Research On The Relationship Between Galanin Positive Neur On And Serotonin In The Dorsal Raphe Nucleus In A Rat Model Of PCPA Intervening Insamnia

【Backgroud】Galanin, a 29 (30 in human) amino acid neuropeptide, is widely distributedin the rodent central nervous system (CNS). Brain areas expressing galanininclude the ventral forebrain, amygdala, hypothalamus (HP), dorsal raphe nucleus(DRN), locus coeruleus (LC), and preoptic area[1,2], suggesting its importance inphysiological actions like feeding, recognition, endocrine response, memory, andsleep[3,4,5]. Galanin and galanin receptors are widely expressed by neurons in ratbrain and affect the synthesize/release of several classical neurotransmitters, suchas gamma aminobutyric acid (GABA), acetylcholine (ACh), noradrenaline (NA),and serotonin (5-HT). Galanin plays a major role in sleep regulation; galaningene expression in the rat brain is enhanced during REM-sleep deprivation[6], andthe intravenous administration of galanin leads to a change in the sleep EEG,evidence suggesting that one effect of galanin is to lower LC electrical activity[7].Serotonergic neurons are widely distributed in the CNS, however, themajority of these are located in the dorsal raphe nucleus (DRN), which lie around the midline of the brainstem and have extensive projections throughout thebrain[8]. Additionally, there are more than 14 serotonergic receptor subtypes in thebrain, implying complex functions in the CNS. The serotonergic system has beenimplicated in depression, the stress of injury, and also in regulating sleep andwaking [9,10]. A recent study has demonstrated that the activation of 5-HT1Breceptors induces and maintains REM, and maintains the suppression of thebehavioral state[11]. The 5-HT2A receptor increases slow wave sleep (SWS) andimproves sleep maintenance. APD125, a potent and selective in verse agonist for5-HT2A receptors may be useful in the treatment of insomnia [12]. Microdialysishas also been used to examine interactions between 5-HT1A and galaninreceptors in the DR by measuring the extracellular levels of 5-HT in the ventralhippocampus of awake rats [13]. Furthermore, in the rat brain, approximately 50%of the DRN is 5-HT neurons, and these neurons are co-expressed with galaninand are responsive to exogenous galanin in vitro [14]. Together, this implies acomplex mechanism of sleep regulation involving 5-HT and galanin neurons inthe DRN.P-Chlorophenylalanine (PCPA) is a 5-HT synthesis inhibitor that acts as acompetitive and irreversible inhibitor of tryptophan hydroxylase, the rate limitingenzyme in 5-HT synthesis. The administration of PCPA result in depletion onbrain and 5-HT leading to depression, and even insomnia [15,16]. Insomnia ischaracterized by difficulty falling asleep, difficulty staying asleep, or noconstraint sleep, accompanied by daytime impairment or distress[12]. Thepathological mechanism of insomnia remains unclear. However increasingevidence suggests that galanin and 5-HT may play a crucial role in sleep andinsomnia.Furthermore, the potential role of galanin in sleep and insomnia is partially based on its co-localization with 5-HT in the DRN. Severalneurochemical studies have provided the first evidence for the possibility ofgalanin interactions with 5-HT and a modulatory action of galanin on brain 5-HTneurotransmission [17]. In vivo, the relationship between galanin and 5-HT in a ratmodel of insomnia was also not yet revealed. Therefore, we investigated theexpression of galanin neurons in the DRN of insomniac rats at varying timepoints,and confirmed the potential relationship between galanin and 5-HT in the DRN.【ObjectiveObjectives】The aims: 1. Using PCPA, a 5-HT synthesis inhibitor, establishing a rat model ofinsomnia , to explore the change of rat behavior (FST , TST), also immobile time(IM) at the different timepoints, to be a method of valuing a rat model ofinsomnia. 2. Observing the difference in the morphology and distribution ofgalanin and 5-HT neurons in a rat model of insomnia,to detect the expressingnumbers of galanin and 5-HT neuron in a rat model of insomnia, to explore thebiology action of galanin at the development of insomnia and the relationshipbetween galanin and 5-HT neurons in a rat model of insomnia. 3. Real-time PCRwas used to quantify galanin mRNA in a rat model of insomnia, to explore andanalyze the trend of galanin mRNA with the behavior of insomnia rat and thenumber of positive neurons.【Methodes】1. Using PCPA of a 5-HT synthesis inhibitor, establishing a rat model ofinsomnia, discovering the change of rat behavior (FST, TST), also immobile time(IM) at the different timepoints.2. Observing the difference in the morphology and distribution of galanin and 5-HT neurons in a rat model of insomnia. Using IPP 6.0 software to detect theexpressing numbers of galanin and 5-HT neuron in a rat model of insomnia. 3. Real-time PCR was used to quantify galanin mRNA in a rat model of insomnia,to analyze the trend with the behavior of insomnia rat and the number of positiveneurons.【ResultResults】1. Through discovering the change of rat behavior (FST , TST) at the differenttimepoints, Thus, immobile time (IM)reached its peak at 4d(p﹤0.01)in bothFST and TST, in another, rat behavior is the shortage at 4d, with the time, ratbehavior basically obtain to the normal level.2. Observation of the green and red fluorescent photographs demonstrated noobvious differences in the morphology and distribution of galanin and 5-HTneurons between the insomnia and control groups. However, the expression ofgalanin neurons in the DRN was brightest at 4d, while expression of 5-HTneurons in the DRN was most reduced at 4d.Therefore, using the IPP 6.0software to analyze the galanin and 5-HT neurons in the DRN of insomnia rat.The results show that the expressing number of galanin neurons is highest at 4d(p﹤0.01)in the DRN of insomniac rats, while the number of 5-HT neurons is theleast at this timepoint, the number span between of galanin and 5-HT neurons isthe maximum, with the time, the number span between of galanin and 5-HTneurons obtain to the normal level.3. Real-time PCR was used to quantify galanin mRNA in a insomnia rat of DRN.The expressed relative quantity (mean±SEM)of galanin mRNA in the DRN ofinsomniac rats was,The expressed relative quantity of galanin mRNA was highestat 4d(p﹤0.01)in the DRN of insomniac rats, it is the same trend with thebehavior and expressing number of neurons. .【Conclusions】1. Using PCPA construct a stabilized rat model of insomnia. therfore, the trend of rat behavioral obtain to the same level.2. The expression of galanin neurons at a rat model of insomnia constructed byPCPA, galanin is crucial role in the insomnia, and take a important peptide atcentral nervous system (CNS).3. To analyze the galanin and 5-HT neurons in the DRN of insomnia rat,preliminary show the negative relationship of galanin and 5-HT neurons in ainsomnia rat of DRN.4. Real-time PCR was used to quantify galanin mRNA in a insomnia rat of DRN,the results is the same as the trend of behavior and the galanin expressing number,deeply displaying galnin biological action at the development of insomnia.