Analysis of an Experimental Cortical Network: i) Architectonics of Visual Areas 17 and 18 After Neonatal Injections of Ibotenic Acid; Similarities with Human Microgyria

Lesions of cortical areas 17 and 18 have been produced in newborn kittens by local injections of the excitotoxin ibotenic acid (ibo). Twenty-four hours after an injection on postnatal days 2 or 3, the gray matter of areas 17 and 18 near the center of the injection appears completely destroyed, with the exception of a one-to-two cell-thick layer at the bottom of layer I. Intact migrating neurons and radial glia can be found light- and electron-microscopically in the region affected. During the following weeks a several hundred micron thick cortex reforms. In the adult, this cortex consists of superficial layers I, II and III as proven by cytoarchitectonics, continuity with the corresponding layers of the normal cortex and cellular composition. We believe that the recovery is due to completion of migration by neurons spared by the ibo injection. More severe destruction of cerebral cortex, i.e. complete loss of the neuronal layers or their reduction to a few cell-thick mantles can be obtained with ibo injections at the end of the second or, respectively, first postnatal week. Severity of lesion also depends on the dose of ibo injected. There are interesting similarities between the ibo-injured cortex and two human neocortical displasias: microgyria and ulegyria.


INTRODUCTION
"Simple" neural circuits, whether created by evolution or tissue culture, have provided a wealth of information on cellular and molecular interactions in development. In a complex system, these mechanisms can provide reasonable a posteriori explanatory hypotheses for a given morphogenetic event, but they are insufficient to predict the outcomes of normal, and even less abnormal development. Thus, the consequences of, for example, early vs. late brain lesions cannot be reliably predicted as indicated by recent debates on the so-called "Kennard principle"/44,52/. VOLUME 2, NO. 1,1991 1 To a large extent, the fact that the development of a neural system cannot be easily reduced to that of its cellular and molecular components in isolation, is a direct consequence of its network properties. In a developing neural network, a localized structural change can have consequences far away from the site where it occurred. For example, experimental or genetic changes in the retinofugal projection can affect the development of visual callosal projections (for references see /19f). Furthermore, the operations a neural network performs, e.g., its input-output transformations, may have regulatory consequences on the local molecular events by which the network itself is assembled.
For example, visual experience can modify the development of geniculo-cortical and callosal projections (for references see/19,63/) probably by affecting molecular mechanisms of synapse formation/57/.
In this study and in two others /1,21/, we explore the potentials of a "simplified" network, produced by neonatal, local injection of ibotenic acid (ibo) in the visual cortex of the cat for the analysis of the relations between structure and function in the developing and adult cortex. In particular, we focus on the regulation of the elimination of transient, juvenile projections to the visual cortex /20,25/. Preliminary findings indicated that the neonatal injection of ibo results in the formation of a cortex consisting exclusively of supragranular layers; some of the normal response properties of visual cortical neurons are maintained although some of the transient projections to the visual cortex, which are normally eliminated in development, are maintained as well/23/.
In a broad sense, the approach used resembles x-irradiation /27,28,51/ or the administration of cytotoxic drugs interfering with cell proliferation /30/, both of which, however, delete the superficial cortical layers. The usefulness of these approaches for the goals outlined above depends on the reproducibility of the results and on the possibility of fully characterizing the effects of the developmental manipulations. Because of its local action, the neonatal injection of ibo appears to be free of complications such as generalized loss of neuronal or glial populations throughout the brain, the production of neuronal ectopias which can, in contrast, result from x-irradiation or cytotoxic drugs/28/, etc. In addition, the cortex produced by neonatal ibo injections (henceforth referred to as microcortex) resembles "microgyria", a congenital malformation of the human cortex often associated with epilepsy, mental retardation and possibly dyslexia /15,34,39,47/; it may therefore provide a model for the study of this malformation.

MATERIALS AND METHODS
Experiments were performed on 39 kittens, born in our breeding colony. About one half of the kittens were obtained from timed pregnancies and were delivered on day 65  Twenty-four to 48 hours before sacrifice, many of the kittens received injections of WGA-HRP alone or combined with [3H]leucine-proline in their visual or auditory areas. In one kitten, single unit recordings were performed in areas 17 and 18 on the side of ibo injection; another one was used for 2-deoxyglucose experiments. Modalities and results of these investigations will be described elsewhere /21/ (Assal et al., in preparation). Kittens were subdivided into four groups, aiming at different experimental questions.
The 15 kittens in the first group (Table 1) were injected with ibo (1-2.5/1; 30/g//zl in 0.1 M phosphate buffer pH 7.4) on postnatal days (pd) 2-3 (day of birth pd 0) and killed either on pd 3-4 or 8-9.5 or 11-27; they were used to study the short-term consequences of the ibo injections.
IBO 73 and 75 received one [3H]thymidine injection each (2.5 mCi), respectively 12 h and 24 h after the ibo injection. The 9 kittens in the second group (Table 2) were injected with ibo (2-2.5 1; 30/g//zl) on pd 1-3 and killed on pd 31-199; these kittens were used to study the long-term effects of the ibo injections.
The 9 kittens in the third group (Table 3) were injected with ibo on pd 2, 3 or 6 and killed between pd 5 and 328. Three concentrations of ibo were used: 10, 20 and 30 /.g//zl and the volume delivered varied between 0.5 and 2.8 These kittens were used to study how the lesion depended on dose and concentration of ibo. Furthermore, in order to determine if in the  CYT, cytochrome oxidase; other abbreviations as in Table 1. All ibo injections were in right hemisphere. Several of these animals also received injections of axonal tracers in areas 17 and 18 of the right or left hemisphere or in areas A1 and A2 of the right hemisphere. VOLUME 2, NO. 1,1991 developing nervous system, as in the adult, the excitotoxic action of ibo is mediated by the Nmethyl-D-aspartate (NMDA) receptor, in two kittens (IBO 93 and 95) the NMDA antagonist phosphonovaleric acid (APV) /10/ was injected together with ibo and at equimolar concentrations in the right hemisphere. The left hemisphere received an identical injection of ibo, without APV.
The 6 kittens in the fourth group (Table 4) were injected with ibo (1-2/xg; 30/g//zl) on pd 6, 7, 14 or 20 and killed between pd 52 and 167; they were used to study if a critical period existed for the effects of ibo.
Injections of ibo were performed in kittens premedicated with atropine and initially anesthetized with Ketalar (30-40 mg/kg i.m.) and subsequently by inhalation of a gas mixture containing 0.5-1.5% Penthrane in 70% N20 and 30% 0 2. Xylocaine (2%) was applied to the wounds and to the periostium. Throughout the experiment, the body temperature of the kittens was controlled by a thermostatically regulated heating pad and the ECG was monitored. Glass pipettes (tip diameter 11-15/zm) were filled with ibo by suction, lowered into the brain with a rnicromanipulator to the depth of 1200/zm in pd 2 3 kittens, 1400/.rn in pd 6 or 7 kittens, 1500 Abbreviations as in Tables 1 and 2. Asterisks denote doses which provoked lesion. In IBO 93 and 95 APV and ibo were injected together in the right hemisphere; ibo alone was injected in the left hemisphere. In all other animals ibo was injected in the right hemisphere. Several of these animals also received injections of axonal tracers in areas A1 and A2 of the right hemisphere.  Table   2) was perfused with 1% paraformaldehyde and 1% glutaraldehyde followed by 5% paraformaldehyde and 4% glutaraldehyde (buffer and sucrose as above). In both cases, the perfusion with fixative was preceded by a rinse with the same phosphate buffer and followed by storage of the brain in the same fixative for 40  week of gestation since at that age ultrasonography showed normal maturation of the skull. The microgyric malformation extended over most of the cortex in both hemispheres although some normal cortex was still found in the occipital region.
The second case (11SN77) was a 53 year old oligophrenic woman, epileptic since the age of 11. Death was caused by acute bronchopneumonia. Neuropathological examination showed slight (not quantified) microcephaly and a small (roughly 1 cm 2) bilateral cortical displasia in the region of the calcarine fissure, which was diagnosed as ulegyria.

RESULTS
Injections of ibotenic acid in the cortex of newborn kittens induce permanent, characteristic cytoarchitectonic changes. These changes are dose and age dependent and result from reorganization of the neocortex following the early destruction.
The size of the lesion depends on the amount of ibo, and the severity of injury typically varies with the distance from the injection center. Near the latter, the cortex consists, in addition to layer I, of a 2-5 cell thick layer although a few wedges of intact neurons can extend further in depth, usually around blood vessels (Fig. 1). This layer is continuous with the cortical plate of the surrounding, intact cortex which, at this age, probably corresponds to the prospective layer II and the top of layer III/55L Progressively deeper cortical layers are intact at increasing distance from the injection center. Characteristically though, the destruction of layer V and of part of layer IV extends further away from the injection center than that of layer VI. At the periphery of the lesion, therefore, an empty layer is interposed between a thinned layer VI and the upper cortical layers (Fig. 8A).
The injured cortex and its underlying white through the postlateral gyrus, near the center of injection; toluidine-blue stained. Area 17 is on the medial (right) and area 18 on the lateral (:left) bank of the gyrus but the precise location of the 17/18 border varies from animal to animal and cannot be determined in this material. Neurons are restricted to a thin superficial layer in most of the gyrus but extend progressively deeper at the transition with normal cortex (arrows). Right: IBO 39, toluidine-blue stained /m thick section through a degenerating portion of the postlateral gyrus (area 17). Pial surface is up. Notice intact neurons at the top, elsewhere pyknotic nuclei (e.g. small arrow), a few intact, probably migrating neurons (e.g. large arrow) and gitter cells (e.g. open arrow) are found. Calibration bars are 500/m on the left, 20/m on the right.
Although this cannot be stated with certainty, the center of injection was probably close to the asterisk. matter have a loose texture. Degenerating elements, most of them presumably neurons, typically show pycnotic nuclei, and severely vacuoled or already disintegrated cytoplasm (Figs. 1 and 2). Electron-microscopically, less advanced signs of degeneration, i.e. empty mitochondria can also be seen in some neurons.
Many axons are vacuolated, or swollen, or disintegrating, but some seem intact and occasionally synapses can be seen.
Many "gitter cells" (brain phagocytes probably related to microglia; see/26/for discussion) are interspersed among the degenerating elements axons directed to the cortex seem to wait for some time, before entering the gray matter (see /22/ for discussion). A similar distribution of GFAP-positive astrocytes is found in the white matter underlying the intact cortex of the iboinjected hemisphere but they disappear under the injured cortex (Fig. 3B).
The radial glia are, at least partially, intact.
Vimentin-positive radial processes can be seen crossing the injured cortex and they terminate with typical end feet in layer I. However, the density of both processes and end feet appears lower than in the normal cortex. Thus, some radial glia have probably been destroyed. End feet of radial glia and processes containing densely packed intermediate filaments typical of radial glia can be observed electronmicroscopically (Fig. 2, small arrows in A, B).
The existence of intact, often elongated elements, oriented perpendicular to the pial surface in the injured part of the cortex and underlying white matter is characteristic (Fig. 1, large arrow). These elements have cytological features typical of neurons; they are probably migrating since they are frequently in contact with radial glia (Fig. 2, large arrows in C, D).
2. Six to eight days after an ibo injection on pd 2 or 2.5 (IBO 40, 97, 47; Table 1) the injected parts of the lateral and postlateral gyri have undergone considerable reorganization and the largest fraction of the degeneration debris has been eliminated. Near the center of injection the cortex is 100 300/m thick and consists of 3 zones (Fig. 4). The outermost zone corresponds to layer I of the surrounding intact cortex. Underneath there is a layer of densely packed, round or triangular cell bodies which is continuous with layers II and III of the intact cortex. Below this cellular layer, the tissue becomes loose and consists of a layer of sparse degenerating cells, "gitter cells" and a few intact neurons. Now, it also contains an accumulation of GFAP positive astrocytes (Fig. 3, C).
Compared with younger animals, astrocytes are more frequent everywhere but particularly in the white matter of the injured cortex. Moving away from the center of the injection this deep nonneuronal layer becomes less conspicuous and is progressively replaced by the normal cortical layers in the same inside-out order described above (Fig. 8  resembling those which are also occasionally found after longer survivals (see next section).
What causes the different evolution of apparently similar initial lesions is unclear. Secondary pathology, superposed on the ibo toxicity, may cause most severe destruction of the white matter, including the radial glia. To the extent that radial glia may be necessary for neuronal migration /46/ the late neuronal migration which in our opinion is responsible for the recovery of cortical structure may be impaired (see Discussion).

Permanent changes after neonatal ibo injections
One month or longer after a neonatal ibo injection of 30 or more (Table 2), narrower lateral and postlateral gyri are found on the injected side (Fig. 5). The main sulci are roughly normal in their position, but near the injection shallow and short new sulci have appeared in about 70% of the experiments. Nissl staining shows that the narrowing of the lateral and postlateral gyri is due to thinning of the gray and white matter (Fig. 6). The severity of the lesion increases towards the center of the injection.
Near the lesion center, the cortex is 500 700 thick. Below layer I, typically two cell layers can be recognized (Fig. 7). The outer layer consists of small, round cell bodies (grains), the inner layer of small and medium-size pyramids. These two layers resemble layers II and III of the surrounding intact cortex and are indeed continuous with them; they are, however, considerably thicker than these layers or the corresponding layers in the intact hemisphere ( Fig. 7, A,B; Fig. 8). Away from the injection center there is a progressive reappearance of the deep layers in an outside-in order. However, destruction of layer V and, in some cases, of the bottom of layer IV, usually extends further away from the center of injection than that of layer VI and creates a neuron-free space between layer VI and the upper cortical layers. Further away from the center of injection only the neurons usually found in the white matter (interstitial neurons/31,37/) have disappeared.
This progressive inside-out loss of the cortical layers approaching an injection center and the thickening of layers II and III are also well documented in cytochrome oxidase preparations (Figs. 8, D and 9). While the histological changes described above can be reliably observed in all the affected brains, others are more rare. In about 10% of the cases, the gray matter is completely destroyed over several hundred microns and the white matter reaches the surface of the brain.
Occasionally and in restricted regions, the bottom of the cortex undulates and the gray matter acquires uneven thickness (Fig. 7, C). The white matter underlying the lesion often contains a higher density of astrocytes than that under normal cortex. Sometimes a glial scar surrounds amorphous material. We assume that these more severe alterations of cortical structure result from the evolution of lesions similar to those in which little or no recovery was found after shorter survivals.
Impregnations with the Golgi method (Figs. 10 12) of parts of the lateral and postlateral gyri corresponding in normal brains to areas 17 and 18 revealed that the outer cell layer of the iboinjected cortex consists of small pyramids with short apical dendrites or of spiny stellates. The inner cell layer contains medium size pyramids and stellate cells, whose axons can occasionally be followed into the white matter. In addition, throughout the thickness of the cortex, nonspinous stellate or bitufted cells with locally arborized axons are seen. All these neurons are normal constituents of layers II and III /5,13,29,35L At the bottom of the cortex, some neurons have an ovoidal cell body, and dendrites oriented tangentially to the cortical surface; they may be layer III stellates whose morphology is modified by the lesion.

Effect of concentration and amount of ibo
The severity of the cytoarchitectonic modifications depends on the amount of ibo injected (Table 3). No changes can be detected when 10/g of ibo have been injected in either 0.5 or 1/1 of solution (IBO 25 and 30; Fig. 5).
Indeed, in these kittens visual areas 17 and 18 of the intact and the injected hemisphere cannot be distinguished by qualitative criteria. This is also the case for one of the two kittens which received 20/g of ibo in 2 1 of solution (IBO 22;      18. Notice the absence of the cytochrome oxidase band corresponding to layer IV on the injected side, as shown in Fig. 8. The cortex on the injected side corresponds to layers III and II of the normal side but it is thicker than the latter. Medial (area 17) is between the two photomicrographs, dorsal is up. Calibration is 500 m.
In two cases (IBO 93 and 95) ibo was injected together with equimolar amounts of APV in one hemisphere, but alone in the other. Only in the latter is there a lesion, indicating that in the former hemisphere, the ibo neurotoxicity was prevented by APV (Fig. 13).

Injections of ibo at different postnatal ages
Injections of ibo on pd 6 or 7 (IBO 49, 95 and 96; Table 4) result in more severe injury to cerebral cortex than earlier injection. Near the center of the injection the cortex consists of a superficial fiber layer, wider than normal layer I and containing sparse neurons, and of a deeper, one or few cells thick layer (Figs. 6, D; 7, D, 13, C). The superficial layer is continuous with layers I and II of the normal cortex, whereas the deep layer is continuous with layer III of the normal cortex and is characteristically undulating. At increasing distance from the center of injection, cortical layers are restored in the following sequence: first layer II, then the granular and infragranular layers following an outside-in sequence similar to that found in kittens injected on pd 2 and 3. As in the latter, the reappearance of layer VI precedes that of layer V.
Injections of ibo on pd 14 or 20 (IBO 61, 65 and 80; Table 4) result in even more severe lesions. In two of these animals (IBO 61  tion. An interrupted line marks the bottom of the gray matter. Location and shape of spines are accurately drawn on enlargements of parts of some neurons, as indicated by arrows. Calibration bars refer to enlargements of the main drawing and of the enlargements. Power of the objectives used for the drawings is also indicated. D dorsal, M medial, refer to orientation of the section outline. Although most of the neurons have normal morphology the tangential orientation of the dendritic arbor of the neuron nearest the white matter may be due to the lesion. VOLUME 2, NO. I, 1991 thick amorphous layer lined outside by a cell layer containing glia and in which no neurons can be recognized in Nissl preparations. This layer is continuous with layer I of the intact cortex. Although ibo occasionally induced the formation of cysts in animals injected at younger ages (see above), a neuronal layer lined the wall of the cyst in those animals. In the third animal (IBO 80) no cyst, but an amorphous tissue containing sparse, non-neuronal cell bodies was found under the superficial glial layer. The complete disappearance of neurons in the ibo-injected region seems to be a characteristic feature of the late injections. The transition from the intact to the injured cortex tends to become sharper the older the animal at injection. Nevertheless, the destruction of the deep layers still extends further away from the injection center than that of the superficial layers.

DISCUSSION
The complexity of cortical networks raises formidable difficulties to the study of structuralfunctional relations in the adult and of cell-cell interactions in development. Against complexity, two approaches have been used: i) the experimental analysis of morphological, functional and developmental properties of single neurons or of selected neuronal populations; ii) the construction of cybernetic models, incorporating features derived from the experimental analysis as well as assumptions concerning principles of connectivity, and developmental rules, both more loosely related to experimental results. Probably, the experimental cortical networks which result from these manipulations will provide useful models for the study of structural functional relations in the adult and cell-cell interactions in development only if they can be characterized in detail and reliably reproduced. This perspective, in addition to the hope of gaining a better understanding of the mechanisms responsible for the formation of cortical connections justified the present detailed description of the structural development of cerebral cortex after neonatal injection of ibotenic acid, as well as further studies of the connections and functional properties of such a cortex/1,21L The effects of ibotenic acid on the developing visual cortex Ibo/12/is an excitotoxin ]53/which we chose as a tool for destroying the neocortical neurons but not the axons afferent to them. Kainie acid has similar neurotoxic "axon sparing" properties in the adult nervous system/9/and also in some immature brain structures/6,7/, but did not show neurotoxicity in two preliminary experiments in which it was delivered in 1 2 injections of 0.6 1 each (4/zg//zl) in the lateral and postlateral gyri of pd 3 kittens. We succeeded in destroying immature cortical neurons with ibo and at least some of the axons were spared. Electron-microscopically, apparently intact axons were found in the injured cortex 24 hours after it had been injected with ibo. Some of these axons may belong to the local intact neurons. Others may be afterents and may be the source of the synapses occasionally found on intact neurons. Twenty-four hours after an ibo injection, and a simultaneous WGA-HRP injection in the contralateral hemisphere, anterogradely labeled axons can be seen at the ibo-injected site/21/.
In the developing nervous system, as in the adult nervous system, the excitotoxic action of ibo seems to be mediated by the NMDA receptor (see ref./59/for discussion). As in the striaturn of 7 day old rats/59/, the neurotoxicity was blocked in both a 3 day and a 6 day old kitten by a NMDA receptor antagonist. Why the activation of the NMDA receptor by ibo kills the neuron is unclear, but one possibility is that Ca 2+ dependent proteases and lipases are activated by the NMDA mediated Ca 2/ entry/38,49/.
Three aspects of the ibo toxicity in the present study will be discussed below.
i) At the doses, concentrations and rates of delivery used here ibo seemed to affect indiscriminately all cortical neurons with the exception of early postmigratory or migrating ones. The presence of intact migrating neurons even near the center of ibo injection sites, 24 hours after the injection on pd 2 or 3, was suggested by the light-microscopical data and unequivocally confirmed electron-microscopically. Their relative insensitivity to ibo was stressed by the proximity of degenerating elements (Fig. 2). In the same animals, at least a few of the most superficia neurons are intact. These neurons have probably completed migration shortly before or, more probably, just after the ibo injection. Why migrating neurons are spared by ibo is unclear.
They may not have developed the NMDA receptor, or one of the steps leading from the latter to death of the neuron may not be working. One may wonder if in addition the contact with radial glia may have a protecting role, for example, by masking the NMDA receptors.
ii) Although all post-migratory neurons seem to be affected by ibo, they may differ in their sensitivity to it. Indeed, at increasing distance from the center of injection the lesion becomes confined to progressively deeper layers; furthermore layer V seems to be affected further away from the injection than layer VI. Since, as discussed above, the latest generated neurons destined to the superficial layers seem insensitive to the neurotoxin, the sensitivity to ibo might develop gradually and with an inside-out order, roughly corresponding to that of neuronal generation.
The action of ibo on the deep layers might be potentiated by that of excitatory transmitter amino acids released by axon endings in the deep cortical layers and in the white matter. Indeed, during the first postnatal week, in the white matter and layer VI there are accumulations of axon endings of cortical origin, many of which are transitory /8,18,22/, and presumably using excitatory amino acids as transmitters. Finally, the differential degeneration of the layers could simply reflect differences in the diffusion and/or accumulation of VOLUME 2, NO. 1,1991 ibo in the cxtraccllular space, duc to the depth of the injections or to inhomogcncitics in the diffusion of substances through the immature cortex.
iii) Following ibo injections, not only neurons degenerate, but also astrocytcs disappear. The possibility that they degenerate is puzzling. Adult astrocytcs appear to have functional receptors to excitatory amino acids (scc/41/for rcf.). However, Ca 2+ entry may not bc regulated through NMDA receptors/43/. The degeneration of the astrocytes may bc secondary to ionic changes in the cxtraccllular space induced by neuronal degeneration/33/. Wc do not know the origin of the astrocytcs which have reappeared in the white matter by six or seven days after the lesion and which probably constitute the glial scar often sccn in the adults. They may bc newly generated or they may have migrated from the surrounding tissue although some probably are transformed radial glia/50/.
In the days following an ibo injection on pd 2 or 3 the cortex seems to recover partially, and, in the adult, near the center of an ibo injection site, the cortex consists exclusively of layers I-III. The latter is strongly suggested by the anatomical continuity between the layers of the ibo-injured cortex and layers I-III of the normal cortex, well documented both in Nissl and cytochrome oxidase preparations. Furthermore, the ibo-injured cortex lacks the projections originating in its infragranular layers such as those to the lateral simplified visual cortex may indeed provide a good model for the study of the circuitry responsible for the emergence of response properties characteristic of the cortical level of visual processing.
The recovery of cortical structure which takes place in the days following the ibo injection is probably due to the fact that migrating neurons are not killed and complete their migration. Three arguments favor this interpretation. First, the neurons destined for layers II and III complete migration during the 1st through 3rd postnatal week/55/. Accordingly, an ibo injection on pd 6 or 7 results in a much thinner layer of intact neocortex than an injection on pd 2-3; no recovery is found after injections on pd 15 or 20. Thus, recovery is obtained when ibo is injected before the end of migration and the extent of recovery seems proportional to the size of the neuronal pool which has not yet completed migration. Second, intact, fusiform elements, resembling migrating neurons, can be seen, both light-and electron-microscopically, in the destroyed cortex, 24 h after an ibo injection. Third, immunohistochemically and electron-microscopically, radial glia appear at least partially intact 24 h after an injection. As one would expect/46/, intact, probably migrating neurons can be seen in contact with radial glia.
A puzzling aspect of the recovery after ibo injections is that layers II and III of the ibo-injured cortex are thicker than the same layers in the intact cortex. A similar observation was reported by Dvorak and Feit /11/ following localized freezing of the newborn rat cortex (see below). Since the density of neurons in the expanded layers II and III is not noticeably less than in the corresponding normal layers, the number of neurons in these layers has probably increased. The absence of labeled neurons following the [3H]thymidine injection after ibo injection suggests that neurogenesis, which is normally finished a few days before birth/36/, is not reactivated by the ibo injections, although negative results such as this are, by their nature, somewhat inconclusive. The most likely explanation for the apparent hypertrophy of layers II and III seems to be that either the partial destruction of glial channels resulted in an accu-mulation of neurons destined to the superficial layers within a reduced surface of cortex, or the loss of the deep layers protects neurons in the upper layers against the neuronal death which may occur in the developing neocortex /14,16,17,45/. Tangential measurements of the ibo-injured cortex could clarify this issue, but are difficult since the lateral border of area 18 in the cat is ill defined by cytoarchitectonic criteria.
Some variability in the severity of the lesion was noticed in the present series of experiments and in a new series in progress/1/in kittens for which the conditions of injection (including amount of ibo, location and age of the animal) appeared very similar. While this variability turned out to be advantageous for some of the studies we performed, we are nevertheless puzzled by it. A possible cause is that the depth of anaesthesia, which was induced with Ketamine, an antagonist of the NMDA receptor/60/, may have differently protected individual kittens. On the other hand, the intensity of inflammatory phenomena following ibo-injection and neuronal destruction may vary and thus modulate the severity of lesions.

Microgyda and ibotenic acid lesions
There are striking similarities between the ibo-induced lesions and two human cortical displasias: microgyria (or polymicrogyria) and ulegyria. Both displasias usually affect circumscribed territories of the cerebral cortex. The typical human microgyric cortex forms numerous small gyri and sulci (hence its name) and is four layered. The most superficial, cell-poor layer is continuous with layer I of the normal cortex. The outer cell layer normally consists of two sublayers: the external one contains densely packed small cells and is continuous with the normal layer II; the internal one contains pyramids and is continuous with normal layer III. In Golgi studies, similar cell types were found in the normal and microgyric layers II and III/64/. Underneath, there is a cell poor layer, continuous with layers IV and V of the intact cortex and another cell layer continuous with layer VI of the intact cortex. At the transition between microgyric and normal cortex another sublayer continuous with layer IV can be found/34,39,47/. This architecture closely resembles that found at the periphery of the ibo injection. However, in the cortex neonatally injured by ibo, layer VI is usually destroyed near to the center of injection. In the microgyric cortex, layer VI may be preserved because it is vascularized differently from the superficial layers and therefore differently affected by ischemia /11/ which is the probable cause of microgyria. On the other hand there are many deviations from the typical microgyric pattern, and layer VI is not always preserved/32,39/. The latter is illustrated in Fig. 14; the similarity between human microgyria and ibo-lesioned cortex is particularly clear at the transition between normal and displasic cortex.
Although ibo can induce the formation of sulci and undulations in the cell layers of the gray matter, the degree of folding is always less than that characterizing typical polymicrogyria. This difference may not be a substantial one. According to a mechanical model of cortical convolutional development /48/, the "convolutional wavelength" (i.e., the intersulcal or intergyral distance) may be dictated by the differential growth of the different layers both in normal and microgyric cortex. There may be species, age and place differences in this differential growth since the degree of folding greatly varies across mammals. Therefore, the loss of the deep layei's may not be a sufficient condition for the development of abnormal convolutions. However, the model predicts that the "convolutional wavelength" in the microgyric cortex should tend to be proportional to the thickness of the cortex. To some extent this seems also to be the case in the ibo-injured cortex, since in the animals injected on pd 6, where the thinning of the cortex is greater than in animals injected earlier, the tendency to the formation of abnormal cortical folding is also greater.
Little or no abnormal folding but severe thinning of the cortex is found in human ulegyria /4,42/. Fig. 15 illustrates the similarity between ulegyria and the lesions provoked in the cat cortex by ibo injections towards the end of neuronal migration (e.g. Fig. 6, D and 13, C). The ulegyric cortex in this case is a thin neuronal layer continuing layer III of the normal cortex. At the transition with the normal cortex, layer VI reappears before layers V and IV.
The pathogenesis of human microgyria may be similar to that of ibo lesions. In both conditions, the cause seems to act before the end of neuronal migration /11,39/. An experimental VOLUME 2, NO. 1,1991 Fig. 14: Two transitions between the normal and the microgyric cortex in the human case 308SN82. Notice the progressive loss of infragranular and granular layers and the similarity with the experimental cases illustrated in Fig. 8. Calibration bar is 300/m. model of microgyria was obtained by Dvorak and Feit /11/ by locally freezing the neocortex in newborn rats. Freezing induced necrosis of variable degree but at least some migrating neurons survived the insult and continued migration, giving rise to microgyric layers II and III. This course of events resembles that described here.
However, in Dvorak's and Feit's model, the neuronal migration stopped at the bottom of the region of total necrosis provoked by the lesion.
Therefore, the microgyric layers II and III were infolded. The association of microgyria with porencephaly, "a deficit of the wall of the cerebral hemisphere without accompanying cranial or facial malformations"/34/, might occur ff the pathological process which destroys postmigratory neurons, or secondary pathology associated with it, also destroys the radial glia, and/or the migrating neurons, thus preventing the partial recovery which leads to the formation of the microgyric cortex. Destruction of radial glia probably occurred in those experiments in which ibo injections evolved towards the formation of cysts accompanied with massive destruction of the white matter and no recovery of cortical structure.
Both microgyria and ulegyria (the latter is more rare and its pathogenesis less well understood than the former) are usually restricted to an identifiable vascular territory and this is a strong argument supporting ischemia as their cause /34,39/. Recent evidence suggests an important excitotoxic component in the neuronal damage due to brain ischemia or hypoglycemia in the adult. Indeed, the glutamate levels increase in the ischemic brain and the damage due to ischemia can be prevented by specific blockers of NMDA receptors /56,62/. Activation of the NMDA receptor may thus be the common step leading to neuronal destruction in both human microgyria/ulegyria and in the present experiments. In both conditions excitotoxicity may be enhanced by the presence in the white matter and the deep cortical layers of high levels of aspartate and glutamate released by the terminals of transitory axons as well as by the especially great efficiency of NMDA receptors in the young cortex/61L