an L R was generated in layer 4. A positive, 'mirror' potential of moderate amplitude accompanied the initial layer 4 LRs as is shown in Fig. 2. Over the course of several minutes from the onset of injection, an enhanced primary response often developed in layers 5-6. This was followed by a gradual decline in the amplitude of the positive potential and the concurrent development of a negative L R component. Note that these deep pyramidal late responses required 2-3 min post-injection to develop, as compared to 30 s or less for the granular and superficial pyramidal LRs, and their amplitudes were much smaller. When present, layer 5-6 LRs also appeared to be dependent upon concurrent layer 4 L R responses. Decline of the L R in layers 5-6 occurred sooner than that in layers 4 or 2-3 and left the positive potential to wane with the layer 4 focus.

Unit recordings Since the laminar recordings were made through micropipet electrodes, extracellular multi-unit activity and occasionally isolated unit discharges could be discernable at the same time as local field potentials by selective high-pass filtering of the raw signals. Due to the sampling properties of the microelectrodes and the selective nature of the physiologic visual stimulus, unit responses might or might not be recorded from a given electrode site in the control condition, whereas field potentials were always present. As has been the experience of previous investigations of feline visual cortex 22, there was a higher background level of response from neurons in layers 4 through 6 than from the superficial laminae. Like the field potentials discussed above, the evolution of an epileptic focus induced orderly and progressive changes in unit activity from the various laminae. In addition, there was a close association between specific evoked potential waveforms (and their alteration) and neuronal burst discharges recorded from the same site. Most unit bursts occurred on the leading edge of the negative evoked potential with which it was associated, as demonstrated previouslyll,18. Fig. 3 is an example of combined multiu-

258

A J

control

5 sec

15

23

9O

120

4-- +-. II (300p)

~

--.,--

~

I50MV J 300pV L ' 30 m s

(llOO,u)

b

LR

~' EPR

eNaPen

B.

control

II ~Mm~Mmlmi~¸

(~ z.2 nt)

155 sec

360

.+__

725

+-W---

IV ,mmwimlummWw

780

845

.+-. -M,--

+

II (300p)

I"lV

~

-

-

(11oo,u)

30~ 50 ms Fig. 3. A: simultaneous field potential and multi-unit recordings from layers 2 and 4 during the onset of an epileptic focus originating in layer 4 induced by the microinjection of approximately 1.2 nl of penicillin. Note cellular burst discharges associated with both EPR and LR potentials from layer 4, and with LR potentials from layer 2. LR bursts from layer 2 are synchronous with those from layer 4. Double, EPR-LR bursts from layer 4 unite into one prolonged burst at the interictal spike (IIS) stage, seen here (120 s) and in B (155 s). A gain change follows the vertical dashed line. B: note the separation of layer 4 EPR and LR burst discharges and their counterpart field potentials with regression of the focus. Layer 2 LR bursts remain synchronous with those from layer 4. Multiple cellular bursts are seen to underlie the layer 4 LR potential toward the end of the focus decline.

nit, extracellular and laminar field potential recording. The typical response relationships between layer 4 and layers 2-3 during the evolution of a layer 4 focus

are depicted. If not discernable in the control condition, synchronous multiunit cellular discharge from layer 4

259 became apparent with the development of an E P R field potential soon after the introduction of penicillin. A second, longer-latency neuronal burst always accompanied the development of the L R field potential. Cellular bursting was usually not recorded from the superficial pyramidal layers until the development of the L R potential in that layer which was synchronous with a comparable potential from layer 4. As layer 4 loci evolved, there was a shortening of the latency between the E P R and LR-related neuronal bursts, just as there was between the respective field potentials. Single prolonged bursts and interictal spike potentials marked the final stage of development. Separate E P R and L R neuronal discharges were again apparent with the decline of layer 4 loci. Single cellular bursts from layers 2-3 remained synchronized to the longer-latency L R bursts from layer 4 throughout this entire evolution. Frequently toward the latter stages of focus decline or transiently at the earliest stages of focus development, multiple L R neuronal bursts and multilobular L R field potentials were evident in layer 4. Concurrent cellular discharges and L R field potentials from the superficial control

64 sec

pyramidal layers usually accompanied only one of the multiple layer 4 L R responses. When layer 4 L R activity ceased, comparable layer 2-3 responses complied. Epileptiform responses, both neuronal bursts discharges and field potentials, from the superficial pyramidal layers always appeared to be dependent upon layer 4 epileptiform late activity, since they were never elicited without it, nor did they ever occur out of synchrony with it. Although not depicted in Fig. 3, multi-unit cellular bursts were also recordable from deep pyramidal layers 5-6 in association with local E P R and L R field potentials when they developed (see Fig. 7A). With layer 4 foci, these changes usually occurred over the course of several minutes rather than simultaneously with the layer 4 alterations, as was the case with responses from the superficial pyramidal layers.

Epileptic foci induced in layers 2-3 Field potentials Following the microinjection of comparable volumes of penicillin (1-2 nl) into pyramidal layers 2-3,

76

88

352

563

'

lit

.

~

.

- -.

-.-..~/'~.----.-~ * LR

. t-.~"--.-

(500,u) IV

~

~-

(1000p)

-

-'~'

15OpV

'~ --'"v' '~

• EPR

685

I

~, ',~ ~'--~ ~

~ tR

- ~'

~'~...

~ ~' ~ ~i':

-'

~

_

-~

¢

L5Oms vt

~

_~

..~..~

.._~/..._

_~/

~,

,.,,.._

_

(1600p) •

LR

• N a P e n ( ~ 1.4 nL}

Fig. 4. Simultaneous field potential and multi-unit recordings from layers 3, 4, and 6 during the evolution of an epileptic focus originating in layer 3 induced by the microinjection of approximately 1.4 nl of penicillin. Note the development of a layer 4 EPR potential prior to that of a layer 3 LR, and the relatively longer time to the onset of both when compared to Fig. 2. EPR cellular bursting from layer 4 is recorded at the same time that LR bursts develop in layer 3. Layer 3 LRs are initially accompanied only by positive potentials from deeper laminae (76 s). Layer 4 and 6 LRs evolve gradually with additional time. Synchronous LR unit bursts are recorded from layer 6 soon after their onset in layer 3 (88 s) and before their appearance in layer 4. At the height of the focus (362 s), EPR-LR double bursts are seen in layer 4, while only LR bursts are recorded from layers 3 and 6. LR field potentials and unit bursts decline in layer 4 before those of layer 6, while EPR potentials and bursts, and a positive potential unassociated with cellular discharge remain (563 s).

260 the development of response abnormalities proceeded more slowly and did not produce the same extent of change as seen with layer 4 injections. Fig. 4 illustrates simultaneous multi-unit and field potential data that are representative of the laminar patterns of epileptogenesis seen in these superficial foci which were induced in 6 animals. Initial effects were noted only after approximately 1 min. These consisted of a modest increase in the control response amplitude from layers 2-3 and a more definite increase in the primary latency response from layer 4 (an EPR). Within 2 min, longer-latency negative potentials were routinely recorded from the superficial injection site. These were often of longer latency and duration than LR potentials evoked from layer 4 in a comparable stage of epileptogenesis within that lamina. With further development over several minutes, LR potentials from layer 2-3 foci would increase in amplitude, but seldom achieve the size of layer 4 LRs, and were frequently smaller than those recorded from superficial sites in experiments with a layer 4 focus. The typical duration of response abnormalities following these upper layer injections was 8--14 min. Early in the development or late in the decline of a superficial focus, LRs were not accompanied by comparable negative potentials from the underlying granular or deep pyramidal layers. Only a positive, 'mirror' potential of this same latency was seen from deeper laminae. These superficial LR responses were not entirely independent of layer 4 activity, however, for by the time LRs evolved in layers 2-3, enough time had elapsed for some penicillin to diffuse into layer 4, as was evidenced by the development of an EPR in layer 4 as we have demonstrated directly elsewhere 4. In fact, little or no change was seen in any response component from layers 2-3 before the layer 4 primary response began to show enlargement. Eventually, concurrent negative LR responses became apparent in layers 4 and 5-6 at a latency slightly longer than the positive potentials. Usually the distinction between EPR and LR potentials was not lost in the responses of the deeper layers, since the latency of the LRs from a superficial focus were longer and the amplitudes somewhat less than in a layer 4 focus. In the decline of epileptic loci originating in layers 2-3, the LR potential from layers 5-6 often per~isted beyond that from layer 4. When larger penicil-

lin microinjections into layers 2-3 were used, the involvement of deeper laminae became more complete and the response profile across layers would take on the appearance of that seen with layer 4 injections.

Unit recordings Multi-unit activity was usually not apparent in recordings from the upper pyramidal layer injection sites until an LR potential developed. Shorter-latency neuronal discharges from layer 4 synchronous with an EPR, were generally recorded at or before the appearance of the first superficial cellular burst. Neuronal bursts were not seen to underlie the positive potentials recorded from the deeper laminae. This was most clearly appreciated in layer 4 early or late in the evolution of superficial loci, when LR responses were not present while positive potentials were. Longer-latency multi-unit discharges synchronous with those from layers 2-3 appeared in recordings from layers 4 and 5-6 when LR potentials developed in these layers. At the height of focus evolution, as illustrated in Fig. 4, LR discharges were seen from all 3 laminar sites, while EPR unit activity was seen only in layer 4. LR cellular bursts persisted in layers 5-6 during the decline of foci originating in layers 2-3 longer than they did in layer 4.

Epileptic loci induced in layers 5-6 Field potentials Microinjection of similar amounts of penicillin into deep pyramidal layers 5-6 was also accompanied byresponse alterations that evolved more slowly than those following a layer 4 injection. The laminar response profiles illustrated in Fig. 5 are representative of the data obtained from 5 animals with deep layer injections. Approximately 30--45 s after the introduction of penicillin, enhancement of the primary latency responses of both layers 4 and 5-6 was routinely evident. Within 60-90 s, these alterations were followed by a late response in the deep pyramidal layers, which evolved in as short as 3 rain into an interictal spike potential. EPRs from layers 5-6 were not as enhanced as those from layer 4. Similarly, the LRs were usually of longer latency than those from a layer 4 focus so that both EPR and LR components were discernable throughout most of the evolution of the focus. This was particularly evident in the decline

261 Pre NaPen

30

80

105

185 sec

10

13

15 m;n

Laver 2 ~ • LR

Layer 4 ~ ~EPR

i

i ~ LR

Layer 5 ~ k EPR •

~ LR

~ 250 uV[_.~ 50 ms

1 nL N a P e n

I **IIS

Fig. 5. Simultaneous 3 layer recordingduring the development of an epileptic focusoriginating in layer 5 after the microinjectionof approximately 1 nl of penicillin. Note the appearance of EPRs in both layers 4 and 5 prior to the onset of LR responses in the deeper layer. LR activity is soon reflected in the layer 4 response, and to a lesser extent superficially. Interictal spike potentials principally from layers 4 and 5 are evident 3 min after injection. Note the continued close association of layer 4 and layer 5 LR responses during the decline of the focus. Multiple, long latency LRs, with some degree of amplitude independence between laminae, are seen in the 13 min responses. Recordingdepths were approximately 300,850 and 1400/~m, respectively.

phase, when multilobular rather than single LR potentials were sometimes seen, as illustrated in Fig. 5. The total duration of layer 5-6 foci, 10-18 min, was less than that of layer 4 foci given comparable injections. There was a close association in the responses recorded from a deep pyramidal focus and from adjacent layer 4. During the longer time to the onset of abnormalities in layers 5-6 (> 30 s compared to < 10 s in layer 4 loci), penicillin diffused into adjacent cortex as could be ascertained from the the development of a layer 4 EPR. The latter was routinely present before LRs developed in layers 5-6. When these deep LRs appeared, a counterpart negative potential in layer 4 rapidly developed. In fact, the layer 4 LR was commonly of larger amplitude than that from the focus. Response alterations from layer 4 otherwise paralleled those of layers 5-6, including the multilobular responses noted during the decline phase. Some degree of independence in activity was evident, however, since the relative amplitudes among the different LR components within a multiple response could differ in the two layers (see Fig. 5, 13 min response). A small negative LR potential was usually recordable from the superficial pyramidal layers in association with a layer 5-6 focus. In some cases a positive potential was seen preceding the negative LR superficially, much the same as was seen in deep layers with a superficial focus. In the case of larger injec-

tions of penicillin into the deep pyramidal layers, with a resultant more extensive diffusion of penicillin, the laminar response profile would take on the appearance of a layer 4 focus except for a larger than usual LR from the deep layers.

Unit recordings The combined laminar field potential and multiunit extracellular recordings of Fig. 6 are representative of the data from deep pyramidal layer loci and illustrate in detail the close relationship between response abnormalities of layers 5-6 and layer 4. Cellular discharges associated with control and EPR field potentials were more commonly recorded from the deep pyramidal layers than from the superficial pyramidal layers. A second, longer-latency neuronal burst accompanied the development of LR potentials in layers 5-6. LR field potentials recorded from adjacent layer 4 were not always associated with cellular discharges in the initial stages of evolution. With further development, LR neuronal bursts synchronous with those from the deep layer foci were recorded from layer 4 as well. During most of the development and decline of layer 5-6 foci, dual EPR-LR discharges were recordable from both stellate and deep pyramidal layers. A single, fused burst could be recorded from layers 5-6 in those instances where the deep focus progressed to an interictal spike stage in which the EPR component was obliterated. Separate

262

control

47 se¢

51

64

IV Vl

LR

(9oop)

EPR

150 pV

L

SOres

VI 07oop)

T

LR

EPR •

186

1.5 nL NaPen

248

300

415

'

I[

i.l~L

....

L .......

IIS Fig. 6. Simultaneous field potential and multi-unit recordings from layers 4 and 6 during the development of an epileptic focus originating in layer 6 induced by the microinjection of approximately 1.5 nl of penicillin. Note that EPR bursts and field potentials appear in both layers prior to the development of LR activity. LR cellular burst discharges from layer 4 develop after those from layer 6. Double EPR-LR bursts are recorded from both layers 6 and 4 during much of the evolution of the focus. Amplitude of the LR field potentials from layer 4 surpasses that of layer 6 at the height of development.

263 E P R and L R bursts were usually still distinguishable from layer 4. Progressive separation of E P R and L R neuronal bursts from both layers 5-6 and layer 4 occurred during the decline phase of these foci. Layer 4 L R discharges persisted until the end of such activity in both layers. Layer 4 E P R potentials and associated unitary activity was often the last response alteration to be evident even though the focus origin was within the deeper layers. Though not illustrated in Fig. 6, cellular discharges seldom acompanied the smaller L R potentials recorded from the superficial pyramidal layers in response to a deep pyramidal layer focus (for an exception, see Fig. 7C).

Spontaneous epileptiform potentials Spontaneous LRs were also noted after penicillin injection, but they appeared later in the evolution of

Ao

a given focus than their evoked counterparts. A differential laminar susceptibility to spontaneous events was also evident and often more pronounced than a comparison of onsets of evoked LRs. Spontaneous LRs from layer 4 foci routinely appeared within 1 min of the first evoked L R and had a recurrence rate of 5-20 per minute during stimulus-free periods. Spontaneous LRs from a layer 2-3 focus were slower to develop, even relative to the prolonged onset of its evoked epileptiform activity, and had a slower intrinsic frequency. Layer 5-6 loci were somewhat more capable of producing spontaneous L R activity than layer 2-3 foci in terms of latency to onset, frequency, and amplitude. The laminar profiles of these spontaneous L R potentials were the same as the evoked responses minus the primary latency component, as illustrated in Fig. 7. The more layer 4 became involved via penicillin diffusion, as measured by

C°

B°

Layer 2 I,i 'I

Layer 4

i!

4

"~,~l"i

,. r~

5

Layer 5

t I

Stimulus Condition

so m~

l Off

--'1 Off

i

O.nJ--

Fig. 7. Simultaneous 3 layer recordings of spontaneous and evoked epileptiform spike potentials and associated cellular discharges recorded during the evolution of epileptic foci induced at 3 different levels (indicated by filled circles in A, B and C). Note that the response configurations for the spontaneous events are similar to those for the evoked events minus the EPR components in the middle

and deep laminae. Time to onset of the first spontaneous potential was routinely longer than that for the first evoked LR, regardless of injection site, and was always shortest in a layer 4 induction. Responses similar to those in A occurred between 1 and 1.5 min following penicillin injection, in B between 5 and 8 rain and in C between 2 and 3 min. Recording depths for conditions A and C were the same as in Fig. 2 and 5, respectively; those for condition B were 200,800 and 1400/~m.

264 changes in its primary evoked potential, the more likely spontaneous LRs were to appear from superficial and deep foci. DISCUSSION By means of simultaneous multil'aminar recordings during the evolution of discrete epileptic loci, we have demonstrated among the various layers of cat visual cortex a diversity of: (1) susceptibilities to epileptogenesis; (2) patterns of response alteration across both space and time; and (3) associations between the activity of specific layers. Differentiation across laminae, rather than homogeneity, was the rule in the functional organization of these discrete epileptic loci. We believe evidence now exists that these distinctive characteristics are a reflection both of differences in intrinsic responsivity among cell types and of variation in connectivity within the layers, and can be used to better understand the respective roles for these neuronal subpopulations in the onset and propagation of epileptiform response abnormalities. The progressive involvement of neuronal aggregates during epileptogenesis was assessed in part by means of intracortical field potentials. These result from transmembrane currents, both active and passive, induced by synaptic activity. Such potentials can have definite localizing value if significant differences are noted in their amplitude and polarity over short distances, as were seen in the laminar recordings of the present experiments. It is unlikely under these circumstances that the field potentials and their alteration reflect volume conduction from distant sources, rather than local generation. Inferences which we will make regarding underlying neuronal activity will be further supported by simultaneous recordings of multi-unit discharges in the present investigation, isolated unit recordings of previous similar experiments TM, and current source density (CSD) analysis of comparable laminar potentials performed by other investigators 38,43-45.

Laminar susceptibility to epileptogenesis This study has confirmed our previous finding that layer 4 of striate visual cortex is most sensitive to the epileptogenic effects of penicillin3a 2. Epileptiform abnormalities, both evoked and spontaneous, were

induced more rapidly and more fully with injection into layer 4 than with comparable injections in the other laminae. Unlike our previous sensitivity study, in which recordings were made from just the injection site, the present multi-laminar monitoring verified the degree and temporal sequence of involvement of adjacent layers as well. The possibility of distant, rather than local, effects occurring first could be ascertained. In the case of a layer 4 focus, both EPR and LR abnormalities originated from the injection site. The same cannot be said of loci from more superficial or deeper injections. Although late responses could be induced independently at these injection sites, latency to the onset of response abnormality was longer and a penicillin effect on the layer 4 primary response was always an initial abnormality. Only in the case of a layer 4 focus is it possible to say that the development of epileptiform abnormalities was due solely to penicillin's effect within that layer. This conclusion has been confirmed experimentally by utilizing [lgc]labelled penicillin2, 4. Autoradiography of sections from a cortical biopsy removed at the onset of LR abnormalities verified that penicillin confined to layer 4 was sufficient for epileptogenesis. Tissue removed at the same stage of epileptiform development from superficial and deep foci, however, revealed penicillin infiltration into layer 4 from these injected layers. Lockton and Holmes27,30,31 have assessed the laminar sensitivity of rat somatosensory cortex to penicillin's influence by means of selective intracortical microiontophoresis. They concluded that deep layer 3 is the most susceptible neocortical stratum, closely followed by layer 4. This divergence from our results may be accurate and related to intrinsic differences in the lamination, organization, and afference of rat somatosensory cortexl0, 58,59 as compared to cat striate cortex 22,32. These results may also reflect differences in methodology, however. Distinguishing penicillin's effects on deep layer 3 from those on layer 4 is technically difficult in the rat preparation, since layer 4 is only a thin, 100+-~m wide band of tissue. It is easily encompassed within the radius of their calculated, critical cortical mass for epileptogenesis (450/~m), when centered in layer 3. This problem of resolution is compounded by the use of spontaneous interictal potentials, whose longer delay to onset is associated with more extensive penicillin diffusion, and by the

265 use of separately positioned electrodes for penicillin administration and recording. By contrast, cortical layer 4 in cat striate cortex is 500-600 pm wide, which is sufficient for our techniques to resolve a true differential laminar susceptibility. Accuracy is enhanced by the use of evoked epileptiform response alterations, rather than spike rate or amplitude. The former develop earlier than spontaneous interictal spikes and provide definite stages of epileptogenesis to monitor. As shown by our multilaminar recordings, LR or interictal spike amplitude is not a good measure of epileptogenic sensitivity, since potentials recorded from a more superficial layer may be as large or larger than those from the focus origin. Long debated has been the concept of the epileptogenic 'critical mass', i.e. the smallest volume of cortex and/or the smallest neuronal population that can support interictal spike generation. Progressively smaller penicillin-soaked pledgets or other applicators wer used initially. A surface area of 0.7 mm2 was finally proposed in experiments which also included surgical division of larger fociS0. Others using small applicators and discrete physiologic stimuli concluded that a single cortical column was the smallest functioning epileptogenic unit 21. Penicillin foci as small as 650 pm in diameter have been observed by radioactive [14C]deoxyglucose measurements of increased metabolic activity5. All these experiments had the same basic limitations, however, namely topical drug application and surface monitoring of responses. By using multicontact laminar recording and a delivery of topical penicillin controlled in its distribution by penicillinase, epileptiform potentials limited to the upper cortex were later shown [19]. With the present investigation, laminar epileptic foci have now been demonstrated. The critical mass for epileptogenesis is thus even smaller than a neocortical column, and may be as small as a single layer within a column in the case of lamina 4 in visual cortex.

Laminar interactions during epileptogenesis Most previous spatial analyses of neocortical epileptogenesis utilized more fully evolved loci at the stage of spontaneous spikes or seizures. Laminar recordings after the topical application of penicillin to cat neocortex revealed negative interictal potentials or seizure-associated DC shifts of varying amplitudes at all depths through the center of the focus. AI-

though maximum negativity was recorded from depths corresponding to layers 4 and 5, little could be said concerning the functional relationships among the layers given the uniformity that was seen 24. More recent laminar studies of small topical penicillin loci began to reveal the differences present at earlier stages of epileptogenesis. Focal interictal epileptiform discharges (FIED) of negative polarity could be restricted to the upper layers of motor cortex, while synchronous positive potentials were noted from the deeper laminae 19. Responses were recorded from the spinal cord, however, only when negative interictal potentials spread to layer 5, where cells of origin for the pyramidal tract are located. The current investigation presents for the first time a multi-layer perspective of the evolution of discrete epileptic foci that are initiated in each of the 3 major laminar divisions of neocortex. In addition, early changes of neuronal excitability during epileptogenesis, that cannot be effectively monitored by awaiting spontaneous IIS potentials, were demonstrated in the alterations of local evoked responses. Significant new information concerning the functional relationships among these layers was gained by observing association between the development of response abnormalities in one layer and their expression in another. The prime example was the response coupling that existed between stellate layer 4 and pyramidal layers 2-3. Epileptiform LRs, when generated in layer 4, were immediately reflected in synchronous field-potentials from the superficial laminae. It is unlikely that these superficial responses were a far-field reflection of the activity below, since 'follower' cellular bursts from layers 2-3 were also evoked simultaneously with the onset of LR discharges from a layer 4 focus. Both LR field potentials and neuronal bursts from the superficial layers were, however, dependent upon and appeared to be due to the projection of abnormal activity from layer 4. This recruitment did not seem to be a function of penicillin diffusion into the upper layers, since LRs were elicited within 20-30 s of the injection, which is too short a time for significant physical spread of the drug out of layer 4, as we have demonstrated autoradiographically4. Furthermore, penicillin injection directly into the superficial layers cannot induce LRs that rapidly and does not induce potentials as large and as short in latency. Since comparable potentials were not seen in

266 the deep layers at a similar distance from a layer 4 injection, diffusion or capillary action along the electrode tract are also improbable explanations for this association. It is more likely that epileptiform LR bursting within layer 4 provided superficial pyramidal layers 2-3 with an intense synaptic barrage carried by existing interlaminar pathways. This functional relationship between the responsivity of layers 4 and 2-3 was not reciprocal. LRs, when initially evoked from layer 2-3 loci, were not associated with comparable responses from layer 4. Often just a positive potential was seen, which did not have a neuronal burst counterpart, and only after one or more minutes did well formed, synchronous LR potentials and unit discharges appear in layer 4. These were preceeded, however, by a gradual enhancement of the primary response in layer 4, which we have shown to be the initial direct effect of penicillin. This activity may have provided the developing superficial loci with an enhanced but otherwise normal synaptic input. These slower alterations in layer 4 responses have a time frame consistent with the physical spread of penicillin from the superficial injection site 4. Diffusion of drug would appear to play a much greater role in the interactions between these layers in this direction. Another close response association existed between deep pyramidal layers and stellate layer 4. LR potentials generated from a focus in layers 5-6 were usually reflected immediately in layer 4. This layer 4 late response was in some experiments evident before a counterpart neuronal discharge. Although this may have been a sampling error, these potentials were probably related to graded excitatory synaptic input from layers 5-6 that was not yet intense enough to elicit wide-spread layer 4 LR bursting. In the case of deep foci, a contribution to epileptogenesis from penicillin diffusion beyond the initiating laminae cannot be ruled out, since the development of LRs in layers 5-6 takes one or more minutes. By that time a penicillin effect was routinely noted in layer 4 in the form of an enhanced primary response. In addition, progressive accumulation of penicillin in the more susceptible stellate layer for several minutes after a deep injection resulted in an additional exaggeration of its LR potentials and neuronal bursts and an eventual degree of independence from the activity of the focus origin. The functional relationship between

layers 5-6 and 4 is not a reciprocal one either. LRs were not seen immediately in the deep pyramidal layers with the onset of layer 4 epileptiform potentials as they were in the superficial pyramidal layers. Several minutes were routinely required for the development of LRs in layers 5-6 following penicillin injection into layer 4. Diffusion of penicillin into this region from the injection site would appear to be the'principal reciprocal influence. A final functional association was evident between the superficial and deep pyramidal layers. Even though separated by the stellate layer, LRs (field potentials and neuronal burst discharges) appeared in layers 5-6 sooner and/or persisted longer than in layer 4 during the evolution of a layer 2-3 focus. LR activity within layer 4 appeared only after sufficient time had elapsed for penicillin to have diffused into it. Again there does not appear to be a close reciprocal relationship between layers 2-3 and 5-6. Although large LR responses were recorded in layer 4 synchronous with those from a deep pyramidal focus, relatively little concurrent activity was noted in the superficial layers. An LR of comparable size originating in a layer 4 focus, rather than that associated with a layer 5-6 focus, would have been accompanied by a much larger layer 2-3 response. The translaminar projection of response abnormalities thus appears to be limited in both extent as well as in direction.

Circuitry mediating laminar interactions during epileptogenesis It is of considerable interest that the closest functional relationships during the translaminar evolution of epileptiform abnormalities exist between cortical layers with subpopulations of neurons known to possess anatomical connections by means of axon collaterals 22,32 (see Fig. 1). The principal efferents from the spiny stellate cells of layer 4, which are the major recipients of geniculo-cortical input, are directed to the more superficial pyramidal layers. It would seem likely that this normal pathway is utilized in the rapid projection of layer 4 response abnormalities to layers 2-3. The close association between the responses of layers 5-6 and layer 4 is likewise probably related to ascending axon collaterals of layer 6 pyramidal cells which are known to terminate in layer 4. Completing a potential loop within the cortex are

267 collaterals of axons of superficial pyramidal neurons, which terminate in the deep pyramidal layers before exciting the cortex. This latter circuit may explain why the response of layers 5-6 often show a closer relationship to those of layers 2-3 than do those of intervening layer 4 in the presence of a superficial focus. In general, it would appear that abnormalities from discrete intracortical loci spread rapidly in the direction of known interlaminar connections, surpassing that attributable to penicillin diffusion. On the other hand, when normal connectivity is sparse or absent, epileptiform response abnormality spreads more slowly and appears to be dependent upon the diffusion of penicillin and the intrinsic sensitivity of the layers being infiltrated. Complementing the above anatomical findings, and consistent with our position concerning the major laminar interactions during epileptogenesis, are current source density (CSD) analyses of normal and epileptiform potentials from visual cortex reported by other investigators3S. 43-45. This mathematical transformation of laminar evoked response data can be used to determine the sites and polarities of gross transmembrane currents (extracellular sinks and sources) which generate field potentials. CSD profiles are thought to reveal principally the basic pattern of excitatory post-synaptic activity in terms of extracellular active current sinks and passive current sources 3s. Excitatory activity underlying the normal visual evoked potential was found to flow along several intracortical pathways which all start in layer 438. The most prominent was from layer 4 via strong local connections to layer 3 and from there via longer and more wide-spread connections to layer 2. Similar current source density analyses of penicillin-induced interictal spikes 44,45 or tonic seizures 43 routinely have shown major and initial current sinks at cortical depths corresponding to layer 4 with subsequent spread of excitatory activity into the superficial layers. Other visual cortex excitatory pathways, determined by CSD methods to be of lesser magnitude, involve polysynaptic activity within layer 4 itself prior to projection to layer 3 and spread from layer 4 to deeper pyramidal layers 38. The former may be involved in the rapid elaboration of epileptiform response abnormalities within layer 4 as well as the spread of these abnormalities to the superficial py-

ramidal layers that we have shown here. Positive, 'mirror' potentials, that were found deep to a more superficial focus or occasionally in superficial layers with deep foci, appeared to result when major connections between layers did not exist or occurred before these connections became fully functional. When recorded in isolation, without an accompanying LR, it was evident that they were not associated with cellular discharges. These positive potentials are probably passive current sources for the active sinks of the epileptic focus. CSD analysis of an early stage of a topical penicillin focus supports this 44. The most likely carriers of this current flow are the long apical dendrites of deep pyramidal cells, which are known to span all neocortical laminae22,32. These positive potentials have been noted by other investigators using superficial foci, but were thought to be an active expression of inhibition [19]. It is noteworthy that spontaneous LR and IIS potentials had the same configuration across layers as did triggered epileptiform potentials minus the EPR component. The eventual form of an epileptiform response seemed more closely related to the location and size of the penicillin focus rather than to whether triggering was internal (spontaneous) or external (evoked). Although called spontaneous, these potentials probably result from population response synchronization and integration initiated by internal thalamocortical triggers. Spontaneous activity may have occurred later in the evolution of a focus than did evoked epileptiform potentials because the visual stimulus is a more powerful initial synchronizer of the neuronal population, which would facilitate LR generation. In terms of spontaneous LRs, layer 4 is again the most susceptible. It also seems to play a significant role in the expression of epileptogenesis in other layers. As the primary receiving lamina, layer 4 is well situated to relay in an enhanced fashion intrinsic thalamocortical activity, which may act as a trigger for loci in other layers. EPR versus L R burst discharges

Two forms of multiunit burst discharge were repeatedly observed in this investigation. They were distinguishable by their latency, association with specific field potential waveforms, prominence in specific layers, order of appearance and disappearance during the evolution of laminar foci, and response to

268 alterations in stimulus parameters. The primary latency, E P R burst induced by penicillin has been shown to be an exaggeration of the normal response of neurons to a physiologic stimulus in both visual and somatosensory cortex 1~.~8~33. All characteristics other than the intensity of the response were maintained and stimulus specificity was necessary. The present results support, at least for layer 4, a previous contention, which was based upon simultaneous recordings of several nearby neurons, that E P R bursts occur in a population of neurons before LR or PDS bursts are evoked in any neuron II. Our data further demonstrated that this ability to respond in an enhanced fashion to normal levels of synaptic input is not uniform among neocortical laminar populations when exposed to penicillin. E P R discharges and field potentials were noted principally in those layers which received monosynaptic geniculocortical input, namely layer 4 and to a lesser extent the deep pyramidal laminae. Substantially less penicillin-induced enhancement was noted in the initial response of populations in the superficial pyramidal layers whose input originates in layer 4. It is unclear whether this difference in response to the drug is related to intrinsic properties of the predominant cell type (stellate versus pyramidal), intralaminar circuitry, type or abundance of synaptic input, or some combination of the three. It is our belief that the E P R burst and its counterpart field potential reflect an action of penicillin upon the responsiveness of individual neurons that does not require population interaction. The E P R is an abnormal response of neurons to a normal synaptic input. To this extent it may be called an 'intrinsic" abnormality, although the basic mechanism is probably a reduction of inhibitory modulation, rather than an effect on neuronal membrane properties as will be discussed later. Layer 4 neurons appear to play an important initiating role in epileptogenesis by being able to respond in this enhanced fashion better than any other neocortical neuronal population. Indeed, since layer 4 is also more sensitive to low penicillin concentrations, E P R bursts of cells in this layer may be an initial effect of the drug, regardless of where it is injected into the cortex. The second type of neuronal discharge was the LR burst. It has been shown to be the early stage of the more commonly described PDS or DS II,l~ just as its

associated field potential represents the early graded form of the interictal spike potential, Unlike the EPR, multiunit L R discharge and field potentials were recorded nearly as well from all cortical layers in one or another experimental condition. Typically, both more penicillin and a longer time period were needed before LRs became evident. These longer latency bursts were never seen in isolation without being preceded by E P R activity locally or at least in layer 4. As shown previously TM, L R discharges show a greater variability in latency and magnitude, have a longer refractory period, and are more susceptible to changes in stimulus parameters than are E P R bursts. LR bursts from individual neurons do not require in this penicillin model the stimulus specificity needed to elicit an EPR. Both present data and previous results continue to suggest that the difference between E P R and L R bursts are a reflection of the latter's neuronalpopulation origin. The L R burst would appear to be based upon excitatory synaptic coupling among the neurons affected by penicillin. Feedback and feedforward interactions could elaborate the EPRs of a smaller 'pacemaker' population of neurons responding to a specific stimulus into a longer-latency synaptic drive for themselves and other 'follower' members of the aggregate, for whom the triggering stimulus was not specific, but whose responsiveness had also been enhanced by the drug. Unlike the E P R , the LR burst is an abnormal neuronal response to an already enlarged, multisynaptic, recurrent or collateral input. It is now generally accepted that penicillin's epileptogenic action is based on a partial blockade of G A B A - m e d i a t e d inhibition 9,37,57,6°. By blocking modulating 1PSPs, a normal excitatory stimulus could elicit an enhanced response, such as the E P R burst in neocortex. The time required for the initial 'pacemaker' bursts to recruit in chain-reaction fashion a larger 'follower' population may be of the order of tens to over a hundred milliseconds, as shown by computer modeling 55,56 and as demonstrated presently at early or late stages of epileptic focus evolution. The degree of disinhibition induced by penicillin in our model, however, does not seem to be sufficient for independent epileptogenesis among the neuronal aggregates of all cortical layers. Development of epileptic foci in the pyramidal layers, particularly those superficial ones, appears to need or at least benefits

269 from enhanced external triggering, which is probably provided by layer 4 EPR activity. The enhanced susceptibility of layer 4 stellate populations to both EPR and LR epileptogenic alteration may be related to reduced and/or different types of inhibition which is more easily overcome by penicillin than inhibition in the non-granular layers. In fact, cartridge-shaped inhibitory synapses of certain aspiny stellate interneurons, which terminate on the initial segment of their target cells and are thought to provide a powerful inhibitory influence, are found in superficial and deep pyramidal layers but not in layer 432.54. A more complete or differently mediated disinhibition, which might be supplied by higher concentrations or volumes of penicillin or by other G A B A antagonists such as bicuculline 41 or picrotoxin 40, or by glycine inhibitors such as strychnine 7 may enable other cortical laminae, besides layer 4, to develop epileptic loci independently 25. These more powerful convulsive agents may induce such extensive change, however, that early or subtle differences in response among the laminae are masked. In vitro correlations In vitro experimentation with the hippocampal slice, and more recently the neocortical slice, has added much to our understanding of neuronal membrane properties in epileptogenesis [6,26,28,29, 36,48,49,51-53,61]. Although the presence of intrinsic neuronal membrane properties which can lead to burst generation was stressed as the most important factor in epileptogenesis after the initial studies of hippocampal neurons48.53, analysis of neocortical neurons has confirmed the importance of enhanced synaptic input to members of a neuronal aggregate by means of excitatory interactions26. It is of interest that neurons with an unusually increased responsivity and the ability to burst to intracellular depolarization have been noted in neocortical slices in the middle layers6, where we have demonstrated an increased epileptogenic susceptibility. The role of these neurons as potential pacemakers has likewise been posited. Two classes of burst discharge have also been recognized in vitro in hippocampal pyrami-

dal cells28.29. The 'endogenous' burst is purported to be an independent, single cell event based upon voltage-sensitive membrane properties, while the 'network' or PDS burst is dependent upon synaptic interactions among a group of neurons and is evoked from individual cells by a 'giant' EPSP. Though these data are derived from a different cell population and experimental model, the conceptual relationship between these burst types and our EPR and LR is similar. CONCLUSION Striate neocortical layer 4 is thus distinguished by both its own increased sensitivity to epileptogenesis and by its involvement in this process in other layers. Penicillin's action within layer 4 appears to be sufficient for epileptogenesis. When constituting the focus proper, layer 4 neurons can project abnormally large and synchronous vollies to other layers over existing intracortical pathways. These vollies originate as epileptiform late responses and are capable of evoking near synchronous discharges from the receiving layers. When peripherally involved with foci originating in other cortical layers, penicillin's action within layer 4 may be necessary or is at least supportive. Resultant stellate EPRs, the most sensitive indicator of local penicillin action, would provide adjacent laminae with an enhanced but otherwise physiologic input. This potentiated afference may synchronize responses sufficiently from the more penicillin-affected, but less susceptible, pyramidal populations to facilitate the elaboration of epileptiform LRs in these layers. This role of layer 4 cannot be fully appreciated by monitoring spontaneous interictal spike potentials. ACKNOWLEDGEMENTS This work was supported by USPHS Grant NS06208, the Veterans Administration, the Esther A. and Joseph Klingenstein Fund and the Swebilius Trust Fund.

270 REFERENCES 1 Ayala, G. F., Dichter, M., Gunnit, R. J., Matsumoto, H. and Spencer, W. A. Genesis of epileptic interictal spikes. New knowledge of cortical feedback systems suggests a neurophysiological explanation of brief paroxysms, Brain Research, 52 (1973) 1-17. 2 Chatt, A. B. and Ebersole, J. S., 'Evidence for the neocortical microcircuitry involved in epileptogenesis, Soc. Neurosci. Abstr., 7 (1981) 812. 3 Chatt, A. B. and Ebersole, J. S., The laminar sensitivity of cat striate cortex to penicillin induced epileptogenesis, Brain Research, 241 (1982) 382-387. 4 Chatt, A. B. and Ebersole, J. S., Identification of penicillin diffusion at the onset of epileptogenesis in the cat striate cortex following differential laminar microinjection, Brain Research, in press. 5 Collins, R. C. and Caston, T. V., Activation of cortical circuits during interictal spikes, Ann. Neurol., 6 (1979) 117-125. 6 Connors, B. W., Gutnick, M. J. and Prince, D. A., Electrophysiological properties of neocortical neurons in vitro, J. Neurophysiol., 48 (1982) 1302-1320. 7 Curtis, D. R., Duggan, A., Felix, D., Johnston, G. A. R. and McLennan, H., Antagonism between bicuculline and GABA in the cat brain, Brain Research, 33 (1971) 57-73. 8 Dichter, M. and Spencer, W. A., Penicillin-induced interictal discharges from the cat hippocampus. I, J. Neurophysiol., 32 (1969) 649-662. 9 Dingledine, R. and Gjerstad, L., Penicillin blocks hippocampal IPSPs, unmasking prolonged EPSPs, Brain Research, 168 (1979) 205-209. 10 Donoghue, J. P., Kerman, K. and Ebner, F., Evidence for two organizational plans within somatic sensory-motor cortex of the rat, J. Comp. Neurol., 183 (1979) 647-664. 11 Ebersole, J. S., Initial abnormalities of neuronal responses during epileptogenesis in visual cortex, J. Neurophysiol., 40 (1977) 514-526. 12 Ebersole, J. S. and Chatt, A. B., The laminar susceptability of cat visual cortex to penicillin-induced epileptogenesis, Neurology, 30 (1980) 355. 13 Ebersole, J. S. and Chatt, A. B., Toward a unified theory of focal penicillin epileptogenesis; an intracortical evoked potential investigation, Epilepsia, 22 (1981) 347-361. 14 Ebersole, J. S. and Chatt, A. B., Cortical laminar epileptic foci from penicllin microinjection: differential sensitivity, interdependance and polarity changes with development, Neurology, 31 (1981) 52. 15 Ebersole, J. S. and Chatt, A. B., Interlaminar projection of epileptiform abnormalities from neocortical layer IV. A pacemaker role? Neurology, 32 (1982) 91. 16 Ebersole, J. S. and Chatt, A. B., Exploitation of interlaminar circuitry by evolving epileptic foci in neocortex, Soc. Neurosci. Abstr., 8 (1982) 1017. 17 Ebersole, J. S. and Kaplan, B. J., Spatial distribution of'intracortical evoked potential components elicited by punctate visual stimuli, Brain Research, 224 (1981) 160-164. 18 Ebersole, J. S. and Levine, R. A. 'Abnormal neuronal responses during evolution of a penicillin epileptic focus in cat visual cortex, J. Neurophysiol., 38 (1975) 250-266. 19 Elger, C. E., Speckman, E. J., Prohaska, O. and Caspars, H., Pattern of intracortical potential distribution during focal interictal epileptiform discharges (FIED) and its relation to spinal field potentials in the rat, Electroenceph. clin.

Neurophysiol., 51 (1981)393-402. 20 Gabor, A. J. and Scobey, R. P., Spatial limits of epileptogenic cortex: its relationship to ectopic spike generation, J. Neurophysiol., 38 (1975) 395-404. 21 Gabor, A. J. and Scobey, R. P., Epileptogenicity and the cortical column, Electroenceph. clin. Neurophysiol., 43 (1977) 461-462. 22 Gilbert, C. D. and Wiesel, T. N., Morphology and intracortical projections of functionally characterized neurones in the cat visual cortex, Nature (Lond.), 280 (1979.) 120-125. 23 Goldensohn, E. S., Zablow, L., and Salazar, A., The penicillin focus: I. Distribution of potential at the cortical surface, Electroenceph. clin. Neurophysiol., 42 (1977) 480492. 24 Gumnit, R. J., Matsumoto, H. and Vasconetto, C., DC activity in the depth of an experimental epileptic focus, Electroenceph. clin. Neurophysiol., 28 (1970) 333-339. 25 Gutnick, M. J., Grossman, Y. and Carlen, P., Epileptogenesis in subdivided neocortical slices, Neurosci. Lett., (Suppl.) 10 (1982) 226. 26 Gutnick, M. J., Connors, B. W. and Prince, D. A., Mechanisms of neocortical epileptogenesis in vitro, J. Neurophysiol., 48 (1982) 1321-1335. 27 Holmes, O. and Lockton, J. W., Penicillin epileptogenesis in the rat: diffusion and differential laminar sensitivity of the cortex cerebri, Brain Research, 231 (1982) 131-141. 28 Johnston, D. and Brown, T. H., Giant synaptic potential hypothesis for epileptiform activity, Science, 211 (1981)294-297. 29 Johnston, D. and Brown, T. H., Mechanisms of neuronal burst generation. In Schwartzkroin, P. and Wheal, H. (Ed.), Electrophysiology of Epilepsy, Academic Press, London, 1983. 30 Lockton, J. W. and Holmes, O., Site of the initiation of penicillin-induced epilepsy in the cortex cerebri of the rat, Brain Research, 190 (1980) 301-304. 31 Lockton, J. W. and Holmes, O., Penicillin epilepsy in the rat: the responses of different layers of the cortex cerebri, Brain Research, 258 (1983) 7%89. 32 Lund, J. S., Henry, G. H., MacQueen, C. L. and Harvey, A. R., Anatomical organization of the primary visual cortex (Area 17) of the cat, J. comp. Neurol., 184 (1979) 59%618. 33 Macon, J. B. and King, D. W., Penicillin iontophoresis and the responses of somatosensory cortical neurons to amino acids, Electroenceph. clin. Neurophysiol., 47 (1979) 52-63. 34 Matsumoto, H. and Ajmone-Marsan, C., Cortical cellular phenomena in experimental epilepsy: interictal manifestations, Exp. Neurol., 9 (1964) 286-304. 35 McCaman, R. E., McKenna, D. G. and Ono, J. K., A pressure system for intracellular and extracellular ejections of picoliter volumes, Brain Research, 136 (1977) 141-147. 36 Mesher, R. A. and Schwartzkroin, P. A., Can CA3 epileptiform discharge induce bursting in normal CA1 hippocampal neurons? Brain Research, 183 (1980) 472-476. 37 Meyer, H. and Prince, D. A., Convulsant actions of penicillin: effects on inhibitory mechanism, Brain Research, 53 (1973) 477-482. 38 Mitzdorf, U. and Singer, W., Prominent excitatory pathways in the cat visual cortex (AI7 and A18): a current source density analysis of electrically evoked potentials, Exp. Brain Res., 33 (1978) 371-394. 39 Noebels, J. L. and Pedley, T. A., Anatomic localization of topically applied [14C]-penicillin during experimental focal

271

40

41

42 43

44

45

46

47 48 49

epilepsy in cat neocortex, Brain Research, 125 (1977) 293-303. Olsen, R. W. and Leeb-Lundberg, F., Convulsant and anticonvulsant drug binding sites related to the GABA receptor/ionophore system. In Morselli, P. L., Loscher, W., Lloyd, K. G., Meldrum, B., and Reynolds, E. H. (Ed.), Neurotransmitters, Seizures and Epilepsy, Raven Press, New York, 1981. Olsen, R. W., Ticku, M. K., Greenlee, D. and Van Ness, P., GABA receptor and ionophore binding sites: interaction with various drugs. In Krogsgaard-Larsen, P., ScheelKruger, J. and Kofod, H. (Ed.), GABA Neurotransmitters: Pharmacochemical, Biochemical and Pharmacological Aspects, Academic Press, New York, 1979. Pedley, T. A., The pathophysiology of focal epilepsy: neurophysiologic considerations, Ann. Neurol., 3 (1978) 2-9. Petsche, H., Pockberger, H. and Rappelsberger, P., Current source density studies of epileptic phenomena and the morphology of the rabbit's striate cortex. In Klee, M. R., Lux, H. D. and Speckman, E. J. (Ed.), Physiology and Pharmacology of Epileptogenic Phenomena, Raven Press, New York, 1982. Pockberger, H., Petsche, H. and Rappelsberger, P., 'Die beeinflussing von interiktalen penicillin-spitzen durch clonazepam', Z. EEG-EMG, 12 (1981) 69-75. Pockberger, H., Petsche, H. and Rappelsberger, P. The action of benzodiazepines on focal penicillin induced interictal activity. In Klee, M. R., Lux, H. D. and Speckman, E. J. (Ed.), Physiology and Pharmacology of Epileptogenic Phenomena, Raven Press, New York, 1982. Prince, D. A. and Wilder, B. J., Control mechanisms in cortical epileptogenic foci, Arch. Neurol., 16 (1967) 194-202. Prince, D. A., Inhibition in 'epileptic' neurons, Exp. Neurol., 21 (1968) 307-321. Prince, D. A., Neurophysiology of epilepsy, Ann. Rev. Neurosci., 1 (1978) 395-415. Prince, D. A., Ionic mechanisms in cortical and hippocam-

pal epileptogenesis. In Jasper, H. H. and van Gelder, N. M. (Ed.), Basic Mechanisms of Neuronal Hyperexcitability, Alan R. Liss, New York, 1983. 50 Reichenthal, E. and Hocherman, S., A critical epileptic area in the cat's cortex and it's relation to cortical columns, Brain Research, 47 (1979) 147-152. 51 Schwartzkroin, P. A. and Prince, D. A., Penicillin-induced epileptiform activityin the hippocampal in vitro preparation, Ann. Neurol., 1 (1977) 463-409. 52 Schwartzkroin, P. A. and Prince, D. A., Cellular and field potential properties of epileptogenic hippocampal slices, Brain Research, 147 (1978) 117-130. 53 Schwartzkroin, P. A. and Wyler, A. R., Mechanisms underlying epileptiform burst discharge, Ann. Neurol., 7 (1980) 95-107. 54 Somogyi, P., A specific axo-axonal interneuron in the visual cortex of the rat, Brain Research, 136 (1977) 345-350. 55 Traub, R. D. and Wong, R. K. S., Cellular mechanism of neuronal synchronization in epilepsy, Science, 216 (1982) 745-747. 56 Traub, R. D. and Wong, R. K. S., Synchronized burst discharge in disinhibited hippocampal slice. II. Model of cellular mechanism, J. Neurophysiol., 49 (1983)459-471. 57 Van Duijn, H., Schwartzkroin, P. A. and Prince, D. A., Action of penicillin on inhibitory processes in the cat's cortex, Brain Research, 53 (1973) 470-476. 58 Welker, C. and Woolsey, T., Structure of layer IV in the somato-sensory neocortex of the rat, J. cornp. Neurol., 158 (1974) 437-454. 59 Wise, S. P., The laminar organization of certain afferent and efferent fiber systems in rat somatosensory cortex, Brain Research, 90 (1975) 139-142. 60 Wong, R. K. S. and Prince, D. A., 'Penicillin blocks inhibitory control of dendrite burst generation in hippocampal neurons, Soc. Neurosci. Abstr.. 4 (1978b) 239. 61 Wong, R. K. S. and Traub, R. D., "Synchronized burst discharge in disinhibited hippocampal slice. I. Initiation in CA2-CA3 region, J. Neurophysiol., 49 (1983) 442-458.