Abstracts

Rationale: Developmental cortical malformations including polymicrogyria and tuberous sclerosis are associated with intractable epilepsy. To understand the pathophysiology of epilepsies associated with cortical malformations, we have utilized glutamate imaging in the freeze-lesion (FL) model of polymicrogyria. In this model neurons that form deep cortical layers are lost, resulting in a microgyrus enriched in layer II/III neurons. Our preliminary findings have shown that the ability of glial cells to remove applied extracellular glutamate is altered in the FL model. Regional differences in glutamate reuptake were measured using glutamate imaging and SR101 staining was used to quantify the location of astrocytes in and around the lesion site. Immunohistochemical analysis of multiple glial cell markers was also used to examine the local density and phenotype of astrocytes in the malformed cortex. Methods: Microgyri were created by briefly placing a freezing probe on the skulls of neonatal rat pups. Neocortical brain slices from sham operated and freeze lesioned rats were prepared 14-128 days later. Brain slices were then loaded with glutamate FRET biosensor and both images and extracellular field recordings were collected simultaneously. Brain slices were also stained with SR101, an astrocyte specific dye for use in live tissue, and maps of glial density were made using in-house imaging software. Results: In order to thoroughly address the regional variability in glutamate reuptake capacity we locally perfused 5 mM glutamate onto the microgyral zone (MZ). Glutamate reuptake capacity was increased in the MZ itself while in areas adjacent to the MZ glutamate reuptake capacity was compromised. As predicted, glutamate reuptake capacity was directly correlated to glial cell density as measured by SR101 staining. In regions where glial cell density was highest there was a higher capacity to remove applied glutamate. This correlation was lost when TBOA, an antagonist of the plasma membrane glutamate transporters, was applied to the tissue before perfusion of glutamate. Immunohistochemical analysis of GFAP, ALDH1L1, a pan-glial marker, and GLT-1, the astrocytic glutamate transporter, was performed. Interestingly ALDLH1 and GLT-1 appear to be increased in the MZ where glutamate reuptake capacity was highest and GFAP staining was more abundant directly adjacent to the MZ, where glutamate reuptake capacity was lower. Conclusions: Our findings indicate that there are anatomical and functional changes in astrocytes in the FL model. The decrease in glutamate reuptake capacity directly adjacent to the MZ and the increased expression of GFAP in this area suggests that reactive astrocytes cannot efficiently clear synaptically released glutamate. This may in turn lead to prolonged glutamate signaling and contribute to the hyperexcitability of the FL. The increase in astrocytic protein expression and regional glutamate uptake within the MZ, on the other hand, suggests that the increased density of glia in the MZ may contribute to more efficient glutamate clearance within this region.