Contribution of K+/Cl− Cotransporters in AT1aR‐Dependent GABAa Inhibition in the MnPO following Chronic Intermittent Hypoxia
Authors: Farmer George Edward , Little Joel T , Marciante Alexandria B. , Cunningham J. Thomas 
Published: June 2021

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 sympathetic tone and reactivity and a persistent elevation in blood pressure during periods of normoxia, similar to that observed in patients with 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 hypertension. Our lab has previously reported that AT1aR activation influences GABAa mediated inhibition within the MnPO. Here we investigate the role of Angiotensin II (Ang II) signaling via the AT1a receptor in the MnPO and the contribution of the K+/Cl− cotransporters KCC2 and NKCC1 on excitatory/inhibitory balance in rats subjected to CIH. 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 (AT1a KD) or an AAV construct 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 borosilicate glass micropipettes containing aCSF as the internal solution (1–3 MΩ). Spontaneous action potentials were recorded in response to focal application of Ang II (100 nM, 30s) or muscimol (100 uM, 30s). Focal application of Ang II produced a transient increase in spontaneous action potential frequency of MnPO neurons from both CIH/Scr (n = 11) and normoxic/Scr (n = 9) rats. The Ang II dependent increase in activity was blocked in neurons from AT1a KD rats from both CIH (n = 12) and normoxic (n = 10) groups. The GABAa agonist muscimol decreased action potential activity (n = 10) of neurons from normoxic/Scr rats. This GABAa mediated inhibition was blunted in neurons from CIH/Scr (n = 13) and normoxic/AT1a KD (n = 11) rats. However, GABAa activation from neurons in the CIH/AT1a KD (n = 17) group produced an increase in spontaneous activity. KCC2 block reduced GABAa mediated excitation in CIH/AT1a KD (n = 7) but had no effect GABAa mediated inhibition in CIH/Scr (n = 9). NKCC1 block also reduced GABAa mediated excitation in CIH/AT1a KD (n = 6) and facilitated GABAa mediated inhibition in CIH/Scr (n = 9). The current study shows AT1aR KD mediated reduction in GABAa inhibition is exacerbated to the point where GABAa activation is excitatory following CIH. KCC2 and NKCC1 contribute to the GABAa mediated excitability in CIH/AT1a KD but only NKCC1 contributes to attenuated GABAa function in CIH/Scr. Future studies will address the influence of reduced AT1a signaling combined with reduced GABAa mediated inhibition on downstream targets of the MnPO in AT1aKD from both normoxic and CIH rats.Support or Funding InformationSupported by P01 HL088052This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.