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

Rats exposed to chronic intermittent hypoxia (CIH), an animal model simulating the hypoxemia associated with obstructive sleep apnea, exhibit persistent elevations in blood pressure during normoxic periods. In MnPO neurons, angiotensin II type 1 receptor function mediates reductions in GABAa inhibition that become excitatory following CIH. Here, we use the ratiometric Cl- sensor, ClopHensorN, to monitor the chloride flux of MnPO neurons in normoxic (Norm) and CIH treated rats following GABAa activation. Using isoflurane anesthesia, male Sprague-Dawley rats (250-350g) received microinfusions of AAV9-Cre in PVN and DIO-ClopHensorN in MnPO. After recovery, rats underwent 7 consecutive days of CIH (6 min cycles of 3 min 21% O2, 3 min 10% O2 repeated 10x/hr for 8 hours) or Normoxia. For ClopHensorN imaging, rats were anesthetized with isoflurane and coronal slices containing MnPO were cut using standard in vitro slice procedures. Images were captured every 3 sec. Cl- flux was determined from ratiometric responses to 10 s focal application of muscimol (100 uM). Twelve rats (6 Norm, 6 CIH) were used for ClopHensorN studies. In MnPO CIH neurons, 20.1% showed decreased fluorescent ratios while 0.3% showed increased ratios indicative of Cl- efflux. In MnPO Norm neurons, 41.9% showed a muscimol dependent decrease in fluorescent ratio with 0 cells showing an increase. The magnitude of muscimol dependent decreases in fluorescent ratios were reduced in CIH treated rats suggesting reduced GABAa inhibition. Results demonstrate CIH alters Cl- flux of PVN projecting MnPO. These changes may contribute to hypertension associated with CIH.