Impact of sustained hypoxia on GABAergic and non-GABAergic neurons of the nucleus tractus solitarii (nTS)

Exposure to hypoxia occurs during various physiological (e.g., high altitudes) and pathophysiological conditions (e.g., obstructive pulmonary disease). During hypoxia, respiration, blood pressure, and sympathetic nervous activity are reflexively altered to optimize oxygen delivery and blood flow to the brain and other critical systems. Hypoxia sensing peripheral chemoreceptors project to the nucleus tractus solitarii (nTS), which is the primary center for integrating sensory afferents and is critical for maintaining cardiorespiratory homeostasis. nTS activity is determined via the balance of excitatory glutamate (Glu) and inhibitory gamma-aminobutyric acid (GABA) signaling, which is critical for generating proper reflexes in response to hypoxia. Therefore, any changes in this excitatory-inhibitory balance, and thus overall nTS activity may directly influence cardiorespiratory homeostasis. Our previous work indicates that nTS Glu signaling is exaggerated following hypoxia, however the contribution of inhibitory GABA signaling to this phenomenon remains largely unknown. This study's objective was to determine the extent to which nTS GABA neurons are altered by exposure to hypoxia. We hypothesize that reduced GABA activity contributes to increased nTS excitability and cardiorespiratory function following hypoxia. Male and female GAD1-EGFP mice [strain FVB-Tg (GadGFP) 45704Swn/J, Jackson Laboratory, 4-5 weeks] were used to differentiate GABA (GAD+) and presumptive Glu (GAD-) neurons in the nTS. Mice were exposed to sustained hypoxia (SH, 10% O2, 24 hours) or normoxia (21% O2, 24 hours) before experimentation. First, plethysmography was used to monitor respiratory parameters in mice before and after SH. We observed increased tidal volume and minute ventilation during an acute hypoxia challenge (15% O2) following SH exposure. Next, immunohistochemistry (IHC) was used to identify recently activated GAD+ or GAD- neurons in the nTS by staining for c-Fos. Preliminary results indicate that SH increases c-Fos within the nTS, but primarily in GAD- neurons. We then employed ratiometric Ca2+ imaging via Fura-2 fluorescence as a readout of neuronal activity in GAD+ and GAD- cells isolated from the nTS of normoxic and SH mice. We observed that GAD+ neurons exhibit a consistent increase in intracellular Ca2+ to repeated high K+ challenges regardless of normoxia or hypoxia exposure. In contrast, GAD- neurons from normoxic mice exhibit a reduction in Ca2+ elevation during repeated high K+ exposures, whereas Ca2+ elevation is maintained in GAD- neurons from SH exposed mice. Lastly, horizontal brainstem slices were generated from normoxic or SH exposed mice, and using whole-cell patch-clamp recordings, we examined synaptic neurotransmission and electrophysiological properties of GAD+ and GAD- nTS neurons. Neurons were further characterized by monosynaptic or polysynaptic connection with the tractus solitarii (TS) via TS stimulation. We found that the amplitude of spontaneous excitatory post synaptic currents (sEPSCs) is greater in GAD+ monosynaptic neurons than GAD- under normoxic conditions, but that SH exposure increases sEPSC amplitude in GAD- neurons. Furthermore, SH increases sEPSC frequency but not amplitude in polysynaptic GAD+ NTS neurons. We also found SH to increase the paired pulse ratio of the amplitude of two TS-EPSCs in GAD+ polysynaptic neurons. Taken together, these data indicate an SH-induced increase in the activity of non-GABAergic NTS neurons likely via alterations in GABA influence on local NTS circuitry. Funding: NIH HL166183 and MU REP Postdoctoral Fellowship supporting HW 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.