Chronic Intermittent Hypoxia Alters the Chloride Gradient in Median Preoptic Nucleus (MnPO) Neurons of Rats

Abstract

Chronic intermittent hypoxia (CIH) is an animal model that simulates the hypoxemia associated with obstructive sleep apnea (OSA). Rats exposed to CIH exhibit increased sympathetic tone and reactivity and a persistent elevation in blood pressure during periods of normoxia, similar to patients with OSA. In MnPO neurons, angiotensin II type 1 receptor knock down (AT1aR KD) mediated reductions in GABAa inhibition are exacerbated to the point where GABAa activation is excitatory following CIH. Here, we use the genetically encoded ratiometric Cl‐sensor, ClopHensorN, to monitor the chloride flux of PVN projecting MnPO neurons in normoxic (Norm) and CIH treated rats in response to GABAa receptor activation.Using isoflurane (2–3%) anesthesia, male Sprague‐Dawley rats (250–350g) received microinfusions (0.4 μL) of a retrograde AAV9‐Cre in the PVN and a DIO‐ClopHensorN in the MnPO. After recovery, rats were subjected to 7 days of CIH (0800–1600 hrs) or Normoxia. The CIH protocol consisted of 6 min cycles (3 min 21% O2, 3 min 10% O2) repeated 10x/hr for 8 hours (during the normal inactive/sleep phase) on 7 consecutive days. For ClopHensorN measurements, rats were anesthetized with isoflurane (2–3%) and coronal slices (300 μm) containing the MnPO were cut using standard in vitro slice procedures. Image sets were captured every 3 sec. Cl‐flux was determined from the ratio of GFP (ex 488 nm, em 500–550 nm) to tdTomato (ex 594 nm, em 650–700 nm) fluorescence in response to 10 s focal application of muscimol (100 uM).Twelve rats (6 Norm, 6 CIH) were used for ClopHensorN studies. In MnPO neurons from CIH treated rats (n = 632), 20.1% showed a decrease in the ratio of GFP to tdTomato fluorescence (n = 127) while 0.3% showed an increase in this ratio (n = 2) indicative of Cl‐efflux. In MnPO neurons from Norm rats (n = 482), 41.9% showed a muscimol dependent decrease in the ratiometric Cl‐signal (n = 202) with 0 cells showing an increase. Furthermore, the magnitude if muscimol dependent decreases in the Cl‐signal were reduced in the CIH treated rats suggestive of reduced GABAa mediated inhibition. In a separate groups of rats, Western blot analysis showed that CIH increased the expression of pKCC2 (n = 6) in punches containing the MnPO compared to MnPO punches from Norm controls (n = 5) injected with Scr. CIH/AT1a KD rats showed decreased pKCC2 (n = 6) compared to CIH/Scr but increased pKCC2 in Norm/AT1a KD rats (n = 6) compared to Norm/Scr rats.The results demonstrate that CIH alters the Cl‐flux of PVN projecting MnPO neurons and these changes are likely due to altered function of KCC2. These changes may contribute to the increased sympathetic tone and reactivity, as well as the persistent hypertension associated with CIH.Support or Funding InformationSupported by P01 HL088052