Molecular mechanisms and therapeutic potential of inhibitory G protein signaling in anxiety disorders

Abstract

Anxiety disorders are common and debilitating. Current medications for treating anxiety disorders carry addictive potential and have adverse side effects, highlighting the need for improved therapeutics. Several commonly prescribed drugs used to treat anxiety disorders enhance inhibitory G protein signaling in neurons, leading to the modulation of multiple enzymes and ion channels. The relative contributions of these individual G protein-regulated effectors to anxiety-related behavior are unclear. My thesis research focuses on one such effector – the G protein-gated inwardly rectifying K+ (GIRK) channel. GIRK channels mediate the postsynaptic inhibitory effect of GABA and other inhibitory neurotransmitters in the central nervous system. There are 4 GIRK subunits (GIRK1-4). Neuronal GIRK channels typically contain GIRK1 and GIRK2. Previous work from our lab found that the GIRK channel activator (ML297), which shows a slight preference for GIRK1/2 channels, reduces anxiety-related behavior in mice without exhibiting addictive potential. My central hypothesis is that activators of the GIRK1/2 channel subtype could treat anxiety-related disorders. My thesis explored three interrelated questions: Which brain regions and neuronal populations underlie the influence of GIRK channels on anxiety-related behavior? While ML297 reduces anxiety-related behavior in mice, the relevant brain regions and neuron populations underlying this effect were unclear. I utilized pharmacologic and viral genetic approaches to manipulate GIRK-dependent signaling in distinct neuron populations in the ventral hippocampus (vHPC) and the basolateral amygdala (BLA), and evaluated the impact of these manipulations on anxiety-like behavior using the elevated plus maze (EPM) test. Intra-vHPC ML297 reduced anxiety-related behavior, akin to the effect observed with systemic ML297. In contrast, ML297 infusion into the BLA increased anxiety-related behavior in the EPM. Chemogenetic neuron-specific manipulations revealed neuronal subtypes within vHPC and BLA mediate these effects. These findings could inform targeted treatments for anxiety-related disorders. Do GIRK channel activators have anxiolytic therapeutic potential? Despite the promise of ML297 in studies of anxiety-related behavior, its modest selectivity for neuronal channels, its poor in vivo stability, and its limited ability to penetrate the blood-brain barrier preclude its clinical utility. I characterized a new GIRK channel activator, VU0810464, which showed improved brain penetration and enhanced selectivity for GIRK1/2 channels. I also demonstrated its in vivo efficacy in the stress-induced hyperthermia test. VU0810464 is a new, important tool for investigating the relevance of GIRK1/2 channels in physiology and behavior. What factors influence GIRK-dependent signaling? The GIRK2 subunit is necessary for neuronal GIRK channel function and has three distinct splice variants that have not been extensively characterized. I demonstrated the influence of these splice variants on three different GIRK-dependent signaling pathways in cultured hippocampal neurons using an electrophysiological approach. We found that these GIRK2 splice variants differed in their subcellular distribution, and this difference impacted their contribution to the processing of inhibitory input and to fear learning behavior. This knowledge provides insight into a key element influencing GIRK channel function, and importantly, opens more opportunities for future studies targeting GIRK-dependent signaling for therapeutics. In brief, I present a body of work in this thesis that contributes to the field’s knowledge of GIRK-dependent signaling and offers the potential for novel treatment for anxiety-related disorders.