GABA transporters on astrocytes regulate neuronal firing rate in the nucleus tractus solitarii (nTS) in a state-dependent manner

The nucleus tractus solitarii (nTS) is the primary integration site for autonomic and respiratory sensory information. Our group has shown that astrocytes in the nTS critically regulate synaptic transmission, in part, via glial transporters and their reuptake of extracellular neurotransmitters. Gamma-aminobutyric acid transporters (GAT) 1 and 3 reuptake GABA. GAT-3 block reduced spontaneous IPSC frequency. However, less is known for the contribution of astrocytic GABA transporters to nTS activity. We hypothesize blocking GAT-3 transporters decreases neuronal activity in the nTS, driven by an increase in extracellular GABA. To test this hypothesis, GAD2 tdTomato mice [GAD2tm2(cre)Zjh/J #010802 + B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze)/J #007914 ] were used to distinguish GABAergic from non-GABAergic neurons in the nTS. Immunohistochemistry for GAT-3 and Glial Fibrillary Acidic Protein (GFAP) was done on nTS slices to quantify GAT-3 expression and visualize astrocyte interactions with GABA neurons. Neuronal activity was recorded using a multielectrode array (MEA), with analysis restricted to electrodes positioned within the nTS. Baseline activity was recorded for 5 min, followed by GAT-3 inhibition (SNAP 5114, 50 μM, 5 min) to assess the extent to which astrocytic GAT-3 influences spontaneous firing. Following a 10-minute wash period, the GABAA receptor antagonist (Gabazine, or SR-95531, 25uM, 5 minutes) was applied alone and in the presence of SNAP 5114 (5 min) to confirm the GABA receptor activated via SNAP 5114 -mediated elevation of GABA. Neuronal activity from the final 2 minutes of each segment was separated into those with low (< 0.3), medium (0.3-1) and high ( >1) number of spikes per second based on our previous reports and normalized to the initial baseline. Immunohistochemistry revealed both GAT-3 and GFAP-labeled astrocytes surround and are closely associated with both GABAergic and non-GABAergic neurons. Analysis of MEA recording demonstrated GAT-3 inhibition was state-dependent, with neurons with a higher baseline firing rate exhibiting the greatest decrease (~47%) in activity from baseline following GAT-3 block. Cells with medium firing rates also decreased ~18%, whereas, GAT-3 block did not alter low firing cells (only a 10% reduction). In the presence of GABAA receptor block, the SNAP 5114-mediated decrease was ablated in the high-firing cells. Together, these data demonstrate astrocytic GAT-3 tempers nTS inhibition and that this occurs in an activity-dependent manner. This abstract was presented at the American Physiology Summit 2026 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.