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Developmental cortical malformations are common in patients with intractable epilepsy; however, mechanisms contributing to this epileptogenesis are currently poorly understood. We previously characterized hyperexcitability in a rat model that mimics the histopathology of human 4-layered microgyria. Here we examined inhibitory and excitatory postsynaptic currents in this model to identify functional alterations that might contribute to epileptogenesis associated with microgyria. We recorded isolated whole cell excitatory postsynaptic currents and GABA(A) receptor-mediated inhibitory currents (EPSCs and IPSCs) from layer V pyramidal neurons in the region previously shown to be epileptogenic (paramicrogyral area) and in homotopic control cortex. Epileptiform-like activity could be evoked in 60% of paramicrogyral (PMG) cells by local stimulation. The peak conductance of both spontaneous and evoked IPSCs was significantly larger in all PMG cells compared with controls. This difference in amplitude was not present after blockade of ionotropic glutamatergic currents or for miniature (m)IPSCs, suggesting that it was due to the excitatory afferent activity driving inhibitory neurons. This conclusion was supported by the finding that glutamate receptor antagonist application resulted in a significantly greater reduction in spontaneous IPSC frequency in one PMG cell group (PMG(E)) compared with control cells. The frequency of both spontaneous and miniature EPSCs was significantly greater in all PMG cells, suggesting that pyramidal neurons adjacent to a microgyrus receive more excitatory input than do those in control cortex. These findings suggest that there is an increase in numbers of functional excitatory synapses on both interneurons and pyramidal cells in the PMG cortex perhaps due to hyperinnervation by cortical afferents originally destined for the microgyrus proper.

Lesions that occur either during fetal development or after postnatal brain trauma often result in seizures that are difficult to treat. We used two animal models to examine epileptogenic mechanisms associated with lesions that occur either during cortical development or in young adults. Results from these experiments suggest that there are three general ways that injury may induce hyperexcitability. Direct injury to cortical pyramidal neurons causes changes in membrane ion channels that make these cells more responsive to excitatory inputs, including increases in input resistance and a reduction in calcium-activated potassium conductances that regulate the rate of action potential discharge. The connectivity of cortical circuits is also altered after injury, as shown by axonal sprouting within pyramidal cell intracortical arbors. Enhanced excitatory connections may increase recurrent excitatory loops within the epileptogenic zone. Hyperinnervation attributable to reorganization of thalamocortical, callosal, and intracortical circuitry, and failure to prune immature connections, may be prominent when lesions affect the developing neocortex. Finally, focal injury can produce widespread changes in gamma-aminobutyric acid and glutamate receptors, particularly in the developing brain. All of these factors may contribute to epileptogenesis.

The presence of developmental cortical malformations is associated with epileptogenesis and other neurological disorders. In recent years, animal models specific to certain malformations have been developed to study the underlying epileptogenic mechanisms. Teratogens (chemical, thermal or radiation) applied during cortical neuroblast division and migration result in lissencephaly and focal cortical dysplasia. Animals with these malformations have a lowered seizure threshold as well as histopathologies typical of those found in human dysgenic brains. Alterations that may promote epileptogenesis have been identified in lissencephalic brains, such as increased numbers of bursting types of neurons, and abnormal connections between hippocampus, subcortical heterotopia, and neocortex. A distinct set of pathological properties is present in animal models of 4-layered microgyria, induced with cortical lesions made during late stages of cortical neuroblast migration. Hyperexcitability has been demonstrated in cortex adjacent to the microgyrus (paramicrogyral zone) in in vitro slice preparations. A number of observations suggest that cellular differentiation is delayed in microgyric brains. Other studies show increases in postsynaptic glutamate receptors and decreases in GABA(A) receptors in microgyric cortex. These alterations could promote epileptogenesis, depending on which cell types have the altered receptors. The microgyrus lacks thalamic afferents from sensory relay nuclei, that instead appear to project to the paramicrogyral region, thereby increasing excitatory connectivity within this epileptogenic zone. These studies have provided a necessary first step in understanding molecular and cellular mechanisms of epileptogenesis associated with cortical malformations.

Polymicrogyria, a developmental cortical malformation associated with epilepsy, can be modeled in rats with a transcortical freeze lesion on the day of birth (P0) or P1. We have used field potential recordings to characterize the incidence, propagation patterns, and distribution of epileptiform activity in slices from rats with experimental microgyri. Interictal-like epileptiform activity was evoked in slices from 85% of freeze-lesioned rats aged P12-P118. These data show age-specific properties of epileptogenesis, including: a delay in onset, a decrease in the incidence of epileptiform activity in rats >P40 that was specific to those lesioned on P0 as opposed to P1, and a shift in the likely site of initiation to areas further from the microgyrus in mature animals. Several observations suggest that the area adjacent to the microgyrus, which appears histologically normal in Nissl stains, contains the necessary epileptogenic neuronal circuits: 1) in 78% of slices, epileptiform activity could be evoked only from a focal zone adjacent to the microgyrus (paramicrogyral zone) and not within the microgyrus proper; 2) epileptiform activity consistently originated from a particular site within this paramicrogyral zone, independent of the location of the stimulating electrode, suggesting that the generator is outside of the microgyrus; 3) evoked epileptiform activities in the paramicrogyral cortex were unaltered after separation of this zone from the microgyrus with a transcortical cut; and 4) the short-latency graded field potential evoked in the paramicrogyral zone contained an additional negativity not seen in control slices. The epileptiform activity was blocked reversibly by N-methyl--aspartate receptor antagonists in slices from mature as well as immature freeze-lesioned rats. These results suggest that aberrant synaptic connectivity develops in rat cortex surrounding the microgyrus and produces a focal epileptogenic zone whose capacity to generate epileptiform activities does not depend on connections with the malformation itself. We hypothesize that afferents, originating from cortical and extracortical sites, lose their targets in the region of the malformation and make appropriate laminar contacts in the cortex adjacent to the malformation, creating an overabundance of excitatory input to this cortical zone. Increased excitatory feedback onto specific cortical elements may be one factor involved in epileptogenesis in this model of a cortical malformation.

Several lines of evidence have suggested that decreases in postsynaptic inhibition may have a role in epileptogenesis in cortical structures. However, other studies have suggested that GABAergic inhibition is spared, or even augmented in some forms of post-lesional epilepsy. In the studies described here, inhibitory events were recorded in two models of post-lesional chronic epileptogenesis. (i) As previously reported (D.A. Prince and G.-F. Tseng. J. Neurophysiol. 69: 1276-1291. 1993), epileptiform activity develops in slices from partially isolated rat neocortical islands 2-3 weeks after the initial in vivo lesion. In this model of post-traumatic epilepsy, large amplitude polyphasic inhibitory postsynaptic currents (IPSCs) in layer V pyramidal neurons are associated with each interictal epileptiform field potential. The frequency of spontaneous IPSCs as well as miniature IPSCs was significantly increased in neocortical slices from the epileptogenic chronically injured cortex versus controls. Immunocytochemical reactions for parvalbumin and calbindin, calcium binding proteins present in subgroups of GABAergic neurons, showed an increased staining of both neuropil and somata within the epileptogenic tissue. Immunoreactivity for glutamic acid decarboxylase (GAD) and GABA also appeared to be increased in the neuropil. (ii) Cortical microgyri resembling human malformations were produced by freeze lesions made transcranially in P0 rat cortex (K.M. Jacobs, M.J. Gutnick, and D.A. Prince. Cereb. Cortex, 6: 514-523. 1996). The boundary between the four-layered microgyrus and surrounding cortex become epileptogenic within about 2 weeks, as judged by evoked extracellular field potentials and cellular activities. Epileptogenesis in the surrounding cortex is not altered when the microgyrus itself is isolated by transcortical cuts. Patch-clamp recordings from layer V neurons in the epileptogenic zone showed that spontaneous IPSCs are larger and more dependent on glutamatergic synapses than in control neurons. The amplitudes of polysynaptic IPSCs evoked by threshold stimulation were also larger than in control cells. Although evaluation of inhibitory events in these models is still incomplete, results to date suggest that GABAergic inhibition may be enhanced in epileptogenic areas associated with chronic cortical injury. Sprouting of axonal arborizations of pyramidal cells onto interneurons, upregulation of GABAergic neurons, and perhaps sprouting of inhibitory axons that make increased numbers of contacts onto pyramidal cells may all contribute to the increased inhibitory drive. Results in these models do not support the disinhibitory hypothesis of chronic epileptogenesis.

The presence of developmental cortical malformations has been associated with the occurrence of epilepsy, and correlative anatomic-clinical electrophysiological studies suggest that microdysgenic lesions may actually initiate epileptiform activity. We have investigated the electrophysiological properties of an animal model of polymicrogyria created by making cortical freeze lesions in rat pups at P0 or P1. Such lesions create microgyri with histological features similar to those of human polymicrogyria. We have determined that there is a focal region of hyperexcitability around the lesion in this rat microgyrus. Field potentials evoked by stimulation within a few millimeters of the microgyrus have characteristics typical of epileptiform activity. This aberrant activity is seen as early as 12 d after the lesion, as well as in animals as old as 118 d. Immunochemical staining for the calcium binding protein, parvalbumin, shows a decrease in neuronal and neuropil staining within the microgyrus. These findings suggest that inhibition might be decreased within the lesion, which may contribute to generation of the adjacent hyperexcitable region. These results demonstrate that this animal model is appropriate for examining the mechanisms contributing to epileptogenesis associated with a cortical malformation.

Calcium-calmodulin protein kinase II and GAP43 are two molecules which have been linked to synaptic plasticity. Localization of mRNA for these molecules identifies the neuronal populations which have the potential to utilize these mechanisms. General descriptions for calcium-calmodulin protein kinase II or GAP43 mRNA have been previously reported. In light of recent evidence that suggests that at some sites these two molecules may interact, we sought to determine the cortical distribution in detail, and to examine the extent of overlap between neuronal populations containing each mRNA. To this end we have used in situ hybridization techniques to study the distribution of calcium-calmodulin protein kinase II and GAP43 mRNA in adjacent sections of adult rat forebrain. Overall, the distribution patterns were distinct but partially overlapping. For both calcium-calmodulin protein kinase II and GAP43, mRNA levels were highest in hippocampus, allo- and neocortex, compared to moderate to low levels in striatum and thalamic nuclei. Within the heavily labeled regions certain populations expressed both calcium-calmodulin protein kinase II and GAP43 mRNA at high levels, while other populations were selective for calcium-calmodulin protein kinase II. In the hippocampus, the stratum pyramidale of CA1-3 expressed high levels of both calcium-calmodulin protein kinase II and GAP43 mRNA. Granule cells of the fascia dentata and the stratum radiatum of CA3 both contained moderate to high levels of calcium-calmodulin protein kinase II mRNA, but near background levels of GAP43 mRNA label. Within the neocortex, deep layers were distinguished from superficial layers by their lack of calcium-calmodulin protein kinase II mRNA expression within the neuropil, and the presence of GAP43 mRNA in neurons located in layer V and the deepest part of layer VI. Thus, layer V and deep layer VI neurons showed high levels of label for both GAP43 and calcium-calmodulin protein kinase II mRNA, while neurons of superficial layers contained only calcium-calmodulin protein kinase II mRNA. These markers differentiate neuronal populations which can also be distinguished on the basis of their ability to undergo specific forms of synaptic plasticity. These different forms of plasticity may be due in part to the laminar-specific patterns of GAP43 and calcium-calmodulin protein kinase II mRNA that we have described.