May 30, 2018 - distinct mechanisms for reduced activity of HMs during REM sleep: ... (arousal latency- 12.6 ± 0.9 sec), and woke-up on every CO2 trial. In.
A. Basic Sleep Science
IV. Neural Circuits
0139
Sections were covered with primary antibodies for 5-HT3 and CB1 receptors or 5-HT3 and CB2 receptors. After rinsing the sections were covered with fluorescent secondary antibodies. The sections were rinsed and coverslipped with anti-fade glycerol. The immunohistochemical specificity was verified by omitting the primary antibodies. The slides were scanned using a Vectra automated quantitative pathology imaging system. Data are presented as mean ± SEM of percentage of fluorescent pixels observed for the entire sectioned ganglion. Results: The NG were 31.37 ± 5.8% fluorescent for 5-HT3 receptors. A tiny percentage of the NG was fluorescent for CB1 (0.03 ± 0.02%) or CB2 (0.03 ± 0.01%) receptors. Colocalization of 5-HT3 and CB1, or 5-HT3 and CB2, was 0.03 ± 0.01% or 0.03 ± 0.01%. In other words, those NG cells that expressed CB receptors also expressed 5-HT3 receptors. Conclusion: Only a small percentage of NG cells express CB receptors, and most of those cells are colocalized with 5-HT3. Support (If Any): This work was supported by the Chicago Biomedical Consortium Postdoctoral Research Grant Program.
COMPUTATIONAL MODEL OF BRAINSTEM CIRCUIT FOR STATE-DEPENDENT CONTROL OF HYPOGLOSSAL MOTONEURONS Komarov M1, Naji M1, Krishnan G1, Malhotra A1, Powell F1, Rukhadze I2, Fenik V3, Bazhenov M1 1 Department of Medicine, University of California, San Diego, San Diego, CA, 2Department of Medicine, David Geffen School of Medicine, UCLA, Los Angeles, CA, 3Department of Veterans Affairs, Greater Los Angeles Healthcare System, Los Angeles, CA Introduction: In patients with obstructive sleep apnea (OSA) compensatory mechanisms keep the upper airway open allowing patients to breathe normally when awake. However, during sleep the pharyngeal muscles become relaxed, which leads to a partial or complete closure of airway. In this study, we developed a computational network model to investigate the impact of monoaminergic drive that is mediated by hypothetical interneurons (Fenik, 2015) on hypoglossal motoneurons (HMs) during REM sleep. Methods: The network model included five distinct populations of cells: serotonergic raphe neurons (RN), noradrenergic A7 neurons, perihypoglossal GABAergic interneurons (PGI) and excitatory interneurons (PEI), and HMs. HMs were indirectly controlled by A7 neurons through the populations of PEI. Medullary/pontine RN provided inhibitory serotonergic projections to PGI that sent GABAergic projections to PEI. Results: The model captured the changes in the firing rate of the studied neuronal groups across sleep stages. The firing rate of HMs was very low (~ 15 Hz) during REM sleep compared to NREM sleep (~ 50Hz) as observed in in vivo experiments. The model predicted two distinct mechanisms for reduced activity of HMs during REM sleep: (i) decrease of firing rate of noradrenergic A7 neurons leading to disfacilitation of PEI, (ii) silencing of serotonergic RN leading to disinhibition of PGI, which, in turn, inhibited PEI. Conclusion: Using a biophysical model of the brainstem neural network, we predict that the state-dependent control of hypoglossal motoneuron excitability can be mediated by perihypoglossal interneurons, which integrate the noradrenergic and serotonergic drives to HMs and may serve as new potential targets for treatment of OSA. Support (If Any): ONR MURI: N000141310672; RO1HL116845.
0141 DORSAL RAPHE SEROTONINERGIC NEURONS ARE PART OF THE NEURONAL CIRCUITRY REGULATING CO2 INDUCED AROUSALS Kaur S, Fuller PM, Saper CB Beth Israel Deaconess Medical Centre and Harvard medical School, Boston, MA Introduction: Serotoninergic (Sert) raphe (Dorsal and medullary) neurons are CO2- responsive, and mice lacking these neurons have impaired arousal to CO2. However, CO2 responsiveness can be restored by 5HT2A agonist, suggesting that serotonin may be a neuromodulator. We have shown previously that the external lateral parabrachial nucleus (PBel) regulate the hypercapnia induced arousal. PBel also receive substantial serotoninergic innervation from dorsal raphae (DR), therefore, we hypothesize that Sert-DR mediate CO2 arousals at least in part by its input to the PBel. Methods: To test the role of Sert-DR, we conducted optogenetic inhibition of DR neurons selectively in Sert-cre mice (n=5) and tested their arousal responses to 10% CO2. We injected an adeno-associated virus containing the gene for Archaerhodopsin TP009 T in a Cre-inducible FLEX cassette (AAV-FLEX-ArchT-GFP), that expressed ArchT in Sert+-DR cells. These mice were also instrumented for sleep and optogenetics. To model cyclic hypercapnia as seen during sleep apnea, we investigated EEG arousals to 10% CO2 given for 30s every 300s and compared them with and without the 593nm laser light that inhibits Sert-DR neurons. Results: Without the laser, mice showed normal responses to CO2 (arousal latency- 12.6 ± 0.9 sec), and woke-up on every CO2 trial. In 3 mice in which we histologically confirmed both the expression of ArchT and the placement of the glass fiber in the Sert-DR nucleus, with the 593nm laser-ON, the arousal latency doubled (33.88 ± 0.76 sec) and in 19.04 ± 1.9% of the trials mice did not wake up to CO2 stimulus. In these mice, a dense fiber and terminal field of ArchT-GFP was also observed in the PBel nucleus. Comparison of Sert-DR inhibition to that of inhibition of CGRP-PBel, shows that the latter is almost 2 fold higher. Conclusion: These results suggest that Sert-DR neurons are part of the neural circuitry that regulates the cortical EEG arousals to hypercapnia, presumably by projecting to the CGRP-PBel. Current studies are underway to dissect the role of Sert-DR on the CGRP-PBel neurons in causing CO2 induced-arousal. Support (If Any): NIH grant- 2PO1HL095491- 06.
0140 CO-LOCALIZATION OF SEROTONIN AND CANNABINOID RECEPTORS IN THE NODOSE GANGLIA Calik MW, Carley DW University of Illinois at Chicago, Chicago, IL Introduction: Pharmacological treatments for obstructive sleep apnea are limited due to incomplete knowledge of the neurochemical properties of respiratory circuits. The afferent vagus nerves provide key respiratory modulating feedback to the brainstem. The nodose ganglia (NG) of the vagus nerves are neurochemically diverse containing many receptors, including serotonin type 3 receptors (5-HT3), and cannabinoid type 1 (CB1) and/or type 2 (CB2) receptors. In anesthetized rats, a cannabinoid (CB) agonist attenuates serotonin (5-HT)induced reflex apnea via activation of CB receptors located on the NG. However, it is unclear how 5-HT3 receptors colocalize with CB1 and CB2 receptors. Here, we examine the localization of these three receptors in the rat NG. Methods: NG were extracted from anesthetized adult male SpragueDawley rats, fixed with 4% paraformaldehyde and 20% sucrose, processed and embedded in paraffin and sectioned using a microtome. SLEEP, Volume 40, Abstract Supplement, 2017
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