Abstracts

We sought to visualize the activity of entire populations of neurons in a rat seizure model. Endogenous excitation in hippocampal brain slices can be mapped with the channel-permeant organic cation 1-amino-4-guanidobutane (AGB), which is highly selective for ionotropic glutamate receptor (iGluR) activation (Marc, 1999). Quantitative immunogold labeling of small amino acids (such as GABA, glycine, taurine, etc) can be performed on the same sections in which AGB mapping is evaluated (Marc and Jones, 2002). We used these approaches to test the hypothesis that functional excitatory neural circuits would be altered in medial entorhinal (mEC)-hippocampal (HC) brain slices obtained from kainic acid (KA)-treated rats. Sprague-Dawley rats were repeatedly injected with KA (5 mg/kg, i.p.) until stage 4/5 seizures were observed. mEC-HC brain slices (400 [mu]m) were prepared 1 wk later from KA-treated or age-matched control rats. Slices were incubated in oxygenated ACSF at room temperature for 35 min, after which 5 mM AGB was added to the solution for 10 min. Slices were fixed, dehydrated, embedded in epoxy resin and serially sectioned at 250 nm onto 12-spot Teflon-coated slides and probed for AGB and other targets. Signals were detected with goat anti-rabbit IgGs adsorbed to 1nm gold particles (Amersham), silver intensified, and captured as 8 bit images. Images were computationally registered with 200 nm precision and visualized as multichannel datasets. AGB signals indicative of iGluR activation could be detected in slices obtained from control and KA-treated animals, although the distribution and intensity of staining was quite different between groups. Sections from control animal brain slices showed minimal AGB signals in the CA3 and CA1 pyramidal cell body layer and the hilar region of the dentate gyrus. In contrast, sections from slices of KA-treated animals displayed high AGB signals in select cells of the hilus, along with heterogeneous expression in the CA3 and CA1 pyramidal cell body layer. In slices obtained from KA-treated animals, AGB signals were observed in layers 2 and 4/5 of the mEC as well, with diminished activity in layer 3. In addition, glutamine, glutamate, GABA, taurine, and aspartate profiling revealed heterogeneous metabolite signatures in CA1 pyramidal cells as well as alterations in those signatures in KA-treated rats. AGB mapping of brain slices from normal and epileptic tissue is a viable and valuable technique with which to assess changes in functional neural circuits. Our data also suggests that combined approaches of AGB mapping and quantitative immunogold labeling of small amino acids will provide insight into classes of neurons participating in seizure generation and propagation. (Supported by R01 EY02576 (REM), RPB Senior Research Scholar Award (REM), RO1-NS44210 (KSW).)