Patterns Of Interaction Between GABA_A And Glycine Receptors: A Study On Their Correlation With Receptor Expression Level

Neuronal inhibition is crucial for normal functions in the central nervous system (CNS). Too much inhibition would cause loss of consciousness and coma; too little inhibition would lead to a seizure. It must be precisely regulated in the CNS. GABAa and glycine receptors (GABA_ARs and GlyRs) are widely distributed inhibitory neurotransmitter receptors throughout the CNS, which are structurally and functionally belong to families of ligand-gated anion channels. GABAaRs and GlyRs gate distinct but homologous classes of chloride-permeable ion channels. In an adult animal, activation of GABAaRs and GlyRs leads to an inward flow of chloride ions and a hyperpolarizing neuronal response. This GABAaR- and GlyR-mediated precise neuronal inhibition is crucial for normal functions in the CNS, such as nociception and the coordination and generation of rhythmic motor activity in the spinal cord, auditory signal processing in the brain.A large body of evidence shows that GABAaRs and GlyRs can interact functionally in the CNS. Interestingly, accumulating evidence indicates that antagonistic interaction, or cross-inhibition, between these two anionic ionotropic receptors has different patterns in the CNS. These previous studies indicate that cross-talk between GABA_ARs and GlyRs in the CNS may be regionally regulated; however, the underlying mechanisms determining this region-dependent cross-talk remain unclear. In the present studies, I investigated cross-talk between GABA_ARs and GlyRs in cultured neurons from the auditory cortex, the inferior colliculus and the spinal cord with plasmid transform/transfection techniques and whole-cell patch clamp recordings. I have found that the cross-talk between GABA_ARs and GlyRs is regionally regulated and shaped by receptor expression level. In this dissertation, I also discussed possible physiological significance and the underlying mechanisms of these region-specific cross-talk patterns.1. Region-specific cross-talk between GABA_ARs and GlyRs in the CNSIn the present study, I examined cross-talk between GABAaRs and GlyRs in three different regions: the auditory cortex, the inferior colliculus and the spinal cord. I found that activations of GABA_ARs and GlyRs inhibit the activities of two receptors in a region-specific manner. The region-specific cross-inhibition pattern is revealed most clearly with investigation of this cross-inhibition in three different regions, which shows that the cross-inhibition has an asymmetric pattern in the spinal cord, a symmetrical pattern in the inferior colliculus and both one-way and symmetric patterns in the auditory cortex. These findings support the notion that cross-talk between GABA_ARs and GlyRs reflects a region-specific feature in the nervous system.2. Region-specific cross-talk between GABA_aRs and GlyRs is shaped by the relative expression levelFurther analysis revealed that the amplitude ratio between GlyR- and GABAAR-mediated currents was increased as patterns of cross-talk between GABAaRs and GlyRs change from one-way, to symmetric and to asymmetric patterns, suggesting that there is a positive correlation between this cross-inhibition pattern and the relative expression levels of these two receptors. Increasing of GlyRs expression level with transfection of GlyRs subunits in cultured inferior colliculus neurons, resulted in an increasing of GlyR- and GABAAR-mediated current amplitude ratio, and changed this cross-inhibition from symmetric pattern to asymmetric or one-way pattern. The results demonstrate that the relative expression levels of functional GABA_aRs and GlyRs shape this region-dependent cross-inhibition pattern, revealing an alternative mechanism for precisely controlled neuronal inhibition.3. The possible role of cross-inhibition between GABA_aRs and GlyRs in the CNSI then examined synaptical coupled cultured neurons from the IC to test for the possible role of this cross-talk during GABA_AR-mediated neurotransmission. Application of 30μM glycine, the firing of the majority of neurons tested completely was inhibited. However, in the minority of neurons tested, both the amplitude and frequency of pure GABAergic miniature inhibitory postsynaptic currents (mIPSCs) was inhibited by application of 30 uM glycine. Similarly, application of 50μM glycine completely inhibited the GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) firing of all the neurons tested. The co-localization of these receptors and co-release of glycine and GABA, as well as GABA or glycine spillover from synaptic release sites to glycinergic or GABAergic synapses, provide the physiological conditions for this cross-talk, which may play a role in modifying precisely the strength of the inhibitory neurotransmission There are several novelties residing in the present studies: 1) in this study, I examined the cross-talk pattern between GABA_aRs and GlyRs in cultured inferior colliculus neurons. I demonstrate that a novel pattern of cross-talk between GABA_aRs and GlyRs in cultured neurons of rat inferior colliculus (IC) is bidirectional and symmetric cross-inhibition; 2) the cross-talk pattern between GABA_aRs and GlyRs is regionally regulated. However, the mechanisms underlying cross-talk between GABA_aRs and GlyRs in the CNS remains unclear. I examined cross-talk between GABA_aRs and GlyRs in three different regions, and also investigated this cross-talk in cultured inferior colliculus neurons with trasfection of GlyR a subunits. I demonstrate that this region-dependent cross-inhibition pattern is shaped by the relative expression level; 3) I also found that glycine inhibites GABA_AR-mediated neurotransmission, and suggest which the modulation of GABA_AR-mediated neurotransmission by glycine may be through the GlyR-GABA_AR interaction.