[PMC free article] [PubMed] [Google Scholar] 53

[PMC free article] [PubMed] [Google Scholar] 53. activation of orexin neurons. Acute ethanol exposure significantly reduced the number of orexin neurons comprising c-Fos, suggesting an inhibition of orexin neurons after ethanol intake. Conclusions: Based on our results, we believe that ethanol promotes sleep by increasing adenosine in the orexinergic perifornical hypothalamus, resulting in A1 receptor-mediated inhibition of orexin neurons. Citation: Sharma R; Sahota P; Thakkar MM. Part of adenosine and the orexinergic perifornical hypothalamus in sleep-promoting effects of ethanol. 2014;37(3):525-533. and studies suggest that AD inhibits orexin neurons via A1R41,42. Activation of A1R in the PFH promotes sleep, whereas blockade of A1R promotes arousal and attenuates recovery sleep following sleep deprivation.43,44 To evaluate whether ethanol-induced sleep promotion is mediated by AD via A1R and entails inhibition of orexin neurons, we performed three experiments: Our first experiment examined whether blockade of A1R in PFH attenuates ethanol-induced sleep promotion. Our Rabbit Polyclonal to FCGR2A second experiment examined the effect of ethanol, locally given into the PFH, on AD launch. Our third experiment determined the effect of ethanol exposure on c-Fos manifestation in orexin neurons. MATERIALS AND METHODS Adult male Sprague-Dawley rats (250-350 g; Charles River, Wilmington, MA) were housed in Harry S. Truman vivarium under standard 12:12 h light-dark cycle, with ambient heat and access to food and water. All experiments were performed according to the Association for Assessment and Accreditation of Laboratory Animal Care guidelines and Guideline for the Care and Use of Laboratory Animals. All protocols were approved by local committees. Experiment 1: To examine ethanol-induced sleep promotion in rats pretreated having a selective A1R antagonist into the PFH. SurgeryUsing standard surgical procedure and under inhalation anesthesia,45 rats were implanted with electrodes for electrographic recording of electroencephalogram (EEG) and electromyogram (EMG) to determine sleep-wakefulness. Intracerebral guideline cannulas (22 gauge; Plastics One, Roanoke, VA, USA) were also implanted bilaterally at a 90 angle above the prospective site in the PFH.44 The prospective coordinates for the tip of the injector cannulas were: AP -3.3, ML 1.5, DV -8.5 [relative to bregma46]. Flunixin TP-0903 (1.5 mg/kg), administered subcutaneously, was used like a postsurgical TP-0903 analgesic. Experimental protocolAll experiments were carried out inside a sound-attenuated chamber with food and water available test, was performed to examine the effect of A1R blockade on ethanol-induced sleep promotion. Experiment 2: Effects of local ethanol perfusion on AD launch in the PFH. SurgeryUsing standard surgical procedure and under inhalation anesthesia, rats were surgically implanted having a unilateral guideline cannula (CMA, Stockholm, Sweden) in the PFH (stereotaxic coordinates as explained for experiment 1). After 5 days of postoperative recovery, microdialysis probe was put through TP-0903 the guideline cannula into the PFH and artificial cerebrospinal fluid [aCSF; NaCl 147 mM, KCl 3 mM, CaCl2 1.2 mM, MgCl2 1.0 mM, pH 7.2) was perfused at a flow rate = 0.7 L/min. Experimental protocolThe experiment was begun at dark onset after permitting 12-16 h for probe insertion recovery. In addition, 4 20 min (14 L/sample) pre-ethanol baseline samples were collected. Subsequently, 30-, 100-, and 300-mM doses of ethanol were perfused. Each dose of ethanol was perfused for 80 min and 4 20 min samples were collected. Finally, aCSF was perfused and 4 20 post-ethanol samples were collected. Samples were stored in snow until analyzed. The flow rate was managed at 0.7 L/min during the entire experiment. On TP-0903 completion, probes were removed and processed for recovery.47 AD separation and quantification was achieved by high-performance liquid chromatography (HPLC) coupled with an ultraviolet (UV) detector.16,47C50 In brief, 10 L of microdialysis sample was injected into the HPLC. The mobile phase contained 8 mM NaH2PO4 and 8% methanol (pH = 4; circulation rate = 80 L/min). AD was separated out having a microbore column (1 100 mm; MF-8949; BASi, Western Lafayette, IN) and recognized by a UV detector (wavelength = 258 nm; Model SPD20, Shimadzu, Columbia, MD, USA). Chromatogram data were acquired and analyzed by PowerChrom 280 system (eDAQ Inc, Colorado Springs, CO, USA). AD maximum in the sample was recognized and quantified by comparing its retention time and area under the maximum to real known amounts of external AD requirements (Sigma-Aldrich Co. LLC)..

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