1 After 8 days in vitro, rat cerebellar granule cells were exposed to 1 mM y-aminobutyric acid (GABA) for periods of 1, 2, 4, 6, 8 and 10 days. The effect of the GABA exposure on GABAA receptor ax, a6 and #2,3 subunit protein expression and al and a6 subunit steady-state mRNA levels, was examined using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. 2 GABA exposure for 2 days decreased al (35+10%, mean+s.e.mean), ,B2,3 (21+9%) and a6 (28 +10%) subunit protein expression compared to control levels. The GABA-mediated reduction in al subunit expression after 2 days treatment was abolished in the presence of the GABAA receptor antagonist, Ru 5135 (10 uM). 3 GABA exposure for 8 days increased al (26+10%, mean+s.e.mean) and ,B2,3 (56+23%) subunit protein expression over control levels, whereas a6 subunit protein expression remained below control levels (by 38+10%). However, after 10 days GABA exposure, a6 subunit protein expression was also increased over control levels by 65+29% (mean + s.e.mean). 4 GABA exposure did not change the al or a6 subunit steady-state mRNA levels over an 8 day period, nor did it alter the expression of cyclophilin mRNA over 1-8 days. 5 These results suggest that chronic GABA exposure of rat cerebellar granule cells has a bi-phasic effect on GABAA receptor subunit expression that is independent of changes to mRNA levels. Therefore, the regulation of the GABAA receptor expression by chronic agonist treatment appears to involve posttranscriptional and/or post-translational processes.

Glutamate is the principle excitatory neurotransmitter in the mammalian CNS [l]. It's actions are mediated by two classes of receptor, namely the ionotropic excitatory amino add (EAA) receptors and the metabotropic EAA receptors. The ionotropic EAA receptors comprise an integral ion channel and are further subdivided into N-methyl-D-aspartate (NMDA), a-amino-3methyl-4-isoxasole propionate (AMPA) and kainate receptors on the basis of their pharmacology. In contrast, the metabotropic receptors are coupled to G-proteins, and their stimulation results in changes in the levels of cellular second messengers [2]. Excessive EAA receptor activation has been implicated in a wide variety of both acute and chronic neurological disorders including stroke, epilepsy and Huntington's disease [3,4]. As yet, the mechanisms which lead to neuronal injury are not fully understood.In order to investigate this, a model system was set up using cultured rat cerebellar granule cells. These neurons express a wide variety of EAA receptor subunits and subtypes [5-71 and have been used previously to study excitotoxicity [8,9]. The majority of these studies have been carried out using cells cultured in the presence of serum which has itself been implicated in the induction of excitotoxicity [lo] and in the regulation of EAA receptor subunit expression [ll]. For this reason, in this study, the neurons were cultured in the presence of a serum-free growth supplement (N,) to prevent any interference by serum. In addition, our technique produces relatively pure cultures of these neurons (>99%) and thus minimizes the effects of other cell types.Cerebellar granule cell cultures were prepared f m Sprague-Dawley rats on postnatal day 8 and cultured in Eaties Basal Medium supplemented with 10% (v/v) ,heat inactivated foetal calf serum, 25 mM KCI, 50 pgml gentamicin, 100 IU/ml penicillin and 100 pgml streptomycin. Cells were seeded into poly-D-lysine coated 96 well tissue culture plates at a density of 0.