Visuocortical epileptiform discharges in rabbits: Differential effects on neuronal development in the lateral geniculate nucleus and superior colliculus

Brain Research, 209 (1981) 61-76 © Elsevier/North-Holland Biomedical Press

61

VISUOCORTICAL E P I L E P T I F O R M D I S C H A R G E S IN RABBITS: D I F F E R E N T I A L EFFECTS ON N E U R O N A L D E V E L O P M E N T IN T H E L A T E R A L G E N I C U L A T E N U C L E U S A N D SUPERIOR C O L L I C U L U S

H. DALE BAUMBACH and KAO LIANG CHOW* Department of Neurology, Stanford University School of Medicine, Stanford, Calif. 94305 (U.S.A.)

(Accepted January 18th, 1980) Key words: epilepsy - - lateral geniculate nucleus - - superior colliculus - - penicillin - - development

SUMMARY We have studied the effects of interictal epileptiform discharges originating from the striate cortex on the development of the receptive field characteristics of neurons in the lateral geniculate nucleus (LGNd) and superior colliculus (SC) in neonatal rabbits. The paroxysmal discharges were generated by twice-daily injections of penicillin into an implanted cannula. Control injections of penicillin ÷ penicillinase were given to the other striate cortex of the same animal. Similar experimental procedures were used to study the effect of such projected discharges on the L G N d neurons in adult rabbit. The results of the first experiment show that cortical epileptiform discharges, initiated in neonatal rabbits 7-9 days of age and continuing to 20-25 days of age, retard the normal development of L G N d cells. There was an abnormal increase of indefinite cells, cells failing to respond to any light stimulation, and a concurrent decrease of cells with concentric or uniform receptive fields. The second experiment shows that the effect of such interictal discharges is age-dependent; they did not cause any changes on the receptive fields of L G N d cells in adult rabbit. That these epileptiform discharges, occurring early in life, had long-lasting effects is demonstrated in the third experiment. An abnormal increase of uniform ceils and a concurrent decrease of concentric cells was still present in adult rabbits which had interictal discharges in the striate cortex limited to the period of 7-9 days to 21-25 days of age. The fourth experiment shows that the interictal discharges in neonatal rabbits do not affect the normal receptive field development of neurons in the SC. The present results demonstrate that asymptomatic interictal epileptiform discharges, produced without focal structural damages in immature brain, can affect * To whom correspondence should be addressed.

62 the development of neuronal connectivity. These results may have some clinical implications in relation to our understanding about the learning and developmental disabilities exhibited in children who had episodic seizure discharges.

INTRODUCTION Historically, the common approach in studying the role of experience in central visual function has been to experimentally alter afferent visual input by either visual deprivation or selective visual experience. Consequent alterations in anatomical connectivity, functional properties of single cells, and visually guided behavior are then attributed to the change in prior visual experience. Such altered rearing techniques have proven particularly useful in elucidating some of the critical factors governing the development of central visual pathways and functions. Although there are a number of persisting uncertainties and ambiguities, the cumulative data indicate a crucial role of afferent visual input during early development in the normal developmental sequence of central visual structures13,1s,26. However, specific sensory input is but one of many relevant factors. Spontaneous activity generated within the developing system, for example, can itself be a source of influence on neuronal development12. This last point may have some relevance to the understanding of certain types of human epilepsy. A significant proportion of individuals with epilepsy display signs of the disorder at a very early age 1. Frequently, these consist only of asymptomatic interictal dicharges evident in the electroencephalographic (EEG) analysis lasting for a few years. While many such children subsequently exhibit various types of learning and developmental disabilities19,22, sufficient experimental data are lacking with which to make any clear assessment as to what extent, if any, such disorders can be directly attributable to the abnormal neuronal discharges. The present study was designed to examine whether disordered signals occurring within the immature nervous system might have detrimental effects on neuronal development. A method was developed in which interictal discharges were induced in the visual cortex of young rabbits by applying penicillin epidurally and maintained throughout the critical period of visual development. Receptive field properties of cells in the dorsal nucleus of lateral geniculate body (LGNd) and superior colliculus (SC) were then examined. An essential aspect of this technique is that only internally generated CNS activity is modified. Afferent visual input remains undisturbed. The rabbit visual system is a convenient model in which to study this. Receptive field properties develop postnatally20,21,25,32 and are sensitive to visual deprivation3, 4, 9,13. In addition, the rabbit visual system is a largely crossed one. Roughly 90-95 ~ of the optic efferents cross at the optic chiasm 1°,11 and there is only a relatively small lateral strip of the visual cortex which contributes interhemispheric connectionsS,34, 35. Corticofugal efferents to the LGNd and SC are ipsilateral only11. In the present study, therefore, we induced an epileptic focus in the visual cortex on one side and examined its effects on receptive field development in the ipsilateral LGNd and SC while using the other side as a control.

63 The present study includes 4 experiments. In the first, epileptiform discharges were initiated in neonatal rabbits at 7-9 days of age and continued to 20-25 days of age. Electrophysiological recordings were made and LGNd receptive fields mapped approximately 24 h after epileptiform discharges had subsided. In the second experiment, the effects of cortical epileptiform discharges on LGNd receptive field organization in fully mature, adult rabbits were examined. Interictal discharges were maintained for 10-15 consecutive days with electrophysiological recordings being made on the 1lth-16th day, after interictal activity had stopped. The third experiment tested the long-term consequences on LGNd receptive field organization of epileptiform discharges occurring during early development. In these animals, epileptiform discharges were begun at 7-9 days of age and discontinued at 21-25 days of age. They were then allowed a 3-5 month period of seizure-free activity prior to electrophysiological recording. The fourth experiment tested the effects of epileptiform discharges occurring during the early neonatal period on receptive field development in the SC. METHODS

Epileptiform focus The cannulation technique preparatory to producing a focus in the visual cortex was as follows. A small (2 mm diameter) opening was drilled into the skull over the monocular region of visual area 1 on each side with extreme care taken not to damage underlying dura. A stainless steel cannula was placed in each opening and two screws were fixed, one on each frontal bone. The entire assembly was then anchored to the skull with acrylic, care was taken to avoid covering the bone sutures. The incision was sutured, a ground clip placed anterior to the screws, and the animal allowed to recover. Rabbits were cannulated at 6--8 days of age, prior to the time of normal eye opening which occurs at 9-11 days of age. Commencing on the day following cannulation, one cannula was filled with about 0.15 ml penicillin (200,000 IU/ml). As a control, the other cannula was filled with a solution of penicillin (200,000 IU/ml) which had been deactivated with respect to its epileptogenic properties by penicillinase. The EEG was monitored for the presence of epileptogenic activity by leads attached to each cannula with the frontal screws as reference. Drug applications were made twice daily separated by at least 6 h. Similar cannulation procedures were used in 3 adult rabbits, each received two injections per day for 15 days.

Electrophysiological recording Since over 90 ~o of the optic fibers are crossed, most of the cells in the LGNd and SC respond exclusively to the eye contralateral to each respective structure. Thus, each animal served as its own control. The normative data of Rapisardi et al. 25 in the LGNd and Fox et alp in the SC served as additional referents to which any possible effects due to surgical trauma during cannulation or drug applications, were compared. The surgical procedures for preparation of the animals for electrophysiological

64 studies and single unit recording techniques were identical to those described previously2, 4. Animals were anesthetized with halothane-nitrous oxide throughout all surgical procedures. All wound margins were coated with a long-acting local anesthetic (Anucaine, Calvin Chemicals). The animals were given ketamine HC1 (Ketalar, Parke-Davis) immediately after the withdrawal of halothane as described previously 2. They were then paralyzed with gallamine hydrochloride and artifically respired. In each case, penicillin applications were discontinued 24 h prior to single unit recording. The EEG was monitored throughout the recording session and no interictal activity was seen. Procedures and criteria for identifying unit activity and mapping receptive field properties were identical to those described previsouly2, 4,9,25. Histological verification of units was made by placing two small electrolytic lesions separated by 1 mm along the electrode track at the end of the recording session. Animals were sacrificed with an overdose of sodium pentobarbital, perfused with formal-saline, the brain removed, sectioned at 52/,m and stained with cresyl violet. The entire extent of the electrode penetration was reconstructed with the lesion marks as referents. Only those units clearly located within the boundaries of the L G N d and those superficial to stratum profundum in the SC were included for analysis. RESULTS

Epileptiform activity Interictal discharges began 3-10 min following the application of penicillin and continued for 6-12 h. The lower panels of Fig. l are EEG records from a 9-day-old animal and illustrate the type of epileptiform activity typically seen. We have made semi-quantitative measurements of spike amplitude, frequency, and duration for all animals studied. Spike duration was measured as the time elapsed between the beginning of the initial positive deflection and the peak of the trailing positive-going slow wave. Spike amplitude was measured as the distance between the peaks of the positive and negative deflections. A random sample of 5-10 spikes was taken on each day. The duration of the slowest and fastest spike and the amplitudes of the smallest and largest spikes were noted. For spike frequency, the number of spikes occurring in each of 6 consecutive 10-sec intervals were taken. All samples were taken in a random fashion 10-20 rain following the initiation of epileptiform discharges at the beginning of each day. These data are illustrated in Fig. 2. The adult values are represented as one point since there were no differences in any of the measures across days of injection. Although data were not obtained from every animal on each day, the values and trends depicted are typical of what we have observed in all animals studied. The amplitude of spikes seen in the EEG range from about 0.5 to 4.0 mV with a mean of 1.6 mV for all days combined. As seen in Fig. 2B, there is only a slight variation in mean spike amplitude across days and ages. Both spike frequency and duration, however, vary with age as illustrated in Fig. 2A. Spike frequency shows a linear increase from a mean of 17.7 spikes/min at 8-9 days of age to a mean of 42.6

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Fig. 1. Upper traces : EEG activity recorded in the rabbit visual cortex, superior colliculus (SC), and dorsal nucleus of the lateral geniculate body (LGNd) following penicillin applications in acute preparations. Note well developed focal discharges in the cortex with projections to both LGNd and SC. Cortical records were made with tungsten gross electrodes applied to the site of focal discharge. LGNd and SC records were made with electrolytically etched and varnished tungsten microelectrodes of low resistance (1-2 Mr2). Positivity up. Lower traces: oscilloscope record at two different sweep speeds of interictal discharges typically seen in the EEG of rabbits following epidural application of penicillin through the cannula. Penicillin, EEG recorded from striate cortex ipsilateral to penicillin application (primary focus). Control, EEG of striate cortex ipsilateral to control injections. Note absence of projected spiking to cortex contralateral to the primary focus. Nine-day-old rabbit. EEG recordings taken from the cannula with frontal bone reference electrodes. Positivity up. spikes/min at 22-23 days o f age. Spike duration shows a linear decrease in an inverse relationship with spike frequency (r = --0.93, P < 0.01) f r o m a mean o f 180 msec on postnatal days 8-9 to a mean o f 119.5 msec on postnatal days 22-23. This difference is statistically significant (t ---- 4.66, d f = 12, P < 0.01). Both duration and frequency a p p r o a c h adult values by 22-23 days o f age. In animals 9 days or older, paroxysmal discharges occur only on the side ipsilateral to the penicillin applications. This was due to the placement o f penicillin on the m o n o c u l a r region which, in the adult, does not have callosal connections. As illustrated in Fig. 1 ( b o t t o m traces), no epileptiform activity occurs in the control cortex on which the penicillin-penicillinase solution was applied. As previously reported 4, in animals approximately 9-10 days or older interictal discharges are confined to a restricted region in and directly adjacent to the site o f penicillin application. We examined this in more detail and f o u n d that, under acute

66 preparation, in animals younger than 9 days of age, interictal activity spreads throughout virtually the entire VI cortex ipsilateral to the application site. In these younger animals, there is also some variable projected spiking to the VI area contralateral to the primary focus, but not to the VII area. This contralateral spiking is also seen in some of the very young animals in the chronic preparation. There is considerable variation in the temporal relationship between the primary and projected spikes. Most typically, the projected spikes follow the primary discharge with a latency ranging from 1 to 50 msec. Projected spiking does not invariably occur in response to a primary discharge, but instead, occurs in an irregular fashion, this irregularity increasing as a function of age. A substantial decrease in the frequency of projected discharges is correlated with eye opening and disappears soon after eye opening. It is curious that, in the chronic preparation, projected spiking is not seen if penicillin applications are not begun until after eye opening. The amplitude of the projected spikes is also quite variable, ranging from less than 0.1 mV to 0.5 mV, and decreasing substantially following eye opening, We have recorded gross activity in the LGNd and SC in young animals under acute preparations and have found that the paroxysmal discharges project to both the LGNd and SC. As previously reported 4 these discharges invade the entire extent of both the LGNd and SC ipsilateral to the cortical focus, but not contralaterally. The upper panels of Fig. I illustrates this projected activity.

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67 Receptive field characteristics

Receptive field p r o p e r t i e s for cells in b o t h the L G N d a n d SC were identical to t h a t previously r e p o r t e d in each structure o f n o r m a l l y r e a r e d rabbits20,32, 38. C o n c e n tric, uniform, m o t i o n a n d d i r e c t i o n a l l y selective units were f o u n d in the L G N d a n d SC. T h o s e with concentric receptive fields r e s p o n d to a s p o t o f light presented in the center o f the field a n d give a n t a g o n i s t i c responses to light in the i m m e d i a t e surround. U n i f o r m cells r e s p o n d to a s p o t o f light p r e s e n t e d in a c i r c u m s c r i b e d a r e a o f the field b u t have no a n t a g o n i s t i c s u r r o u n d . M o t i o n - s e n s i t i v e cells r e s p o n d best to a spot o f light m o v i n g within the receptive field, preferring either a fast m o v i n g small spot o r a slowly m o v i n g large spot. D i r e c t i o n a l l y sensitive cells r e s p o n d best to a s p o t o f light or s h a d o w m o v i n g in a p a r t i c u l a r direction across the receptive field. A n a d d i t i o n a l cellt y p e t e r m e d o r i e n t e d - d i r e c t i o n a l was f o u n d in the SC. T h e y r e s p o n d only to an o r i e n t e d b a r m o v i n g a l o n g the long axis o f the receptive field which lies parallel to the visual streak. A n u m b e r o f cells in b o t h the L G N d a n d SC, while r e s p o n d i n g to either whole eye i l l u m i n a t i o n o r light in local areas o f the visual field, l a c k e d any consistency or clearly definable receptive fields a n d were thus classified as indefinite. Cells which gave no response to light were classified as non-responsive. E p i l e p t i f o r m discharges d i d n o t change the qualitative n a t u r e o f receptive fields in either the L G N d o r SC. A l l cell-types were those f o u n d in n o r m a l l y developing a n d a d u l t rabbits. H o w e v e r , the p r o p o r t i o n a l d i s t r i b u t i o n o f cell types in the L G N was significantly altered b y the presence o f e p i l e p t i f o r m activity in the visual cortex at an early age. The d a t a are s u m m a r i z e d in Table I.

TABLE I Lateral geniculate nucleus. Percentage of cells in each receptive field category sampled in the LGNd of normal rabbits and rabbits with a cortical epileptiform focus

'Focus' denotes cells in the LGNd ipsilateral to the cortical focus and 'control', cells ipsilateral to the control injections (e.g. contralateral to the cortical focus) from the same animals. 20-25 days: focus, control ~ rabbits in which chronic interictal discharges were initiated in the striate cortex at 7-9 days of age and recorded at 20-25 days of age; 20-25 days: normal = cells recorded in normally developing 20-25 day old rabbits, data taken from Rapisardi et al.2~. Adult: focus = adult rabbits with a cortical focus begun at 60-100 days of age and recorded at 73-115 days of age; adult: normal = cells recorded in normal adult rabbits, data taken from Stewart et al. 3z. Recovery: rabbits in which chronic focal discharges were initiated at 7-9 days of age, terminated at 20-25 days of age but not recorded until 107-149 days of age. No. rabbits

No. cells Non-responsive Indefinite Concentric Uniform Motion Directional

20-25 Days

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20 Focus

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25 Normal

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100 18 13 39 12 12 6

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68

Effects of epileptiform discharges on LGNd development Rabbits' eyes normally open at 9-11 days of age. At this age, only about 80-90 of the LGNd cells respond to visual stimulation. Approximately 10-20~ are nonresponsive. Approximately 25 % of the cells, although responding to visual stimulation are classified as indefinite. As the animal matures, the percentage of non-responsive and indefinite cells progressively decreases while the percentage of cells which respond to visual stimulation with clear receptive field properties increases until at about 19-20 days of age virtually all cells display adult-like response characteristics. In this experiment, penicillin applications were begun at 7-9 days of age and visuocortical epileptiform activity maintained until 19-24 days of age, the day prior to electrophysiological recordings. A total of 100 cells were sampled in the LGNd ipsilateral to the cortical focus and 79 cells in the LGNd on the control side in 20 rabbits. The percentage of cells found in each receptive field category are presented in the 20-25 days column in Table I. The proportional distribution of cells sampled in the control LGNd was not significantly different (X~ test) from that found in the normal 19-20-day-old rabbit. Inspection of the distribution of receptive field types sampled in the LGNd ipsilateral to the cortical focus, in contrast, clearly reveals a significant disruption of the normal developmental sequence. A high percentage of the cells sampled were either indefinite or non-responsive. Together these comprised fully 31% of the total cells sampled, significantly (P < 0.001) more than the 4 % found in the control LGNd of the same animals. There is, in addition, a concomitant decrease in the percentage of cells with concentric receptive fields; 39 % as compared to 59 % in the control LGNd. This difference is statistically significant (P < 0.01). Fig. 3 compares these data (20-25 days column) with the developmental curves obtained by Rapisardi et al. z5 in normally developing rabbits of various ages. For clarity, motion and directional cells are omitted. It should be noted that in the LGNd ipsilateral to the cortical focus the percentage of cells which respond to visual stimulation, those with concentric and uniform receptive fields, are all virtually identical to that found in the normal 9-11day-old rabbits but considerably less than the normal 20-25-day-old rabbits. This is about the time the eyes normally open and is about 2-3 days after the beginning of penicillin applications. The percentage of indefinite cells, however, is more like that of a normal 14--15-day-old rabbit. The indefinite and non-responsive cells found in the LGNd ipsilateral to the cortical focus were not restricted to any particular area of the LGNd. Nor were they restricted to the area of the LGNd representing the same topographic area as that of the cortical focus. This is consistent with our observations that the projected proxysmal discharges from the cortical focal area invade the entire ipsilateral LGNd.

Epileptiform activity in the adult The above data clearly indicate that interictal epileptogenic discharges occurring in the visual cortex of young rabbits significantly disrupts the development of receptive field properties of LGNd cells. This second experiment was conducted to determine whether this disruption was a unique consequence of epileptiform activity

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Fig. 3. Percentage of cells in each receptive field category recorded in the LGNd of rabbits. The graphs in the left of the figure are data from normally developing animals taken from a previous study (Rapisardi et al.25). The bar graphs to the right are data from the present study. Epilepsy focus, 20-25: percentage of cells recorded in the LGNd ipsilateral to the cortical focus in the 20-25-day group; 107-149: percentage of cells recorded in the LGNd ipsilateral to the cortical focus in the recovery group at 107-149 days of age following 3-5 months of seizure-free activity. Note the lack of recovery in concentric cell development and the increased percentage of uniform cells found in the recovery group.

occurring during a critical period in receptive field development or whether it represented a general consequence o f a b n o r m a l cortical activity irrespective o f the stage o f neuronal maturation. Three y o u n g adult rabbits aged 60-100 days were cannulated as described above except that a slightly larger cannula was used (3 m m diameter). F o r some o f these animals penicillin concentrations ranging f r o m I00,000 I U / m l to 200,000 I U / m l were used since the 200,000 I U / m l concentration used in the experiments with y o u n g rabbits frequently led to continual ictal-type discharges (high frequency repetitive spiking) in pilot studies. This dose administered to adults occasionally caused very stereotyped behavioral seizures which usually began with slight twitches o f the eye contralateral to the cortical focus. After several hours to a day, these twitches would generalize to include head and neck twitches such as to force the head upright and backwards. During these episodes the animal appeared totally unresponsive to visual, auditory or

70 tactile stimulation. In the interval between the most intense seizures animals were very skittish, hyperexcitable, and hyperresponsive. Typically, they would respond with exaggerated escape behaviors (e.g. run or jump around cage) at the slightest provocation or touch. However, they never appeared aggressive and were never observed to attack or threaten the experimenters while approaching or handling them. Such behavioral seizures did not evolve to general tonic-clonic seizures, being restricted, rather, to involvement of the eye, head and neck. Unless large dosages of anticonvulsants (phenolbarbital) were administered, death would follow in a day or two. We therefore titrated the concentrations of penicillin according to the type of interictal activity observed in the EEG. Applications typically began with a concentration of 100,000 IU/ml and were gradually increased until a steady level of epileptiform activity was achieved. All other procedures were identical to those described in the first experiment with younger animals. A total of 41 cells were sampled in the LGNd ipsilateral to the cortical focus. These data are summarized in Table I (adult, focus) along with data obtained in the normal adult rabbit by Stewart et al. 33. It is clear that epileptiform discharges occurring in the mature brain had no detrimental effects on LGNd receptive field organization. All cells were responsive to visual stimulation and only one indefinite cell-type was found. The proportional distribution of receptive field types was within the normal range and did not differ significantly (jr2 test) from the normal adult.

Recovery from the effects of epiletiform discharges This experiment was directed at examining the extent to which the disruptions in L G N d receptive field development produced by interictal discharges during early development represented irreversible alterations in LGNd connectivity. Five 6-8-dayold rabbits were cannulated as previously described and treated in exactly the same manner as the animals in the first experiment. Penicillin applications, however, were discontinued at 19-24 days of age. The animals were then allowed to live in their home cages for an additional 3-5 months. Penicillin applications were not administered during the recovery period. The EEG was periodically monitored and no epileptogenic activity was observed after approximately a day following the last drug application. At 107-149 days of age, the animals were prepared for single unit recordings. A total of 69 cells were sampled in the L G N d ipsilateral to where the cortical focus had been. A systematic sample was not obtained in the control LGNd. However, a sufficient number was sampled at the end of each recording session to verify the general physiological condition of the animal. Without exception, all cells thus sampled in the control LGNd showed normal responsivity with clear receptive field characteristics. In the LGNd ipsilateral to the focus site, however, a number of abnormalities were evident. The percentage of cells found in each receptive field category are listed in Table I along with data from the normal adult rabbit L G N d for comparison. As can be seen, there continues to be a few non-responsive cells found; 6 ~ as compared to 0 ~ in the normal adult LGNd. Of the cells sampled, 9 4 ~ showed clear receptive field

71 properties. However, the proportional distribution of receptive field types differs significantly (P < 0.001) from that of the normal rabbit LGNd. Only 32 ~ of the cells sampled in the LGNd ipsilateral to where the cortical focus had been displayed concentric receptive fields. This is a clearly significant decrease (P < 0.001) as compared to the 68 ~ found in the normal adult LGNd. In addition, there is now a dramatic increase in the percentage of cells with uniform fields. Fully 48 ~ of the cells sampled in the LGNd ipsilateral to the prior focus displayed uniform receptive fields. This is significantly higher (P < 0.01) than the 21 ~ present in the normal adult LGNd. These data are included in Fig. 3 as the 107-149-day group. The curves in the left of Fig. 3 are the developmental data of Rapisardi et al. 25. The adult data are taken from Stewart et al. 33. The striped bar on the right of the figure is the data for the animals in the present experiment which were allowed a 3-5 month recovery period and the stippled bar is the data from the 20-25-day animals in which recordings were taken immediately after cessation of interictal activity. As compared to the 20-25-day group, the percentage of cells which respond to visual stimulation has increased in the recovery group (P < 0.001) while the percentage of indefinite cells has decreased (P < 0.01). Both of these values closely approach normative levels. The percentage of uniform cells, in contrast, has increased substantially, far exceeding normative levels for either 19-20-day-old or adult rabbits (P < 0.001). The percentage of concentric cells remains unchanged, continuing at the decreased level found in the 20-25-day experimental group. These percentages of concentric cells are virtually identical to that found in normal 9-11-day-old rabbits at about the time of eye opening. As noted above, this is also about the time focal discharges began in the present experiment. The general impression is that focal interictal epileptiform discharges block the development of concentric-type fields. Whether the cells which would normally develop concentric fields now have uniform fields as reflected in the striking increase in percentage of uniform cells in the recovery group and whether these effects would eventually reach normal adult proportions require further investigation.

Effects of epileptiform discharges on SC development This study was conducted to examine whether receptive field development in the superior colliculus was disrupted in the presence ofinterictal discharges as was the case in the LGNd. The SC also receives a projection from the visual cortex although it does not itself provide afferents directly to the visual cortex. That the SC is subjected to corticofugal discharges associated with interictal cortical spiking is clear. We have examined this in acute preparations and have found that projected spike discharges coincident with cortical interictal activity invade the entire mediolateral and rostrocaudal extent of the SC, including all layers of the SC, down to but not including the stratum profundum. Receptive field properties of rabbit SC cells are not fully developed at birth. About 75-80 ~ of the cells respond to visual stimulation at the time of eye opening but only about 50 % display distinct receptive field properties. The proportion of these cell

72 types increases as the animal grows until at about 23-25 days of age the full compliment of receptive field types are found in adult proportions 9. Unlike the LGNd, a sizable number of indefinite and visually unresponsive cells are found in normal adult SC. The 4 receptive field types of concentric, uniform, motion and directional found in the SC are categorically identical to those same cell-types described in the LGNd. A fifth type, the oriented-directional cell, constitutes approximately 20 ~ of the cells sampled in the normal SC. A total of 84 cells was sampled in the SC ipsilateral to the cortical focus in 8 rabbits 23-26 days old. (This is about the age at which previous studies have shown SC receptive field types to be fully mature.) These data are summarized in Table II as compared to the percentages of cells in each receptive field category found in normal rabbit SC of equivalent ageL Visuocortical epileptiform discharges apparently did not disrupt the development of receptive field properties in the SC. All types of receptive fields were found in roughly normal proportions and did not differ significantly (Zz test). No deficits or abnormalities were evident. All cells with clear receptive fields showed brisk responsivity to light and all oriented-directional cells were very highly tuned. DISCUSSION The data from the present study d e m o n s t r a t e that interictal discharges i n d u c e d by focal application o f penicillin occurring in thevisual cortex at a n early age result in severe a n d long-lasting detrimental alterations in n e u r o n a l d e v e l o p m e n t in the L G N d , a visual structure remote from b u t a n a t o m i c a l l y related to the area of focal discharge. N e u r o n a l development in the SC, in contrast, is n o t affected by such paroxysmal discharges.

TABLE II Superior colliculus. Percentage of cells in each receptive field category sampled in the SC of normal rabbits and rabbits with an epileptiform focus

Focus - cells recorded in the SC ipsilateral to the cortical focus in 22-27-day-old rabbits in which chronic interictal discharges were initiated in the striate cortex at 7-9 days of age. Normal -- cells recorded in normally developing 23-26-day-old rabbits. Normal data taken from Fox et al.9.

Age No. rabbits No. ceils Non-responsive Indefinite Concentric Uniform Motion Directional Oriented-directional

Focus

Normal

22-27 days 7 74 9 12 23 7 26 8 15

23-26 days 16 91 11 8 2l 4 21 13 22

73 One uncertainty regarding the interpretation of these results is whether the detrimental effects on L G N d cells are indeed caused by the persistent cortical epileptiform discharges. It could be argued that penicillin itself exerts a toxic effect on LGNd, perhaps after retrograde axonal transport from the cortical focus. Although the possibility has not been rigorously ruled out, it seems more likely that the epileptiform discharge was itself the crucial factor. Diffusion of penicillin after cortical application is quite limited, and there is no evidence for neuronal uptake or axonal transport of the antibiotic 8. Moreover, the developmental effect is penicillin-sensitive, as is the epileptogenic action of penicillin 16,z4. Also, we could find no sign of histological changes in L G N d at the light microscopy level. Nevertheless, electron microscopic investigation, and studies similar to this but using other epiletogenic agents are currently in progress in order to resolve this question unequivocally. Assuming that cortical discharge is the critical factor for the present results, this is the first demonstration that corticofugal activity, independent of afferent activity, is as important as normal visual stimulation for LGNd development. Visual deprivation following lid suture in neonatal rabbits similarly affected the normal development of receptive field properties of L G N d cells 2. However, alterations in corticofugal activity disrupt different aspects of L G N d neuronal development than does monocular visual deprivation. Monocular deprivation affects primarily the development of L G N d cells which display uniform receptive fields. The frequency of such cells showed a significant decrease in the deprived LGNd. There is, in addition, a more subtle effect on cells with concentric receptive fields, the size of these receptive fields being significantly smaller in the deprived L G N d than normal. In contrast, epileptiform activity primarily disrupts the development of concentric cells. There is a significant decrease of such cells in the L G N d ipsilateral to the cortical focus in the 20-25-day age group, while the percentages of all other receptive field types are within normal limits. In addition, analysis of receptive field size also revealed a secondary effect of paroxysmal discharges on cells with uniform fields. The mean diameters of the receptive fields for all mapped cells in the 20-25-day age group are presented in Fig. 4. Analysis of variance revealed that field diameter for uniform cells in the L G N d ipsilateral to the 10

[]CONT~ROL~

u,J n"

~1 FOCUS

8 UJ ,,,,,-

6

*p< .05

LU

P-- 4 Ill

<

u,J

2 TOTAL CELLS

CON

UNIF

MOT

Fig. 4. Mean diameter in degrees of visual field of receptive fields of cells recorded in the LGNd of 20-25-day-old rabbits which had unilateral chronic interictal focal dischargesin the striate cortex from the time just prior to normal eye opening. Focus: cells in the LGNd ipsilateral to the cortical focus. Control: ceils in the LGNd contralateral to the cortical focus. Total cells: all cells for which receptive field maps were obtained; CON, concentric; UNIF, uniform; MOT, motion.

74 cortical focus were significantly larger than those in the control LGNd (F ---- 6.6, df 1/23, P < 0.05). Finally, that the effects of early paroxysmal discharges are longlasting is indicated by two findings: (1) the persistent decrease in the percentage of concentric-type cells found in the recovery group following 3-5 min of seizure-free visual experience; and (2) the concomitant and dramatic increase in the percentage of uniform type cells encountered in the LGNd of this group. The mechanisms whereby either monocular deprivation or cortical interictal paroxysmal discharges alter LGNd receptive field development in the rabbit is not known. In other species such as the cat and monkey, binocular competition has been invoked as an explanatory mechanism for the monocular deprivation effects14,15. However, as we have discussed in a previous report 2, binocular competition is not a sufficient explanatory factor for monocular deprivation effects in the predominantly monocular visual system of the rabbit or for deprivation effects seen in the monocular segment of the monkey LGNd. Sherman et al. 3° have suggested that altered corticogeniculate inputs accompanying monocular deprivation may he additionally involved. The present data along with that reported by us in a prior report 4 provide some support for this suggestion. However, the present results indicate an involvement of other factors. As shown by a number of workers in a variety of preparations and described in detail in the work of Prince and his associates, penicillin-induced interictal paroxysmal discharges are associated with large amplitude cell membrane depolarization shifts which are accompanied by bursts of spikes in neurons within the epileptiform focus4,23. Associated complex thalamocortical interactions have been described in the cat somatosensory and visual system17,23,27-a0. This involves both corticofugal effects and antidromic discharges initiated in thalamocortical relay (TCR) cells. Both excitatory and inhibitory thalamocortical interactions have been described with inhibitory effects being ascribed to corticofugal influences and excitatory effects to antidromic bursting in TCR cells. It is possible that such antidromic bursting occurring in geniculocortical relay cells in the present experiment may contribute to the abnormal LGNd development. The differential effect on LGNd and SC development found in the present study may reflect anatomical differences related to these processes between the LGNd and SC and their respective anatomical and functional relations to the visual cortex. In the rabbit the corticofugal afferents to the SC play a relatively minor role in SC receptive field organization20. Removal of these inputs by cortical aspiration does not appreciably alter receptive field organization of cells in the SC. As compared to the LGNd, anatomical studies in the rabbit 11 also suggest a lower density of corticofugal afferents to the SC than is found in the LGNd. More recently, Collins et al. 6 have reported a substantially greater density of first-order corticofugal afferents to the LGNd in the rat as compared to the SC. A reverse relationship was found for retinal afferents to each structure. These differences were reflected in substantially higher rates of glucose utilization in the LGNd as compared to the SC following bipolar electrical stimulation of the cortex, but a reversed effect was again found following visual stimulation. The differential effects of visuocortical paroxysmal discharges may thus reflect increased intensity of functionally significant synaptic input to the LGNd arising from the

75 corticofugal p a t h w a y o r visual radiations. In addition, the SC does n o t c o n t a i n relay cells p r o j e c t i n g directly to the visual cortex. A n t i d r o m i c impulses w o u l d therefore n o t o c c u r in o r affect SC cells. T o w h a t extent the present d a t a are a t t r i b u t a b l e to either c o r t i c o f u g a l interactions o r a n t i d r o m i c spiking in T C R cells c a n n o t be d e t e r m i n e d at this time. T a k e n together, the d a t a f r o m the present e x p e r i m e n t a n d o u r earlier r e p o r t suggest t h a t a b n o r m a l visuocortical activities m a y exert an i m p o r t a n t a n d long-lasting influence on n e u r o n a l d e v e l o p m e n t in the L G N d . T h a t n e u r o n a l d e v e l o p m e n t in a structure r e m o t e from, b u t a n a t o m i c a l l y related to an epileptogenic focus can be affected by the persistent a b n o r m a l discharge m a y have clinical implications. Despite the obvious differences between the features o f mild f o r m s o f epilepsy in children and as the c o n s t a n t discharges in o u r experiments, o u r d a t a at least p o i n t to the possibility t h a t altered b r a i n d e v e l o p m e n t m a y be a factor in the intellectual deficiencies observed in these children. Finally, it s h o u l d be n o t e d t h a t this is the only e x p e r i m e n t a l m o d e l available to address the p r o b l e m o f the effects o f n e o n a t a l epilepsy on b r a i n development. ACKNOWLEDGEMENTS W e t h a n k Dr. B a r r y G o r d o n a n d D a v i d G l a n z m a n for their help in some o f the experiments, Mr. R o b i n L a w s o n for p r e p a r i n g the histological material, a n d Ms. Cheryl J o o for secretarial assistance. This research was s u p p o r t e d by N I H G r a n t s N S 18512, EY00691 a n d N S 12151 to K . L . C . a n d U S P H S P o s t d o c t o r a l F e l l o w s h i p E Y 05176 to H . D . B .

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