Intracellular Ca ²⁺ mobilization in NTS neurons and astrocytes of adenosine A _2A receptor knockout mice exposed to 24 hours of sustained hypoxia

In a previous study using Balb-C [wild type (WT)] and adenosine A 2A receptor knock-out (A 2A KO) mice, we documented that the amplitude of evoked AMPA current in nucleus tractus solitarius (NTS) neurons of WT exposed to 24 hours of sustained hypoxia (24h-SH) was significantly increased, while in NTS neurons of A 2A KO exposed to SH this increase was not observed, suggesting a key role for A 2A receptor in the modulation of AMPA current. To evaluate the mechanisms underlying the increase in AMPA current in WT in response to SH, in the present study we evaluated changes in the intracellular Ca 2+ mobilization in NTS neurons and astrocytes of WT and A 2A KO mice exposed to 24h-SH. Male WT and A 2A KO mice (6-7 weeks old) were maintained in normoxia (FiO 2 : 0.21) or exposed to SH (FiO 2 : 0.1, 24h). Brainstem slices (250 µm thickness) were obtained and neurons and astrocytes loaded with Fluo-4 to visualize the intracellular Ca 2+ concentration. For identification, astrocytes were labeled with sulforhodamine 101. Non-NMDA receptors were activated by AMPA superfusion (50 µM). The images were acquired using multiphoton microscopy (FV1000-FluoView FVMPE-RS, Olympus) and processed by Image J software (NIH/USA). The data are presented as the peak of fluorescence (%ΔF/F 0 ) obtained from several regions of interest (ROIs) of NTS slices. This analyze was restricted to ROIs located the commissural NTS. The data show that the increase in Ca 2+ mobilization induced by AMPA superfusion was similar in NTS neurons from WT and A 2A KO mice maintained in normoxia [WT: 206 ± 23% (31 ROIs/5 slices/4 mice); A 2A KO: 193 ± 7% (43 ROIs/6 slices/4 mice)] or exposed to 24h-SH (WT: 154 ± 12%(15 ROIs/3 slices/3 mice); A 2A KO: 200 ± 8% (58 ROIs/12 slices/8 mice)]. With respect to astrocytes in the NTS of mice exposed to 24 hours of SH, the increase in the intracellular Ca 2+ mobilization was significantly higher in astrocytes from A 2A KO [normoxia: 200 ± 18% (14 ROIs/ 6 slices/ mice); 24h-SH: 244 ± 21% (35 ROIs/ 12 slices/ 8 mice), p=0.0089] than in WT mice [normoxia: 161 ± 12% (25 ROIs/ 5 slices/ 4 mice); 24h-SH: 169 ± 13% (14 ROIs/ 3 slices/ 3 mice)]. In summary, the data show that theintracellular Ca 2+ mobilization induced by AMPA stimulation in neurons is not affected by 24h-SH or by the lack of A 2A receptors. In addition, the increase in intracellular Ca 2+ mobilization induced by AMPA stimulation in NTS astrocytes in response to SH in A 2A KO mice was greater than in WT. We conclude that the observed an additional increase in the AMPA current of NTS neurons after SH is not due to the changes in the intracellular Ca 2+ mobilization and that A 2A receptors modulate the calcium signaling in astrocytes in response to 24h-SH. FAPESP (2018/15957-2; 2020/00201-0) and CNPq (309338/2020-4). 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.