DISTRIBUTION AND FUNCTION OF GABAA RECEPTOR SUBUNITS IN THE AMYGDALA

Abstract

The amygdala has an important role in processing emotional information. Inappropriate processing within the amygdala is thought to induce anxiety disorders. Benzodiazepines, which act specifically on GABAA receptors, are commonly used as anxiolytics. This implies that GABAergic synapses within the amygdala may have an important role in inducing or compensating for these disorders. This thesis investigates the distribution and function of the GABAA receptor subunits in the amygdala. This study has made use of the real-time PCR, western blotting, and molecular cloning methods to determine expression levels of GABAA receptor subunits mRNAs and proteins and making recombinant GABAA receptors. We also used whole cell patch pipette recording to record the currents from transfected HEK 293 cells and acute brain slices containing amygdala. We examined the benzodiazepine pharmacology and subunit composition of the GABAA receptor subunits in the lateral and central amygdala. We examined the pharmacology of GABAA receptors by expressing different subunit combinations in HEK 293 cells and comparing the pharmacology with specific GABAergic inputs in the amygdala. Realtime PCR results showed that the GABAA receptor subunits are distributed differentially within the amygdalar nuclei with α2, α4, β2, β3, γ1 and γ2 abundant. In the case of the γ subunits, the expression level of the γ1 subunit was higher in the CeA whereas the expression level of γ2 was at high level in the LA. The α1 subunit was at very low level in the CeA and α2 was expressed at high level in the LA than in the CeA. The differences in the expression of α1, α2, γ1 and γ2 subunits in the amygdala suggest a diversity function for these subunits. Whole cell recording of HEK 293 cells showed that diazepam, zolpidem had different effects in these receptors depending on their compositions. Diazepam and zolpidem had low affinity for γ1-containing receptors. DMCM blocked the actions of GABA at α1β1γ2 and α2β1γ2 combinations (75 % current reduction) but had no effect at α1β1γ1 or α2β1γ1. In slice recordings DMCM blocked IPSCs by 70% in the lateral amygdala. In the central amygdala, DMCM had variable effects. Diazepam enhanced the amplitude of IPSC in the LA whereas the response in the CeA was either reduction or enhancement of currents. Similar results were obtained with zolpidem. Real-time PCR and whole cell recording from acute brain slices revealed a difference in the distribution of GABAA receptor subunits between the lateral and central amygdala. Our investigation showed there are sex differences in localisation of the GABAA receptor subunits in the rat amygdala in which the expression level of the α2 and γ2 subunits might be higher in female rats, whereas the expression level of γ1 was higher in males than in females. We conclude that in the lateral amygdala all inputs have γ2 subunits whereas in the central amygdala some inputs contain γ1 subunit while others contain γ2 subunits