The Effect Of OrexinA In Entorhinal Cortex On Sleep-wake Cycle And Spatial Memory

Orexin, also known as hypocretin, is a kind of neuropeptide produced by orexinneurons in the lateral hypothalamus. The two monomers orexin-A and orexin-B areprocessed from a single pre-pro-orexin polypeptide synthesized from one single gene.Orexin-A consists of33amino acids with two disulfide bonds. Both orexin-1receptor(OX1R) and orexin-2receptor (OX2R) are G-protein coupled receptors and underlie theorexin-mediated physiological roles. The affinity between OX1R and orexin-A is higherthan that of orexin-B, whereas the affinity between OX2R and orexin-A is identical withthat of orexin-B. orexin neurons project broadly throughout the whole brain, which is ofgreat significance in the maintenance of wakefulness. Furthermore, the narcolepsy can befound in the orexin knockout (KO) or orexin receptor transcription-Disrupted mice. At thesame time, the projection from orexin neurons to hippocampus (Hp) was found，whichsuggested that orexin might has an important role in the spatial learning and memory.The entorhinal cortex (EC) has been regarded as a nodal point between hippocampusand neocortical cortex. Generally, superficial layers of EC(layers II–III) deliveries amultitude of inputs from other brain regions to hippocampus,and the deep layers (layersV–VI)transports outputs from hippocampus, mainly the CA1and subiculum, to theneocortical cortex. The medial entorhinal cortex (MEC) conveys the spatialinformation,while the lateral entorhinal cortex (LEC) transfers non-spatial information likeolfactory and somatosensory signals to hippocampus. These anatomical properties make theEC indispensable in the neocortical cortex-hippocample network–mediated encoding,consolidation and retrieval of memories, especially the spatial memory.The polysomnography recording and micro-injection techniques have been applied inour experiments. Firstly, we explored the effects of exogenous orexinA on rat sleep, then wemanaged to determine the effects of orexinA on the special memory of rat. The potentialinterruptions of the experimental operations, the microinjections for example, were excluded through open field tests.1. The expression of Orexin receptor in the EC and its co-expression with reelinand calbindin in superficial layers of ECThe expression of OX1R and OX2R in EC were determined with immunofluorescencehistochemical detection, we found that both orexin receptors are expressed in EC, and theirexpression in superficial layers are denser than that of deep layers. In addition, theco-expression of OX1R with reelin and calbindin were identified, which suggested thatorexin may have an important role in special memory, taking into account of the importanceof Reelin and calbindin signals of the EC in spatial memory.2. The effect of Orexin A on sleep-wake cycleOrexinA at different concentration of (0.04nmol0.06nmol,0.1nmol) was microinjectedinto EC respectively, nothing significant effect was found on the sleep-wake cycle. Therewere no significant differences among the3hours, namely the11:00~12:00,12:00~13:00,13:00~14:00(P>0.05), in terms of the wakefulness, NREM and REM duration. So doesthe whole wake-sleep cycle of24h (P>0.05). These results showed that the micro-injectionof orexinA in rat’s entorhinal cortical has no effect on sleep-wake cycle.3. The effect of orexinA receptor antagonist SB-334867on sleep-wake cycleOrexinA receptor antagonist SB-334867at different concentration (0.1nmol,5μl) wasbilateral microinjectioned into EC respectively, which resulted in no significant changes ofwake-sleep cycle. There were no significant differences among the3hours, namely the11:00~12:00,12:00~13:00,13:00~14:00(P>0.05), in terms of the wakefulness, NREMand REM duration. So does the whole wake-sleep cycle of this three hours (P>0.05). Theseresults showed that the micro-injection of orexinA receptor antagonist SB-334867in rat’sentorhinal cortical has no effect on sleep-wake cycle.4. The effect of orexinA in EC on the spatial memory1) The effect of orexin A receptor antagonist SB-334867After the orexin A receptor antagonist SB-334867was microinjected into EC, twoT-maze modes (hold mode, acquisition mode) were exerted to define the effect of SB-334867in EC on spatial memory. The SB-334867and placebo DMSO were microinjected intoEC after the continuous training of rats, we found that the accuracy rate of rat in T-mazewas decreased by78%(P <0.05). That is, the SB-334867exerts a significant influence on the spatial memory, suggesting that the endogenous orexin signal in EC plays an importantrole in spatial memory.2) The effect of the knockdown of the orexinA receptor expression(RAAV2/1-OR1-shRNA-EGFP)The control group SCR (RAAV2/1-SCR-shRNA-EGFP) and the interference groupOR1(RAAV2/1-OR1-shRNA-EGFP) were microinjected into EC of rats. Then rats in bothgroups were trained21days in T-maze. Compared with SCR, microinjection ofRAAV2/1-OR1-shRNA-EGFP which knockdown the expression of OR1in EC resulted in areduction of accuracy rate in T-maze by48%(P <0.05). It pointed out that the knockdownof orexinA receptor expression in EC could damage the spatial memory function. That is,orexin A receptors in entorhinal cortex are involved in spatial memory.5. The effect of Postoperative micro-injection of drugs on exploratory behaviorand motor functionAfter recovery from the casing embedded operation, the rats were given microinjectioninto EC of the ACSF, DMSO, orexin A and its receptor blocker SB-334867. Then theexploratory behavior and motor function of rats were observed. We found that these fourexogenous drugs had no significant effect on the number of standings of rat in the openfield and the number of grids the rat walked through. The orexinA was microinjected at twodifferent doses(0.1nmol,0.5μl) and the SB-334867was microinjected at the dose of6mMand0.5μl, with the corresponding doses of ACSF and DMSO respectively. These resultsrevealed that the Postoperative micro-injection of drugs has no significant effect on theexploratory behavior and motor function of rat.In summary, the results of this study showed that:1. The OR1expressed in EC exist inboth reelin-positive and calbindin-positive neurons.2. There was no effect of orexinA in ECat different doses on the sleep-wake cycle.3. There was no effect of orexin A receptorantagonist SB-334867in EC on the sleep-wake cycle.4. Endogenous orexinA in EC playsan important role in the spatial memory of rat.5. Postoperative micro-injection of drugs hasno significant effect on the exploratory behavior and motor function of rat.