This study evaluated hippocampal inhibitory function and the level of expression of y-aminobutyric acid type A (GABAA) receptor mRNA in an in vivo model of epilepsy. Chronic recurrent limbic seizures were induced in rats using injections of pilocarpine. Electrophysiological studies performed on hippocampal slices prepared from control and epileptic animals 1 to 2 months after pilocarpine injections demonstrated a significant hyperexcitability in the epileptic animals. Reduced levels of mRNA expression for the CY2 and a5 subunits of the GABAA receptors were evident in the CA1, CA2, and CA3 regions of the hippocampus of epileptic animals. No decrease in mRNA encoding al, 182, or y2 GABAA receptor subunits was observed. In addition, no change in the mRNA levels of a CaM kinase II was seen. Selective decreases in mRNA expression did not correlate with neuronal cell loss. The results indicate that selective, long-lasting reduction of GABAA subunit mRNA expression and increased excitability, possibly reflecting loss of GABAergic inhibition, occur in an in vivo model of partial complex epilepsy.-y-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter system in mammalian brain (1) and has been implicated in playing an important role in the induction and maintenance of epileptogenesis in several experimental models (2-6). In brain tissue from epileptic patients, decreased levels of GABA receptor-binding and of GABA-synthesizing enzymes have been documented (3). Furthermore, reduced benzodiazepine receptor binding in patients with temporal lobe epilepsy has been demonstrated in vivo by positronemission tomography (7). In animal models of epilepsy, changes in the level of GABA type A (GABAA) receptor binding have also been observed in specific hippocampal regions in kindled rats (8,9). Studies from our laboratories have shown a decrease in GABAA receptor binding in rat brain membranes (10) and a reduction in the physiological response to GABA in acutely isolated CAl hippocampal neurons (11) after the induction of epileptogenesis and persistent seizure discharge by pilocarpine injection. Thus, a considerable body of evidence indicates that decreased GABA receptor function and/or expression may play an important role in the induction and maintenance of epilepsy.Although GABA systems are altered in epilepsy, the specific molecular mechanisms mediating these changes have not been clearly elucidated (4,6). Long-term changes in gene expression produced by the induction of epileptogenesis have been suggested to play an important role in the persistent hyperexcitability observed in chronic epilepsy (12,13

Specialized plasma membranes from the endocytic complex of ileal epithelial cells of suckling rats were isolated by differential flotation. Thin-section and negativestain electron microscopy showed the luminal surfaces of these membranes to be covered by an ordered array of particles -7.5 nm in diam joined together with 14.5-nm separations in long rows. This particulate coating was released from the membrane surfaces by 10 mM CaC12 and recovered free of membranes after dialysis against 0.5 mM EGTA and high-speed centrifugation. Two proteins were resolved by gel filtration to be in the supernate: n-acetyl-/3-glucosaminidase and a filamentous protein which attaches n-acetylglucosaminidase to the membrane surface thereby providing bidirectionality to the array of enzyme.We believe that the filamentous protein has not been previously described.Therefore we have called it ligatin from the latin ligare, which translates "to bind together." Furthermore, we suggest that the membranes of the endocytic complex contain sites for the extracellular digestion of carbohydrate moieties in the maternal milk.

Several models that develop epileptiform discharges and epilepsy have been associated with a decrease in the activity of calmodulin-dependent kinase II. However, none of these studies has demonstrated a causal relationship between a decrease in calcium/calmodulin kinase II activity and the development of seizure activity. The present study was conducted to determine the effect of directly reducing calcium/calmodulin-dependent kinase activity on the development of epileptiform discharges in hippocampal neurons in culture. Complimentary oligonucleotides specific for the α subunit of the calcium/calmodulin kinase were used to decrease the expression of the enzyme. Reduction in kinase expression was confirmed by Western analysis, immunocytochemistry, and exogenous substrate phosphorylation. Increased neuronal excitability and frank epileptiform discharges were observed after a significant reduction in calmodulin kinase II expression. The epileptiform activity was a synchronous event and was not caused by random neuronal firing. Furthermore, the magnitude of decreased kinase expression correlated with the increased neuronal excitability. The data suggest that decreased calmodulin kinase II activity may play a role in epileptogenesis and the long-term plasticity changes associated with the development of pathological seizure activity and epilepsy.

To test the hypothesis that repeated subconvulsive stimulations required to induce kindling can permanently alter gene expression of hippocampal neurons, we used Northern and in situ hybridization analyses to measure steadystate mRNA levels encoding several phenotypic proteins. mRNA encoding a membrane-bound protein, ligatin, was significantly reduced in kindled brains relative to naive and sham control animals. This change in gene expression persisted for over 4 months after kindling, was associated with a decrease in ligatin protein expression, was not produced by single or multiple seizures that did not induce the kindling phenomena, and was blocked by MK801. These results provide direct evidence that kindling can cause persistent changes in the expression of specific genes in hippocampal neurons and suggest that N-methyl-D-aspartate receptor-activated changes in gene expression may be a basic molecular mechanism underlying some of the long-lasting plasticity changes seen in kindling or models of long-term memory.

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