Contribution of K+/Cl- Cotransporters in AT1aR Dependent GABAa Inhibition in the MnPO Following Chronic Intermittent Hypoxia




Little, Joel
Marciante, Alexandria B.
Cunningham, J. Thomas


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Purpose: Chronic intermittent hypoxia (CIH) is an animal model that simulates the hypoxemia seen in obstructive sleep apnea (OSA). Rats exposed to CIH exhibit an increase in blood pressure during periods of normoxia, similar to that observed in OSA. The median preoptic nucleus (MnPO) exhibits increases in Angiotensin type 1a receptor (AT1aR) mRNA following CIH and blocking this increase in AT1aR mRNA prevents the sustained increase in blood pressure. Here we investigate the role of AT1aR in the MnPO and the contribution of the K+/Cl- cotransporters KCC2 and NKCC1 on excitatory/inhibitory balance in rats subjected to CIH. Methods: Under isoflurane (2-3%) anesthesia, male Sprague-Dawley rats (250-350g) received microinfusions (0.4 µL) of recombinant AAV construct containing GFP reporter and shRNA against AT1aR (AT1aKD) or an AAV containing the GFP reporter and a shRNA scramble (Scr) targeted to the MnPO. After recovery, rats were subjected to 7 days of CIH (0800-1600 hrs). The CIH protocol consisted of 6 min cycles (3 min 21% O2, 3 min 10% O2) repeated 10x/hr for 8 consecutive hrs (during the normal inactive/sleep phase) on 7 consecutive days. After 7 days CIH, the rats were anesthetized with isoflurane (2-3%) and coronal slices (300 µm) containing the MnPO were cut using standard in vitro slice procedures. Loose patch recordings were obtained from GFP labeled neurons using glass micropipettes containing aCSF as the internal solution (1-3 MΩ). Spontaneous action potentials (APs) were recorded in response to muscimol (100uM, 30s). Results: The GABAa agonist muscimol decreased AP activity of neurons from normoxic/Scr rats. GABAa inhibition was blunted in neurons from CIH/Scr and normoxic/AT1aKD rats. However, GABAa activation from neurons in the CIH/AT1aKD group produced an increase in spontaneous activity. KCC2 block reduced GABAa mediated excitation in CIH/AT1aKD but had no effect GABAa mediated inhibition in CIH/Scr. NKCC1 block reduced GABAa mediated excitation in CIH/AT1aKD and facilitated GABAa mediated inhibition in CIH/Scr. Conclusion: The current study shows AT1aKD mediated reduction in GABAa inhibition is exacerbated such that GABAa activation is excitatory following CIH. KCC2 and NKCC1 contribute to GABAa mediated excitability in CIH/AT1aKD but only NKCC1 contributes to attenuated GABAa function in CIH/Scr. Future studies will address the influence of reduced AT1a signaling and reduced GABAa mediated inhibition on downstream targets of the MnPO.