Spirits in distress: Converging plasticity of ventral tegmental area (VTA) gamma amino-butyric acid (GABA) neurons evoked by chronic ethanol and stress controls anxiety symptomatology

Abstract

The ventral tegmental area (VTA) and associated signaling has been historically implicated in reward evaluation and seeking. Due to this, dysfunction of the VTA has been largely linked to substance use disorders, including alcohol use disorder (AUD). Recent evidence has also identified a role of the VTA in stress-related anxiety disorders, highlighting its diverse functional role. The majority of work in this structure has focused on dopaminergic (DA) neurons, as well as associated DA release. However, the VTA is host to a variety of neuron populations defined by neurotransmitter content, including gamma amino-butyric acid (GABA)- releasing neurons. Existing evidence positions VTA GABA neurons as potent regulators of anxiety, fear, and aversion through regulation of neighboring VTA DA neurons as well as discrete long-range projections. Lines of evidence indicate that VTA GABA neurons exhibit enhanced activity following chronic ethanol or stress exposure. Therefore, the goals of this dissertation were to further identify plasticity evoked in VTA GABA neurons in these contexts that could contribute to enhanced activity and determine the functional role of VTA GABA neuron plasticity within these disease states. Ethanol's effects in the central nervous system and the expression of AUD has been extensively linked to plasticity in GABAergic signaling, including presynaptic GABA release and postsynaptic GABAA receptors (GABAAR) and GABAB receptors (GABABR). Moreover, G- protein gated inwardly-rectifying K+ (GIRK) channels, a primary effector of GABABR signaling, also plays a critical role in the context of AUD. While it has been partially assessed, sites of GABAergic signaling plasticity evoked by chronic ethanol in VTA GABA neurons have yet to be thoroughly assessed. In Chapter 2, we utilized slice electrophysiology to identify several forms of GABAergic plasticity. Firstly, VTA GABA neurons exhibit a significant reduction in presynaptic GABA release that was dependent on the action of GABABR's, implicating plasticity in GABABR-dependent presynaptic regulation of GABA release. We surprisingly found no effect in GABAAR- dependent currents, suggesting that postsynaptic GABAAR signaling may be largely unaffected. On the other hand, postsynaptic GABABR/GIRK-dependent currents were significantly diminished, which was found to be associated with increased intracellular localization of the GABABR1 subunit as well as GIRK2. Utilizing a constitutive knockout approach as well as VTA GABA specific CRISPR/Cas9 mediated ablation, we identified GIRK3 as necessary for this plasticity in postsynaptic GABABR/GIRK signaling. These data, in conjunction with existing evidence, suggest that GABAergic signaling plasticity in VTA GABA neurons following chronic ethanol exposure could contribute to the expression of symptoms associated with AUD. Utilizing VTA GABA specific CRISPR/Cas9 mediated ablation of GIRK2, we found that loss of GABABR/GIRK currents was sufficient to induce anxiety symptomatology, suggesting a role of VTA GABA neurons in ethanol withdrawal-induced anxiety. Stress has been demonstrated to increase the activity of VTA GABA neurons, as well as evoke plasticity that suggests VTA GABA neurons exhibit enhanced excitability. Interestingly, enhanced activity of VTA GABA neurons is required for restraint stress-induced anhedonia, suggesting a role of VTA GABA neurons in other stress-induced affective symptoms. In Chapter 3, we utilized an unpredictable footshock (uFS) protocol to assess the effect of stress on VTA GABA neurons due to its ability to reliably evoke anxiety symptomatology. uFS was sufficient to enhance the excitability of VTA GABA neurons, as assessed via increased baseline firing, increased input resistance, and decreased rheobase. Furthermore, this hyperexcitability was associated with enhanced presynaptic glutamate release, as well as diminished postsynaptic GABAAR and GABABR signaling, suggesting that stress evokes several mechanisms of intrinsic and extrinsic plasticity of VTA GABA neurons. Utilizing a chemogenetic approach, we found that enhanced excitability of VTA GABA neurons in behaviorally-naïve mice was sufficient to evoke anxiety, while diminishing excitability of VTA GABA neurons was sufficient to block the expression of uFS-induced anxiety. These data posit that VTA GABA neuron excitability is necessary for stress-induced affective symptomatology, suggesting that VTA GABA neuron dysfunction may underlie disorders associated with this symptomatology. Altogether, this work contributes to a growing body of evidence identifying the functional role of VTA GABA neurons, as well as provides insights into novel therapeutic targets for the treatment of anxiety in the context of AUD and stress-related anxiety disorders. Furthermore, this work positions VTA GABA neurons as an overlapping site of plasticity between chronic ethanol and stress that could contribute to reciprocal exacerbation of each disease state.