The effects of epileptic cortical activity on the development of callosal projections

Det~elopmental Brain Research, 77 (1994) 251-255

251

© 1994 Elsevier Science B.V. All rights reserved 0165-3806/94/$07.00

BRES 51750

The effects of epileptic cortical activity on the development of callosal projections Antony M. Grigonis a, E. Hazel Murphy b., " Department ofAnatomy, Hahnemann Unicersity, Broad and Vine, Philadelphia, PA 19102-1192, USA t, Department of Anatomy and Neurobiology, Medical Colh'ge of Pennsylcania / EPPL 3200 Heno' At'enue. Philadelphia, PA 19129, USA (Accepted 7 September 1993)

Key words: Epilepsy; Corpus callosum; Development; Neural activity

The effect of epileptic neural activity on the postnatal development of the corpus callosum was studied. Epileptiform activity was induced in the visual cortex of postnatal rabbits by continuous infusion of penicillin. Callosal projections of the occipital cortex were studied in rabbits aged at least 4 weeks. In these penicillin-exposed rabbits, the visual callosal projections extended through most of area 17 in a projection pattern characteristic of neonatal rabbits, rather than being restricted to a narrow callosal zone at the lateral border of area 17, as they usually are by this age. The results indicate that epileptic cortical activity stabilizes immature callosal projections which are normally eliminated during development. The maintenance of such immature and non-specific projections in the mature CNS may interfere with normal cortical functions and could underlie the cognitive deficits which have been observed following childhood epilepsy.

INTRODUCTION Epilepsy is a disorder characterized by recurrent seizures, which are accompanied by hypersynchronous neuronal discharges over a wide area of cortex 16. The frequency of seizures is much higher in children than in adults, particularly in the first year of life. Epilepsy in childhood may result in a broad spectrum of cognitive deficits, but no characteristic changes in CNS structure or function have been identified which are associated with childhood epilepsy and which may underlie such cognitive deficits t'z'31.3v. In the present study, we show that epileptic activity can prevent the normal maturation of CNS projection patterns. In order to achieve the exquisitely precise pattern of connections characteristic of the mature CNS, axons must grow towards and establish synaptic contact with their appropriate target. In many areas of the CNS, there is evidence that this is achieved by a two stage process: first, a relatively imprecise projection pattern is established; second, a process of fine-tuning occurs

* Corresponding author. Fax: (1) (215) 843-9082.

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during which only appropriate projections are maintained and inappropriate projections are selectively eliminated. This second process is activity dependent and may involve Hebbian synapses, which are stabilized by correlated activity of pre- and postsynaptic elements 4"19~2~'24'25"2~'3°'34-3~''4t. Since epileptic activity drastically alters normal patterns of neural activity, its occurrence during development may interfere with this second, fine-tuning process. In this study, we used the callosal projections connecting the visual cortices to test the effects of early epileptic activity. In newborn rabbits, as in most mammals, visual callosal projections are immature at birth, extending throughout area 17 v'~4'~'2~''3°.Most are eliminated so that, by the end of the second postnatal week, the organization of the callosal projections resembles that of the mature adult, in which the callosal projections are restricted to a narrow, 2 mm wide region at the border of areas 17 and 187'1~. This is the only region within area 17 in which neural activity is normally synchronized in both visual cortices 2° (see Fig. 1). Thus, summation of thalamic and callosal afferent activity could activate the postsynaptic cell and selectively consolidate these restricted callosal projections. In contrast, in more medial regions of area 17, unsyn-

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Fig. 1. Diagrammatic representation of the visual afferent and calIosal projections of the rabbit. An object at the vertical visual meridian will simultaneously activate adjacent and homotopically located neurons in each retina, producing correlated activity in the regions of the dorsal lateral geniculate nucleus (LGN) and visual cortex (VC) at which the vertical meridian is also represented. The increased activity of these neurons is indicated by the solid circles. The (solid) callosal neurons in the cortex contribute axons to the corpus callosum (CC) in the normal adult. Note that callosally projecting neurons are not interconnected, and the lines drawn merely represent axons projecting through the corpus callosum. In the adult, these callosally projecting neurons are located at the lateral border of area 17 and are co-activated by geniculate and callosal afferents in response to a stimulus at the vertical meridian. Other (open circle) neurons in the cortex contribute axons to the corpus callosum in the neonate. They are located medially in area 17 and are activated independently by visual stimuli in the right or left visual fields. Their callosal axons, represented by dotted lines, are normally eliminated postnatally. Penicillin results in synchronous activation of neurons throughout the cortex to which it is applied. Neurons in the contralateral cortex may also be synchronously activated via callosal connections.

penicillin-containing bead contained 2.5 mg oi i~en~cdlin t; am! dispensed approximately 2000 IU per day for approximately 21 days In control animals, beads containing only vehicle were imphmted. 1, some animals, 1-14 days after bead implantation. EEG ~ecordings were made in order to assess the efficacy of the beads in inducing epileptic activity. In these animals, silver ball electrodes were placed. under anesthesia, on the dural surface of each posterior cortex. :~ screw was attached to the skull over the frontal sinus tot a reference electrode and dental cement was used to attach the electrodes to lhc skull. E E G recordings were made during 4 h sessions after animals had recovered fully from anesthesia but within 2 days of the d e c trode implantation. During recording sessions, animals were wrapped in a towel to keep them warm and to minimize movement. Since the electrode implantation sometimes caused minor cortical damage. these animals were not used for histological analysis. When rabbits were aged least 4 weeks, H R P injections were made unilaterally throughout one posterior cortex. Injections of 0.5 ~zl 10% H R P were spaced at approximately 1 mm intervals throughout all of the striate and occipital cortex, and the total volume injected was approximately 8 12 ~zl. Thus, the injections included and surrounded the location where the penicillin bead was placed. Following a 24 h survival, animals were sacrificed and retrogradely labelled callosal cells were visualized in the cortex eontralateral to the injection site (see Fig. 2), using horseradish peroxidase (HRP) histochemistry. The callosal cell zone, i.e. the zone containing the somata of neurons with axons which projected via the corpus callosum to the contralateral visual cortex, was measured at the 17//18 border and its width was expressed as a percentage of the mediolateral extent of area 17. M e a s u r e m e n t s were taken from every 5th section throughout visual cortex. At least 10 sections were measured in each animal. In order to ensure that outlying neurons did not bias the results, the medial limit was defined as the point at which density of callosal cells fell below 10% of the mean. Methods for standardizing H R P injections, and determining the validity and reliability

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chronized activity of thalamic and callosal afferents does not summate and may even depress activity 25. As a result, callosal axons linking medial area 17 in the immature animal fail to establish stable synaptic contacts and are normally eliminated. We hypothesized that if epileptic activity was induced in one cortex, the resulting hypersynchronous discharges of the caliosaUy projecting cells in that cortex would summate and synchronously activate their postsynaptic target neurons in the contralateral cortex. Thus, even in the absence of correlated activity in the thalamo-cortical afferents, these immature callosal projections could establish stable synaptic contacts and survive into adulthood. MATERIALS

AND METHODS

Penicillin-containing beads, 1.5 m m in diameter and 1 m m thick (Innovative Research of America), were implanted unilaterally, under k e t a m i n e / a c e - p r o m a z i n e anesthesia, on the dural surface of the left visual cortical surface of rabbits aged 1-5 days. Local application of penicillin, an antagonist of the G A B A a receptor, induces epileptic activity which propagates throughout the cortex 3'5'6'11'12'28'3s'39. Each

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Fig. 2. Bar histogram, with standard error bars, showing the mean tangential extent of the callosal cell zone, expressed as a percentage of the mediolateral extent of area 17, in each animal. The age at sacrifice (days postnatal) of each animal (from left to right) was: normal: 70, 45, 33; control: 69, 69, 38; penicillin ipsitateral: 65, 72, 64; penicillin contralateral: 43, 90, 87, 86, 88. See text for details of the method of measuring the callosal zone. The diagram above each group of histograms shows the location of retrogradely filled callosal cells at the lateral edge of area 17 (control and normal) or extending medially in area 17 (penicillin animals). As shown, beads were always placed in the left cortex. H R P was injected in the right cortex in all animals except the penicillin contralateral group. The solid circles represent neurons filled retrogradely with HRP. The letters PEN, P L A and H R P indicate the hemisphere in which the pencillin (PEN) and placebo (PLA) beads were placed and into which the H R P was injected.

253 of our methods of calculating cell density and tangential extent have been previously described in detail 1s'27. The M a n n - W h i t n e y test was used to determine the significance of differences between normal, control and penicillin animals. Adjacent Nissl-stained sections were used to identify the borders of area 17 and also to determine if the bead implant had caused significant cortical damage. The neonatal dura and cortex are easily damaged. Animals in which histological examination revealed cortical damage resulting from the bead implantation were excluded from the data sample. H R P injections were made in four groups of animals (see Fig. 2). Control data were obtained from normal rabbits (normal: n = 3) and rabbits with vehicle beads implanted (control: n = 3). In penicillinexposed animals, by placing the H R P injections either in the cortex in which the bead was placed or in the other cortex, we determined the dimensions of the callosal cell zone both in the cortex ipsilateral to the bead (penicillin ipsilateral: n = 3) and in the cortex contralateral to the bead (penicillin contralateral: n = 5).

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RESULTS

Electroencephalography Typical recordings revealed epileptiform activity on the side on which the penicillin bead was placed. Spike activity typically recurred approximately every 1.5 to 2 h, with 4 Hz spikes occurring in bursts with a duration of 10 to 20 min. In some animals, similar epileptiform activity was also observed in the contralateral cortex, but it occurred independently of seizure activity on the penicillin bead hemisphere. Typically, in these animals, seizure activity alternated between the 2 hemispheres with quiescent periods of 1.5 to 2 h interspersed between seizure activity. No spike activity was observed in animals in which control beads had been implanted or in normal animals. Behavioral seizures were rarely seen; the presence of spike activity in the absence of behavioral evidence of seizures is a commonly observed p h e n o m e n o n in childhood epileptic syndromes 32.

Histochem&try Data from individual animals are shown in Fig. 2 and photomicrographs of HRP-labelled callosal cells at the area 17/18 border in penicillin-exposed and control rabbits are shown in Fig. 3. The overall size of area 17 was unchanged across groups, but the mediolateral extent of area 17 occupied by the callosal cells differed significantly in penicillin animals. In normal animals, the mean width of the callosal cell zone was approximately 2 mm and was 25% of the total mediolateral extent of area 17 (S.E.M. = 0.97). In control animals in which a bead containing only vehicle was implanted, the mean width of the callosal cell zone in the cortex ipsilateral to the bead was 22% of the total mediolat~ eral extent (S.E.M. = 1.25), and did not differ from normal ( P > 0.05). The normal extent of the callosal cell zone in these vehicle control animals indicates that any local damage to the cortex caused by the bead does not alter callosal development. In contrast, in peni-

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iiiiiiiiiiil C!i CI¢!II:III: Fig. 3. Photomicrographs from 3 rabbits of callosal cells retrogradely labelled with HRP. Beads were implanted on day 5 and H R P injections made in the 5th postnatal week. A: control vehicle bead implant; H R P injection contralateral to the bead; (B) penicillin bead; H R P injection contralateral to the bead: (C) penicillin bead; H R P injection ipsilateral to the bead. The large arrow is placed at the 17/18 border, and the small arrow is placed at the medial limit of the zone containing callosal cells. Bar = 500 # m .

cillin-treated animals, the mean width of the callosal zone in the cortex in which the penicillin bead was placed (penicillin ipsilateral) was twice as large as normal and extended though more than half of the mediolateral extent of area 17 (mean percent of total mediolateral extent = 56%; S.E.M. = 2.7; P < 0.01). Thus, penicillin-induced epileptic neural activity prevents the normal narrowing in the spatial distribution of cells which maintain callosal axons into adulthood. In the cortex contralateral to the bead in penicillinexposed animals, the mediolateral extent of the caIlosal cell zone was variable. In some animals, it was normal; in other animals, it was greatly expanded and similar in extent to that seen in the cortex ipsilateral to the penicillin bead. In observations from a sample of more than 30 animals in this and in our previous studies ~s'27, we have never observed a callosal projection this large in control or normal animals. Thus the results observed in this group of animals indicate a clear bimodal distribution and not merely an increased variability (see Fig.

254 3). Data from this group of animals indicate that callosally transmitted epileptic activity can but does not invariably increase the mediolateral extent of the callosal zone in the cortex contralateral to the region in which epilepsy is directly induced by topical application of penicillin. DISCUSSION The mechanism by which callosal projections connecting medial regions of area 17 are eliminated during development appears to involve selective retraction of callosal axon collaterals rather than cell death z~. Our data indicate that when visual cortex is exposed to penicillin, these callosal axon collaterals are maintained, resulting in a callosal cell zone which extends through much of area 17. Since exuberant callosal axons are usually eliminated by postnatal day 15 7, and since the earliest age at which we examined callosal projections was 4 weeks, it is unlikely that our results represent a delay of retraction rather than a stabilization of these projections. This interpretation is supported further by the observation of expanded projections in the longest surviving animals which we studied (see Fig. 2 legend). Such an expanded callosal cell zone was observed in all three animals examined (penicillin ipsilateral group). The effect of penicillin-induced epileptic activity on the callosal cell zone in the cortex contralateral to the penicillin bead was more variable; it was much larger than normal in two of the five animals studied, but within normal limits in the remaining three animals. It is of interest to note that in our E E G recordings, although spike activity was sometimes recorded contralateral to the bead, it occurred independently of spike activity ipsilateral to the bead, and was not observed in all animals. Thus, both our E E G data and our H R P data indicate some interanimal variability in the extent to which the cortex contralateral to the penicillin bead is abnormal. The variability of the callosal zone seen in these animals may relate to the extent to which synchronous activity of neurons in the cortex infused with penicillin results in callosally transmitted synchronous postsynaptic activation of neurons in the contralateral cortex, a factor which may be dependent on the precise location of the bead. Whether the callosal cell zone is bilaterally expanded, or expanded only in the cortex directly exposed to penicillin, the callosal projections link cortical areas representing quite disparate regions of the visual field. Although we did not record physiologically from these animals, it is probable that penicillin-induced anomalous projections are functional, since we have

previously demonstrated the functional integrity ot anomalous callosal projections induced by manipulation of neural activity in the afferent visual pathway ~~'. Connections in the mature CNS between such disparate cortical areas are likely to result in significant dysfunction m,3~ Penicillin blocks the G A B A a receptor. Other studies have implicated G A B A in epilepsy ~5'w'3s'3'~ but the effects of manipulating G A B A during development have not previously been studied. Our results indicate that synchronous cortical activity induced by blocking the receptor for this inhibitory neurotransmitter results in the maintenance of immature diffuse callosal projections into adulthood. Other pharmacological manipulations of cortical neuron excitability, involving blocking or depletion of norepinephrine, acetylcholine and N M D A receptors have been shown to interfere with the normal refinement of axonal projections which subserve the organization of ocular dominance columns in the developing cat cortex, and these manipulations result in functional deficits 1~'22"4°'41. Our results suggest the possibility that the cognitive deficits which result from childhood epilepsy may be attributable to a widespread failure of refinement of cortical projections. Supported by NINDS Grant NS 26989, and NIDA Grant DA 06871. We thank Drs Itzhak Fischer and Alan Tessler for their critical comments on the manuscript. We thank Anne O'Brien who performed all the histological processing, and Dr Robert Clarke who carried out some of the EEG recordings. Acknowledgements.

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