Abnormal morphological and functional organization of the hippocampus in a p35 mutant model of cortical dysplasia associated with spontaneous seizures.

Cortical dysplasia is a major cause of intractable epilepsy in children. However, the precise mechanisms linking cortical malformations to epileptogenesis remain elusive. The neuronal-specific activator of cyclin-dependent kinase 5, p35, has been recognized as a key factor in proper neuronal migration in the neocortex. Deletion of p35 leads to severe neocortical lamination defects associated with sporadic lethality and seizures. Here we demonstrate that p35-deficient mice also exhibit dysplasia/ heterotopia of principal neurons in the hippocampal formation, as well as spontaneous behavioral and electrographic seizures. Morphological analyses using immunocytochemistry, electron microscopy, and intracellular labeling reveal a high degree of abnormality in dentate granule cells, including heterotopic localization of granule cells in the molecular layer and hilus, aberrant dendritic orientation, occurrence of basal dendrites, and abnormal axon origination sites. Dentate granule cells of p35-deficient mice also demonstrate aberrant mossy fiber sprouting. Field potential laminar analysis through the dentate molecular layer reflects the dispersion of granule cells and the structural reorganization of this region. Similar patterns of cortical disorganization have been linked to epileptogenesis in animal models of chronic seizures and in human temporal lobe epilepsy. The p35-deficient mouse may therefore offer an experimental system in which we can dissect out the key morphological features that are causally related to epileptogenesis.

Do glia have heart? Expression and functional role for ether-a-go-go currents in hippocampal astrocytes.

Potassium homeostasis plays an important role in the control of neuronal excitability, and diminished buffering of extracellular K results in neuronal Hyperexcitability and abnormal synchronization. Astrocytes are the cellular elements primarily involved in this process. Potassium uptake into astrocytes occurs, at least in part, through voltage-dependent channels, but the exact mechanisms involved are not fully understood. Although most glial recordings reveal expression of inward rectifier currents (K(IR)), it is not clear how spatial buffering consisting of accumulation and release of potassium may be mediated by exclusively inward potassium fluxes. We hypothesized that a combination of inward and outward rectifiers cooperate in the process of spatial buffering. Given the pharmacological properties of potassium homeostasis (sensitivity to Cs(+)), members of the ether-a-go-go (ERG) channel family widely expressed in the nervous system could underlie part of the process. We used electrophysiological recordings and pharmacological manipulations to demonstrate the expression of ERG-type currents in cultured and in situ hippocampal astrocytes. Specific ERG blockers (dofetilide and E 4031) inhibited hyperpolarization- and depolarization-activated glial currents, and ERG blockade impaired clearance of extracellular potassium with little direct effect on hippocampal neuron excitability. Immunocytochemical analysis revealed ERG protein mostly confined to astrocytes; ERG immunoreactivity was absent in presynaptic and postsynaptic elements, but pronounced in glia surrounding the synaptic cleft. Oligodendroglia did not reveal ERG immunoreactivity. Intense immunoreactivity was also found in perivascular astrocytic end feet at the blood-brain barrier. cDNA amplification showed that cortical astrocytes selectively express HERG1, but not HERG2-3 genes. This study provides insight into a possible physiological role of hippocampal ERG channels and links activation of ERG to control of potassium homeostasis.

Characterization of heterotopic cell clusters in the hippocampus of rats exposed to methylazoxymethanol in utero.

Cortical disorganization represents one of the major clinical findings in many children with medically intractable epilepsy. To study the relationship between seizure propensity and abnormal cortical structure, we have begun to characterize an animal model exhibiting aberrant neuronal clusters (heterotopia) and disruption of cortical lamination. In this model, exposing rats in utero to the DNA methylating agent methylazoxymethanol acetate (MAM; embryonic day 15) disrupts the sequence of normal brain development. In MAM-exposed rats, cells in hippocampal heterotopia exhibit neuronal morphology and do not stain with immunohistochemical markers for glia. In hippocampal slices from MAM-exposed animals, extracellular field recordings within heterotopia suggest that these dysplastic cell clusters make synaptic connections locally (i.e. within the CA1 hippocampal subregion) and also make aberrant synaptic contact with neocortical cells. Slice perfusion with bicuculline or 4-aminopyridine leads to epileptiform activity in dysplastic cell clusters that can occur independent of input from CA3. Taken together, our findings suggest that neurons within regions of abnormal hippocampal organization are capable of independent epileptiform activity generation, and can project abnormal discharge to a broad area of neocortex, as well as hippocampus.

Hippocampal dysplasia in rats exposed to cocaine in utero.

Prenatal cocaine exposure can result in neurobehavioral disturbances and structural modifications of the central nervous system. In the present study, cocaine was injected into pregnant rats and the brains of their offspring were examined at the light microscopic level. As adults, cocaine-exposed offspring exhibited subtle, but consistent, hippocampal abnormalities. In particular, the stratum pyramidale (particularly the CA1 region) was interrupted by frequent gaps in lamination, and ectopic pyramidal cells were found in stratum oriens and radiatum.

Estradiol increases the frequency of multiple synapse boutons in the hippocampal CA1 region of the adult female rat.

The effect of estradiol to increase the density of dendritic spines and axospinous synapses on hippocampal CA1 pyramidal cells in the adult female rat has been well-documented. However, presynaptic involvement in this process of synapse elimination and formation in the adult is unknown. To address this issue, we have reconstructed 410 complete presynaptic boutons through coded serial electron micrographs of CA1 stratum radiatum to determine the: (1) frequency of multiple (MSB) vs. single (SSB) synapse boutons; (2) number of synaptic contacts per MSB; (3) bouton volume and surface area; and (4) types of spines in synaptic contact with MSBs and SSBs in ovariectomized, estradiol-treated animals (OVX + E) versus ovariectomized oil-treated controls (OVX + O). Quantitative analysis of this tissue revealed that, in OVX + E animals, 45.0% of presynaptic boutons form multiple synaptic contacts with dendritic spines compared to 27.3% in controls (P < 0.01); the average number of synapses per dendritic spines compared to 27.3% in controls (P < 0.01); the average number of synapses per MSB was 2.7 in OVX + E animals compared to 2.3 in controls (P < 0.05). This represents a 25.5% increase in the number of synapses formed by a given number of presynaptic boutons in estradiol-treated animals (P < 0.01) which largely accounts for the previously observed estradiol-induced increase in axospinous synapse density. There was no treatment effect on bouton size; however, because MSBs are larger than SSBs, the increased frequency of MSBs in estradiol-treated tissue results in a trend toward an estradiol-induced increase in average bouton size. Additionally, MSBS were found to be more irregular in shape, i.e., significantly less spherical, than SSBs. Our results indicate that estradiol-induced dendritic spines form synapses primarily with preexisting boutons in stratum radiatum and that these boutons enlarge and change shape as they accommodate new synapses. Such findings suggest a relatively active role for dendrites in the process of adult synapse formation.

Loss of the p53 tumor suppressor gene protects neurons from kainate-induced cell death.

The tumor suppressor gene p53 recently has been associated with the induction of cell death in response to some forms of cellular damage. A possible role for p53-related modulation of neuronal viability has been suggested by the finding that p53 expression is increased in damaged neurons in models of ischemia and epilepsy. We evaluated the possibility that p53 expression (in knockout mice) is required for induction of cell damage in a model of seizure activity normally associated with well defined patterns of cell loss. Subcutaneous injection of kainic acid, a potent excitotoxin, induced comparable seizures in both wild-type mice (+/+) and mice deficient in p53 (-/-). Using a silver impregnation technique to examine neurodegeneration in animals killed 7 d after kainate injection, we found that a majority of +/+ mice exhibited extensive cell loss in the hippocampus, involving subregions CA1, CA3, the hilus, and the subiculum. Apoptotic cell death, as identified with an in situ nick end labeling technique to detect DNA fragmentation, was confirmed in CA1- but not CA3-degenerating neurons. In marked contrast, a majority of p53 -/- mice displayed no signs of cell damage; in the remaining p53 -/- mice, damage was mild to moderate and was confined almost entirely to cells in CA3b of the dorsal hippocampus. In +/+ mice, but not in -/- mice, damaged neurons also were observed in the amygdala, piriform cortex, cerebral cortex, caudate-putamen, and thalamus after kainate treatment. The pattern and extent of damage in mice heterozygous for p53 (+/-) were identical to those seen in +/+ mice, suggesting that a single copy of p53 is sufficient to confer neuronal vulnerability. These results demonstrate that p53 influences viability in multiple neuronal subtypes and brain regions after excitotoxic insult.