Abstracts

The conversion of glycogen to glucose in astrocytes is thought to supply essential metabolic substrates to neurons during periods of heightened activity or stress. The breakdown of glycogen is catalyzed by glycogen phosphorylase (GP), which is found in 2 forms: active, phosphorylated (GP[italic]a[/italic]) and inactive, dephosphorylated (GP[italic]b[/italic]). We asked if status epilepticus, which involves extreme periods of neuronal activation, followed by gliosis in several brain areas, would produce alterations in glial metabolism that would be reflected by changes in GP. We focused on the entorhinal cortex (EC) because GP has striking pattern of expression, and our method to induce status produces highly reliable degeneration and gliosis in this area. Male Sprague-Dawley rats (30-34 days old) were injected with 380 mg/kg pilocarpine i.p. 30 min after 1 mg/kg atropine methylbromide s.c. Controls received the same treatment but saline instead of pilocarpine. Status epilepticus was truncated by 5 mg/kg i.p. diazepam after 1 hr. Either 1 hr, 1 wk or 1 month after status, brains were flash frozen (^-50 [deg]C). Horizontal sections (35 [mu]m) cut on a cryostat were processed concurrently for GPa and total GP ([italic]t[/italic]GP; GP[italic]a[/italic]+GP[italic]b[/italic]) or were double-labeled immunocytochemically for neurons (NeuN) and astrocytes (GFAP). All procedures were performed on frozen, non-fixed tissue because perfusion fixation denatures GP protein. A clear pattern of reactivity was revealed by GP[italic]a[/italic] and [italic]t[/italic]GP in control rats that was highest in the superficial layers (see Fig. 1A for example of control condition; n=17/17). GP[italic]a[/italic] and [italic]t[/italic]GP reactivity was reduced in a layer-specific pattern when examined 1 hr after status (n=6/6) and irregular patches of apparent GP depletion occurred at 1 hr throughout the EC (n=5/6). At later times GP reactivity was enhanced (1 wk, n=5/5; 1 month, n=4/6) and localized hot spots punctuated superficial layers, parasubiculum and to a lesser extent, deep layers (1 wk, n=5/5, see arrows in Fig. 1B; 1 month, n=6/6).[figure1] GP histochemistry delineates the superficial layers of the EC in normal rats, and status epilepticus produces immediate (reduction) and long-term (enhancement)alterations in the pattern of reactivity. These data highlight the potential importance of GP to normal EC function, and its response to severe seizures. (Supported by an AES-Milken award to SGW and NS 16102.)