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.

Neural correlates of long-term sensitization of defensive withdrawal reflexes in Aplysia occur in sensory neurons in the pleural ganglia and can be mimicked by exposure of these neurons to serotonin (5-HT). Studies using inhibitors indicate that transcription is necessary for production of long-term facilitation by 5-HT. Several mRNAs that change in response to 5-HT have been identified, but the molecular events responsible for long-term facilitation have not yet been fully described. To detect additional changes in mRNAs, we investigated the effects of 5-HT (1.5 hr) on levels of mRNA in pleural-pedal ganglia using in vitro translation. Four mRNAs were affected by 5-HT, three of which were identified as calmodulin (CaM), phosphoglycerate kinase (PGK), and a novel gene product (protein 3). Using RNase protection assays, we found that 5-HT increased all three mRNAs in the pleural sensory neurons. CaM and protein 3 mRNAs were also increased in the sensory neurons 4Corresponding author.by sensitization training. Furthermore, stimulation of peripheral nerves of pleural-pedal ganglia, an in vitro analog of sensitization training, increased the incorporation of labeled amino acids into CaM, PGK, and protein 3. These results indicate that increases in CaM, PGK, and protein 3 are part of the early response of sensory neurons to stimuli that produce long-term facilitation, and that CaM and protein 3 could have a role in the generation of long-term sensitization.

Serotonin (5-HT) shifts the phase of the circadian oscillator of the eye of Aplysia californica in a phase dependent manner. This indicates that 5-HT acts, either directly or through some intermediaries, on a component of the oscillator. Since our goal is to identify the components of the oscillator, we are following the pathway through which 5-HT has its effect on the rhythm. The effect of 5-HT on the rhythm has been shown to be mediated by an increase in intracellular cyclic adenosine-3',5'-monophosphate (cAMP). The most likely action of cAMP is to activate cAMP-dependent protein kinase. Therefore, we used two-dimensional polyacrylamide gel electrophoresis to investigate changes in 32P labelled phosphoproteins which occur with 5-HT and other treatments. Fourteen proteins showed increased incorporation of 32P when eyes were exposed to treatments of 5-HT from CT 06 to 12. Two proteins showed decreased incorporation. 8-bt-cAMP mimicked all but one of the increases and both decreases in incorporation produced by 5-HT. 8-bt-cAMP increased incorporation into three additional proteins and decreased incorporation into three others that were not affected by 5-HT. Incorporation into one protein was increased by 5-HT but decreased by 8-bt-cAMP. By comparison, light, which has little or no effect on the rhythm at this phase, only affected one protein. The protein increased by light was also increased by 5-HT. Tetradecanoic phorbol acetate (TPA), administered during CT 06-12, also had little effect on the rhythm at this phase. TPA increased incorporation into twenty proteins and decreased incorporation into three.(ABSTRACT TRUNCATED AT 250 WORDS)