Effects of Kainic Acid on mRNA Expression of GABA, Receptor Subunits in Rat Hippocampus

Abstract

Purpose: Kainic acid (KA)‐induced seizures in rats have been studied as an animal inodel for human temporal lobe epilepsy. After KA induced acute status epilepticus, neuropathological examination shows neurodegeneration in the CAI and CA3 sector 01 the Ammon's horn and or interneurons i n the hilus of the dcntatc gyrus. Thc GABA, receptor is a ligand‐gated chloride channel consisting of live membrane‐spanning proteins for which al least five different gene families have been identified in the mammalian brain. We investigated changes in inRNA expression of 13 GABA, receptor subunits i n the hippocampus of rats at viirious time intervals after acutc KA‐induced status epilepticus. We also studied scizurc‐related neuronal cell loss during and after acquisition of spontaneous recurrent scizures. Methods: Male Sprague‐Dawlcy rats (250–350 g) were injected with 10 mg/kg of KA in buffered salinc or the corresponding amount of saline without KA as a control. Rats were decapitatcd 6 hours, 12 hours, 24 hours, 7 days, and 30 days after KA injection. The brains were cut into coronal sections (20 pm) and mountcd on chrome‐alumgelatin‐coated slides. For in situ hybridization, synthetic oligonucleotides were labeled with I'SI‐a‐thio‐ATP. Thc oligonucleotide sequences were mostly derived from those used by Wisden and coworkers. The frozen sections rapidly were immersed in 2% paraCormaldehyde, PBS for 10 minutes, immersed in 0.25% acetic anhydride in 0. IM triethylamine hydrochloride for 10 minutes, dehydrated by an ethanol series, and de‐lipidized with chloroform. They were hybridized with about 2nM of labeled oligonucleotide probe in Soy1 hybridization buffer at 42°C for 16–24 hours. Alter thc incubation, the slides were briefly rinsed twice in IxSSC, SO% formamidc, then they were washed four times in SO% formamidc i n 2xSSC (42°C 15 minutes), rinsed in IxSSC, dipped briefly into watcr then into 70% ethanol, dried, and exposed to 13‐max X‐ray film for one to two weeks. Autoradiographical images were digitalixd using an Appligenc Imager and evaluated using the public domain NIH‐image program. For each time interval, sections ohtained from thrcc to six cxperimenlal animals and from 14 controls were examined. Statistics were pel‐formed using the two‐tailed Student's T‐test. Results: In the granule cell layer, initial (after 6 to 12 hours) decreases in a2. a3, 015, P I, p3, y2, and 6 mRNAs (by about 25%) to 50%) were accompanied by increases (by about 50%) in a I, a4, and p2 mcssagcs. At later intervals (alter 7 to 30 days), cxpression of α12, α4, β3, and β2 mRNAs recovered to control values, w i h a5 and 6 mKNA still being reduced (by 15% and 40% below control levcls, respectively). Concentrations of the transcripts encoding for OL I, α3, (3 I, and p2 became markedly enhanced (between 20% and 50% of controls). Within the pyramidal cell layers CAI and CA3, decrcases in (α2, α4, α15, β 1–3, and γ2 mRNAs were detected after 7 to 30 days, reflecting pronounced neurodegcneration in these areas. The a I transcript W Z I decreased in CA3 after 24 hours and increased to control levcls indicating compensatory up‐regulation of this message after 7 days. mRNAs encoding for α3, βI, and y3‐subunits were detected at rather low levels.ot6 was not present in the hippocampus. Conclusions: Our data suggest fast, transient changes in thc expression of mRNAs encoding for different subunits of the GABA, receptor in the granule cell layer of thc dentate gyrus in response to KA‐induced seizures. Changes in the expression of GABA, receptor subunits may reflect altered GABAergic transmission during development of the epileptic syndrome. Changes within the pyramidal cell layer are mostly determined by concomitant neurodegencrative processes.