Increased glutamate spillover within the retrotrapezoid nucleus of heart failure rats drives central chemoreflex potentiation and disordered breathing

Heart failure (HF) is a chronic disease characterized by a high prevalence of respiratory disorders and irregularities in the breathing pattern, which are closely associated with central chemoreflex potentiation. In preclinical HF, we have shown that elimination of chemoreceptor neurons within the retrotrapezoid nucleus (RTN) normalizes central chemoreflex and eliminates irregularity in the breathing pattern. The mechanisms involved in the potentiation of chemoreflex function during HF has not been fully studied but recent evidence support a role for astrocyte metabolic inhibition on RTN-induced chemoreflex potentiation in the setting of HF. Since astrocytes play a pivotal role in glutamate clearance, we hypothesized that reduced astrocytes activity in HF will increase glutamate spillover in the vicinity of RTN chemoreceptor neurons, increasing their activity and enhancing chemoreflex drive. Therefore, we aimed to determine: i) the levels of extracellular glutamate in the RTN and its relation to high RTN chemoreceptor neuron activity in HF and, ii) to determine the contribution of the glutamate transporter GLT-1 on chemoreflex function and disordered breathing in HF. Adult male Sprague Dawley rats (~250 g) underwent volume overload to induce HF. Whole-body plethysmography was used to evaluate central chemoreflex and resting breathing patterns. RTN extracellular glutamate levels were measured using microdialysis and high-performance liquid chromatography. RNAseq data was used to define specific cell cluster localization for GLT-1 in the RTN followed by co-localization analysis using RNAscope. RTN chemoreceptor neurons activity was determined in acute brainstem slices using the genetic encoded Ca 2+ sensor (GCaMP6s) previously transfected into RTN neurons. Finally, overexpression of GLT-1 in the RTN was performed using an adeno-associated virus to determine the contribution of GLT-1 on glutamate spillover, enhanced chemoreflex function and disordered breathing in HF rats. Compared to Sham-operated rats, HF animals exhibited increases in glutamate levels in the RTN (3.2±0.2 vs 8.6±0.9 μM, Sham vs HF) and this was closely related to the potentiation of the central chemoreflex in HF animals (r 2 =0.954; p=0.02). Also, we found that RTN chemoreceptor neurons from HF animals showed increased activity (3.8±0.3 vs 7.0±0.3 spikes/min, Sham vs HF). In addition, we found that endogenous GLT-1 expression was confined to astrocytes residing within the RTN region and that HF animals showed a significant downregulation of GLT-1 (139.7±8.5 vs 71.9±4.9 a.u., Sham vs HF). Finally, overexpression of GLT-1 in RTN astrocytes of HF animals restored normal extracellular glutamate levels in the RTN (8.6±0.9 vs 3.7±0.8 μM, HF vs HF+GLT-1), normalized the activity of RTN chemoreceptor neurons (7.0±0.3 vs 3.7±0.4 spikes/min, HF vs HF+GLT-1), decreases central chemoreflex drive (HCVR: 6.1±0.1 vs 3.2±0.6 ΔV E /%F i CO 2 , HF vs HF+GLT-1) and restored normal breathing patterns in animals with HF. Our results strongly support a role for RTN astrocyte GLT-1 on central chemoreflex potentiation and the development/maintenance of disordered breathing during the progression of HF. Fondecyt 1220950 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.