Neuroscience
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/29935
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Browsing Neuroscience by Author "Cunningham, J. Thomas"
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Item AT1R sniffer cells detects spontaneous and evoke release of AngII in the AP-NTS pathway(2020) Paundralingga, Obed; Farmer, George; Cunningham, J. Thomas; Gusson Shimoura Almeida Lima, CarolineAlthough angiontesin II (AngII) has multiple actions in the brain, the existence of a brain RAS is still controversial. Our previous studies have used angiotensin sensitive sniffer cells to test whether angiotensin peptides are released from subfornical organ projections to the median preoptic nucleus. In these studies, we examined another pathway involving the area postrema (AP) and nucleus of tractus solitarius (NTS). The AP is angiotensin sensitive and projects to the NTS, so the purpose of this study was to test for the release of angiotensin peptides in the NTS after stimulation of AP. Sniffer cells were produced by transfecting Chinese Hamster Ovary cells with commercially available plasmids for the angiotensin 1a receptor (Origene Tech.) and R-GECO (Addgene #32462). These sniffer cells are sensitive to AngII and III but not angiotensin 1-7, bradykinin, or neurotransmitters such as glutamate or acetylcholine. Sniffer cells were placed on coronal brainstem slices containing both AP and NTS from adult male Sprague and Dawley rats. Changes in fluorescent intensity of sniffer cells in the NTS was determined following electric stimulation of the AP (100Hz, 10ms, 1mA). Electrical stimulation increased fluorescence intensity 134 ± 11%, n=13 of sniffer cells on the NTS with a mean response latency of 4 ± 0.7sec, n=13. Some cells demonstrated spontaneous changes in fluorescence intensity 2±0.1, n=28 that were not observed in cells located outside of the NTS. The results indicate that sniffer cells placed on the NTS demonstrated evidence of spontaneous and evoked release of angiotensin peptides.Item Chronic Intermittent Hypoxia Alters the Chloride Gradient in Median Preoptic Nucleus (MnPO) Neurons of Rats(2020) Cunningham, J. Thomas; Little, Joel; Bachelor, Martha; Rybalchenko, Nataliya; Yuan, Joseph; Farmer, GeorgeRats exposed to chronic intermittent hypoxia (CIH), an animal model simulating 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 reduced GABAa inhibition that becomes 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 the PVN and DIO-ClopHensorN in the MnPO. After recovery, rats underwent 7 consecutive days of CIH (6 min cycles of 3 min 21% O2, 3 min 10% O2 repeated 10x/h for 8 hours) or Normoxia. For ClopHensorN imaging, rats were anesthetized with isoflurane and coronal slices containing the MnPO were cut using standard in vitro slice procedures. Images were captured every 3s. Cl- flux was determined from the ratiometric response to 10s 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 in PVN projecting MnPO. These changes may contribute to hypertension associated with CIH.Item PVN-projecting NTS Neurons is Involved in Sympathetic Long-Term Facilitation After Acute Intermittent Optogenetic Stimulation(2020) Cunningham, J. Thomas; Shimoura, Caroline; Paundralingga, ObedAcute intermittent optogenetic (AIO) stimulation to the caudal NTS, a visceral sensory nucleus with projections to, among others, the paraventricular nucleus (PVN), induces sympathetic long-term facilitation (LTF), which is a progressive increase of sympathetic nerve activity. The contribution of the NTS to PVN pathway in sympathetic LTF is unknown. We investigated the effects of AIO stimulation of PVN-projecting NTS neurons in rats that were previously injected with rAAV2/Ef1a-DIO-hchR2(H134R)-mCherry in the NTS and retrograde AAV9.hSyn.HI.eGFP-cre.WPRE.SV40 in the PVN. We measured splanchnic sympathetic nerve activity (SSNA), mean arterial pressure (MAP), and heart rate (HR) in anesthetized (urethane and cloralose) and mechanically ventilated adult male Sprague-Dawley rats. Twenty minutes after AIO, defined as train of 10 stimuli each consisting of pulse of light of 10-ms duration delivered at 20 Hz for 1 min every 6 min, SSNA amplitude increased by 26.8 ± 7.09% (n=7), MAP increased by 16.9 ± 11.72% (n=7). This increase in SSNA and MAP occurred whether singular optogenetic train of stimulation increased (n=3) or decreased (n=4) of SSNA. These results suggest that the splanchnic sympathetic LTF induced by acute intermittent stimulation of NTS neurons might be mediated by the PVN-projecting neurons, independently of baroreflex or chemoreflex circuits. Further histological study is needed to clarify whether the differential responses to single optogenetic stimulation is due to activation of collaterals from PVN-projecting NTS neurons that are connected to different circuits or due to activation of different downstream projections from the PVN.