Chronic Intermittent Hypoxia Alters Chloride Gradients in Median Preoptic Nucleus (MnPO) Neurons of Rats: Comparing ClopHensorN and Perforated Patch Recording

Abstract

Chronic intermittent hypoxia (CIH) is an animal model that simulates the hypoxemia associated with sleep apnea. Male rats exposed to CIH exhibit increased sympathetic tone and reactivity and persistent elevations in blood pressure. In MnPO neurons, CIH induces reductions in GABAa inhibition that are, in some neurons, exacerbated to the point of GABAa mediated excitation. The genetically encoded ratiometric Cl‐ sensor, ClopHensorN, was used to monitor the Cl‐ flux of PVN projecting MnPO neurons in normoxic (Norm) and CIH treated rats in response to GABAa receptor activation. In some experiments, ClopHensorN imaging was combined with perforated patch recordings.Using isoflurane (2‐3%) anesthesia, male Sprague‐Dawley rats (250‐350g) received bilateral infusions (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 hr) or normoxia. CIH 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 recordings, rats were anesthetized with isoflurane (2‐3%) and coronal slices (300 µm) containing the MnPO were cut using standard in vitro slice procedures. All cells were tested in standard artificial cerebrospinal fluid for their responses to 10 s focal application of muscimol (100 uM). ClopHensorN images 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. For some cells, voltage‐clamp recordings using gramicidin (100 ug/ml in 140 mM K‐gluconate) perforated patch were also collected (in labeled and unlabeled MnPO neurons).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 increases 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 increases. Muscimol dependent decreases in the Cl‐ signal were reduced in the CIH treated rats suggesting reduced GABAa inhibition. During perforated patch recordings, MnPO neurons from Norm rats exhibited mIPSCs and outward currents in response to muscimol. MnPO neurons from CIH treated rats show variable mIPSC frequencies and heterogeneous responses to muscimol.The results demonstrate that CIH alters the Cl‐ flux of PVN projecting MnPO neurons in response to GABAa stimulation. These changes may contribute to the increased sympathetic tone and reactivity, as well as the persistent hypertension associated with CIH.