Contribution of GABAA(δ)R to Heart Rate Regulation by Nucleus Ambiguus and Plasticity Under High Fat Diet Disease State

Nucleus ambiguus (NA) contains cardioinhibitory cardiac vagal neurons (CVNs) that decrease heart rate (HR) through direct axonal projections to the epicardial fat pad ultimately altering cardiac pacemaker potential. Despite robust evidence for the role of glutamate to regulate CVN activity, the role of the primary fast inhibitory neurotransmitter, gamma-aminobutyric acid type A receptors (GABAAR), is less clear. GABAAR ligand binding and kinetic properties are determined by the subunit composition of an individual receptor. GABAARs containing a δ-subunit (GABAA(δ)Rs) are typically seen in the extrasynaptic space and possess a high affinity for GABA with slow rates of desensitization, making them particularly well suited to contribute to long duration, or tonic current. Given the critical signal integrating nature of GABAA(δ)Rs, we hypothesized that GABAA(δ)Rs in NA contribute to resting HR regulation. To determine this, we used pharmacological manipulations of GABAA(δ)Rs in NA through bilateral microinjections (60nl; 10nL/s; from bregma: AP: -7.0, ML: 1.3 DV: -4.5). All experiments were under urethane in adult mice (6-12 weeks) with equal sex distributions. Initial experiments microinjected THIP (4,5,6,7-tetrahydro isoxazole[5,4-c]pyridin-3-ol); a GABAA(δ)R superagonist) in C57/Bl6J mice. THIP (20uM; n=4/group) produced significant tachycardia (PBS; 30.7 BPM ± 9.6; p=0.05) compared to vehicle (-1.62 BPM ± 6.12). To specifically isolate GABAA(δ)R contributions to HR for the first time, we utilized a unique transgenic mouse line, δ*KI, which possesses GABAA(δ)Rs insensitive to picrotoxin, a GABAAR antagonist (n=8). Disinhibition of NA with picrotoxin significantly (5.5 mM) decreased HR (97.6 ± 100.7 BPM) compared to vehicle (4% DMSO in PBS; 21.2 ± 8.58 BPM; p=.0008). Sequential microinjection of gabazine (a general GABAAR antagonist; GBZ; 1 mM) further decreased HR (76.3 ± 50.4 BPM) compared to vehicle (PBS; 18.52 ± 25 BPM; p=0.042). Critically, blood pressure did not significantly change after any of these drugs, suggesting that baroreflex was not involved in HR changes. Application of scopolamine, a muscarinic acetylcholine receptor antagonist, after induction of GBZ-sensitive bradycardia confirmed that this response is vagally mediated. Taken together, total GABAAR inhibition contributes to ~30% of resting HR in murines with GABAA(δ)R specifically making up ~50% of the total impact of inhibition of CVNs on resting HR. Immunohistochemical analysis confirmed GABAA(δ)R protein expression in CVNs. To understand the plasticity of GABAA(δ)R signaling in CVNs, mice were fed high fat diet (HFD) for 15 days, where disease pathology causes a decrease in cardiac vagal drive previously theorized to involve GABAA(δ)R. C57/Bl6J mice on HFD for 15 days had significantly greater tachycardic responses to 20 uM THIP (39.9 ± 19.9 BPM), compared to animals placed on normal fat diet (NFD; 8.9 ± 5.6 BPM; p=0.0218). δ* KI mice on fed HFD had no significant changes to picrotoxin, but significantly larger bradycardias to disinhibition with gabazine (HFD; n=4, -270 ± 30.7 NFD; n=3, -68.7 ± -28.4; p= 0.0003), further implicating the role of these receptors in the pathology of HFD. Future studies will follow-up these results using electrophysiology and molecular biology in CVNs to identify mechanism(s) of GABAA(δ)R on CVN plasticity. R01HL157366 NIHLB to CRB This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.