束缚—浸水应激大鼠内侧前额叶皮质对胃机能的调控作用及机制的研究

发布时间：2018-05-03 09:22

本文选题：内侧前额叶皮质 + 边缘前皮质　；参考：《山东师范大学》2015年博士论文

【摘要】：前额叶(Prefrontal cortex,PFC)又称额前皮质,为额叶初级运动皮质和次级运动皮质之前的全部额叶皮质,属最高级别的联合皮质。该结构在“系统发生中最晚出现,个体发育时最迟成熟”,是唯一与丘脑背内侧核有交互纤维联系的新皮质。大鼠PFC依据其形态学特征分为背外侧前额叶、眶额叶和内侧前额叶。其中,内侧前额叶(Medial prefrontal cortex,MPFC)主要包括背侧部和腹侧部,背侧部范围较小,与灵长类背外侧部同源,主要参与各种运动行为的调节(如眼球运动等);腹侧部占据MPFC大部分,与边缘系统间存在大量的纤维投射,参与情感、认知、内脏活动的调控,依其结构与功能可分为边缘前皮质(Prelimbic cortex,PL)和边缘下皮质(Infralimbic cortex,IL)两部分。其中,PL主要与认知(注意力调控、工作记忆等)、情感(条件性恐惧、焦虑等)及执行控制等功能相关,又称“认知-情感活动皮质”;IL主要与内脏自主神经功能活动相关,可发出纤维广泛投射到皮质下参与调控内脏自主神经功能活动的核团(如下丘脑、孤束核、迷走背核、疑核等),参与心血管、胃肠功能活动的调节等,故又称“内脏运动皮质”。另外PL和IL二者相互之间还存在密集的纤维投射。大鼠束缚-浸水应激(Restraint water-immersion stress,RWIS)为生理学、医学、心理学研究中常用的应激模型。RWIS是一种强度较大的复合应激,既包含生理性应激成分(如饥饿、制动、冷水等刺激因素),又有心理性应激成分(如大鼠因RWIS而产生的愤怒、恐惧、焦虑等情绪变化刺激因素)。两类应激成分共同作用可致严重的胃肠机能紊乱(如胃运动亢进、胃酸分泌增多等),甚至胃黏膜损伤。本实验室前期研究证实,RWIS所致胃肠机能紊乱的外周神经机制,主要是因为“副交感神经系统活动加强”所致。关于其中枢神经机制,经多年研究已知,初级中枢—延髓胃肠中枢、较高位中枢—下丘脑都参与了这一生理过程,但对于最高位中枢—大脑皮质的作用及其调节机制,目前尚未确定,鲜见文献报道。本实验室前期工作发现,大鼠RWIS不同时间段(0min、30 min、60 min、120 min、180 min),除延髓胃肠中枢和下丘脑神经元有强烈的Fos阳性表达外,大鼠MPFC(主要是PL和IL)内神经元的Fos蛋白的表达也显著增加,表明PL和IL参与了RWIS反应。那么PL和IL在RWIS所致胃粘膜损伤中是否参与对胃功能活动的调控?分别发挥何种作用?与非应激状态下相比,这种调控作用是否相同?其可能的调控机制是什么?PL和IL二者之间在功能上有无内在联系?目前均未见相关文献报道。本研究重点以RWIS大鼠为研究对象,同时将非应激大鼠作为参照,研究MPFC(PL和IL)与胃功能活动之间的调控关系及PL和IL二者之间的相互关系。本论文共设计了五部分实验:实验一:DA作为一种重要的神经递质,可与MPFC内的多种受体(如D1、D2受体)结合,广泛参与大鼠的情感、认知及自主功能的活动过程;L-Glu为常用的谷氨酸受体激动剂,MPFC内大部分神经元胞体膜上皆有其受体。本研究利用中枢微量给药技术,研究了向PL和IL微量注射DA和L-Glu,对比观察给药前后胃运动、胃酸分泌的变化。结果:1)向PL中段和后段微量注射DA(0.2 mol/L,200 n L),在注药后的三个连续5 min内,大鼠胃运动的频率显著降低(P㩳0.05),同时伴随短时(5 min)的收缩波平均时程延长(P㩳0.05),但对胃运动其他指标,则几乎没有影响;对胃酸分泌也无影响。2)将DA注射到大鼠IL的前、中、后段,均未引起胃收缩活动及胃酸分泌的显著性变化(P㧐0.05)。3)向PL和IL内微量注射L-Glu,对大鼠的胃运动、胃酸分泌、呼吸频率及心率均未产生显著影响。小结:1)大鼠麻醉状态下,PL中段和后段神经元受DA调控,产生的综合信息下行对大鼠胃运动有一定的抑制作用,但对胃酸分泌无影响;IL内的DA敏感神经元不参与对胃功能活动的调控作用;2)大鼠麻醉状态下,PL和IL内L-Glu所兴奋的神经元与大鼠胃功能活动之间无功能上的联系。实验二:RWIS致胃肠机能紊乱的同时,可使MPFC内神经元的Fos蛋白表达增多,表明MPFC参与了RWIS反应,那么MPFC在RWIS所致的胃黏膜损伤中发挥何种作用?本研究以胃黏膜损伤指数为指标,以清醒大鼠为实验对象,比较观察单侧、双侧MPFC损毁大鼠与假手术大鼠,RWIS对其胃黏膜损伤程度的不同。结果:1)与假手术组相比,单侧、双侧损毁MPFC皆可有效降低由RWIS引起的胃黏膜损伤程度(P㩳0.01);2)双侧MPFC损毁降低胃黏膜损伤的效果优于单侧损毁(P㩳0.05)。小结:大鼠MPFC在RWIS所致胃黏膜损伤中,对胃功能活动具有重要的调节作用。实验三:结合实验一和实验二结果,为进一步探究大鼠在RWIS过程中,MPFC的两个部分—PL和IL,各自对胃功能活动(胃运动及胃酸分泌)的调控作用,本研究以清醒大鼠为实验对象,将大鼠分为假手术组、双侧PL损毁组、双侧IL损毁组、双侧PL和IL同时损毁组。比较观察四组大鼠在清醒非应激状态下和清醒RWIS 4 h过程中的胃运动(重点关注胃运动的平均幅度、胃运动指数及收缩分数)、胃酸分泌变化情况。结果:1)大鼠处于清醒非应激状态下,实验组大鼠胃运动的平均幅度、胃运动指数及收缩分数较假手术组均有明显增加,增加程度:双侧PL和IL同时损毁组双侧IL损毁组双侧PL损毁组;2)当大鼠处于RWIS 4 h过程中,假手术组大鼠的胃运动表现出了显著的亢进:各项统计指标与自身应激前相比均存在显著性差异(P㩳0.05或P㩳0.01)。双侧PL损毁组大鼠的胃运动平均幅度、胃运动指数在整个应激过程中,与自身应激前相比,并无显著性变化。双侧IL损毁组大鼠和双侧PL和IL同时损毁组大鼠的胃运动平均幅度、胃运动指数及收缩分数则皆受到了显著的抑制(P㩳0.05或P㩳0.01)。该结果与实验二的结果前后呼应,提示PL和IL的损毁对RWIS所致的胃运动亢进有抑制作用,抑制程度:双侧PL和IL同时损毁组双侧IL损毁组双侧PL损毁组;3)大鼠在RWIS 4 h过程中,与假手术组相比,双侧PL和IL同时损毁组大鼠的胃液分泌量及H+分泌量均显著增多(P㩳0.05)、同时碳酸氢盐含量显著下降(P㩳0.05)。小结:1)大鼠在清醒非应激状态下,MPFC的传出信息对胃运动起抑制作用,且IL的抑制效果强于PL,二者联合作用效果更强;2)大鼠在清醒RWIS应激过程中,MPFC的传出信息对胃运动起促进作用,且IL的促进效果强于PL,二者联合作用效果最强;同时MPFC的传出信息对胃酸分泌也有一定影响。实验四:结合实验三结果,为进一步探明PL和IL内不同类型的神经元在大鼠清醒RWIS过程中分别所扮演的角色,以及PL和IL二者之间神经元活动的相互关系,本研究利用在体多通道神经信号采集技术,同时监测两种不同模态的神经信号—锋电位(Spike)和局部场电位(Local field potentials,LFPs),进而研究大鼠在清醒非应激状态下和清醒RWIS过程中PL和IL内锥体神经元、中间神经元Spike电活动规律及LFPs活动规律。结果:1)大鼠在清醒非应激状态下,PL内的锥体神经元爆发式放电率显著高于IL(P㩳0.05),而IL内中间神经元的爆发式放电率则显著高于PL(P㩳0.05);2)大鼠在RWIS 4 h过程中,PL内的绝大部分锥体神经元和所有的中间神经元皆表现出了抑制效应(P㩳0.05或P㩳0.01),且随应激时间延长,其放电率、爆发式放电率及爆发式放电百分比均显著下降(P㩳0.05或P㩳0.01)。同时,RWIS导致PL内LFPs中的低频段分量信号(0.5-3 Hz,3-7 Hz)显著增多而中高频段分量信号(7-12 Hz,12-30 Hz,30-100 Hz)显著减少(P㩳0.05或P㩳0.01),且LFPs的功率谱密度分析及时域分析结果均与该结果的变化趋势相一致。提示大鼠在RWIS过程中,整个PL表现出了兴奋程度降低的趋势。3)大鼠在RWIS 4 h过程中,IL内所有的中间神经元和少部分锥体神经元表现出了抑制效应(P㩳0.05或P㩳0.01)。其余大部分锥体神经元皆因RWIS表现出了兴奋效应,但兴奋仅在应激第1 h、第2 h较明显,与应激前相比有显著性差异(P㩳0.05或P㩳0.01)。同时,与PL的LFPs活动规律相反,RWIS导致IL内LFPs中的低频段分量信号(0.5-3 Hz,3-7 Hz)显著减少而高频段分量信号(12-30 Hz,30-100 Hz)显著增多(P㩳0.05或P㩳0.01),且LFPs的功率谱密度分析及时域分析结果均与该结果的变化趋势相一致。提示大鼠在RWIS过程中,整个IL表现出了兴奋程度升高的趋势。小结:1)大鼠在清醒非应激状态下,PL内锥体神经元的兴奋程度相对较高,使得整个PL表现出了相对兴奋状态;而IL内中间神经元的兴奋程度相对较高,且多数可能是抑制性神经元,所以使得整个IL表现出了相对抑制状态;2)RWIS可对大鼠PL内的锥体神经元产生抑制作用,而对IL内的锥体神经元产生兴奋作用。3)大鼠无论在清醒非应激状态下,还是在RWIS过程中,其PL和IL内的神经元活动规律均不同,并表现出了相对立的活动状态。实验五:考虑到PL和IL在认知、情感及执行控制中的重要作用,结合RWIS中的心理性应激因素与MPFC的关系,本研究设计了旷场实验,观察损毁MPFC后大鼠的情绪、情感及认知能力的变化。结果:与假手术组相比,损毁MPFC可导致大鼠的焦虑情绪和条件性恐惧情绪加重,对外界刺激更敏感,更胆小怯懦;同时伴随认知能力的下降。小结:MPFC在大鼠情绪、情感的表达中主要执行抑制功能。
[Abstract]:The prefrontal cortex (Prefrontal cortex, PFC), also known as the prefrontal cortex, is the highest level of the frontal cortex, the highest level of the frontal cortex, before the primary frontal cortex and the secondary motor cortex. The rat PFC is divided into the dorsolateral prefrontal lobe, the frontal lobe and the medial prefrontal lobe according to its morphological characteristics. The medial prefrontal lobe (Medial prefrontal cortex, MPFC) mainly includes the dorsal and ventral parts. The dorsal part is small and is homologous with the primate dorsolateral part. It is mainly involved in the regulation of various kinds of movement behavior (such as eye movement, etc.); the ventral side occupies M. Most of PFC, with a large amount of fiber projection between the edge system, participates in the regulation of emotional, cognitive, and visceral activities. According to its structure and function, it can be divided into two parts: the Prelimbic cortex (PL) and the marginal cortex (Infralimbic cortex, IL). Among them, PL main needs and cognition (attention control, working memory, etc.), emotion (conditioned fear,) Anxiety, etc., and the function of executive control, also known as "cognitive - affective active cortex"; IL is mainly related to the autonomic autonomic function of the viscera, and can emit a wide range of fibers projecting into the cortex under the cortex (the following thalamus, the nucleus of the solitary tract, the nucleus of the vagus, the nucleus, and so on), involved in cardiovascular and gastrointestinal function activities. It is also known as the "visceral motor cortex". In addition, there are dense fibrous projection between PL and IL two. Rat binding immersion stress (Restraint water-immersion stress, RWIS) is a physiological, medical, and psychological stress model.RWIS is a kind of stronger compound stress, including physiological stress. Components (such as starvation, braking, cold water and other irritation factors), and psychological stress components (such as anger, fear, anxiety, and other irritation factors in rats caused by RWIS). The combination of two kinds of stress components can cause serious gastrointestinal dysfunction (such as hyperactivity of stomach, gastric acid secretion, etc.), even gastric mucosal injury. The study has confirmed that the peripheral nervous mechanism of the gastrointestinal dysfunction caused by RWIS is mainly due to the "enhancement of the parasympathetic nervous system activity". In the study of the pivot mechanism, the primary center, the medullary gastrointestinal center, and the higher central hypothalamus are involved in this physiological process. The role of cortex and its regulatory mechanism are not yet confirmed, and there are few literature reports. Earlier work in the laboratory found that rats RWIS (0min, 30 min, 60 min, 120 min, 180 min) had strong Fos positive expression in the medullary gastrointestinal center and hypothalamus neurons, and the expression of Fos protein in MPFC (mainly PL and IL) neurons in rats. Also significantly increased, indicating that PL and IL are involved in the RWIS response. Then, what is the role of PL and IL in the regulation of gastric mucosal damage induced by RWIS? What are the same regulatory effects compared with non stress states? What is the possible regulatory mechanism? There is a functional relationship between the PL and IL two. This study focuses on RWIS rats and studies the relationship between MPFC (PL and IL) and gastric function activities and the relationship between PL and IL two. This paper has designed a total of five experiments: Experiment 1: DA as an important neurotransmitter, Combined with a variety of receptors (such as D1, D2 receptor) in MPFC, it is widely involved in the emotional, cognitive and autonomic activities of rats; L-Glu is a commonly used glutamate receptor agonist, and most of the neurons in MPFC have their receptors. This study used central microinjection techniques to study the microinjection of DA and L-Glu to PL and IL, compared with PL and IL. The changes in gastric motility and gastric acid secretion were observed before and after the administration. Results: 1) DA (0.2 mol/L, 200 N L) was injected into the middle and posterior segments of PL, and the frequency of gastric motility in the rats decreased significantly (P? 0.05) in the three consecutive 5 min after injection, while the average time history of short time (5 min) was prolonged (P? 0.05), but the other indexes of gastric motility were almost no more. There was no effect on gastric acid secretion and no effect on.2). Before DA injection into IL of rat, no significant changes in gastric contraction activity and gastric acid secretion (P? 0.05).3) were injected into PL and IL, and L-Glu was injected into PL and IL, and no significant effect on gastric motility, gastric acid secretion, respiratory frequency and heart rate. The neurons in the segment and the posterior segment were regulated by DA, and the integrated information was inhibited, but it had no effect on gastric acid secretion, and the DA sensitive neurons in IL did not participate in the regulation of gastric function. 2) under the anesthetic state of the rats, there was no function between the neurons excited by L-Glu in PL and IL and the function of gastric function in rats. The connection. Experiment two: RWIS induced gastrointestinal dysfunction at the same time, can increase the expression of Fos protein in MPFC neurons, indicating the participation of MPFC in the RWIS reaction, and what role of MPFC in the gastric mucosal injury induced by RWIS? This study took the index of gastric mucosal injury as the index, and compared the unilateral and bilateral MP in conscious rats. FC damage of rats and sham rats and RWIS to the degree of gastric mucosal damage. Results: 1) compared with the sham group, unilateral and bilateral damage of MPFC could effectively reduce the degree of gastric mucosal injury caused by RWIS (P? 0.01); 2) bilateral MPFC damage reduction of gastric mucosal damage was better than unilateral damage (P? 0.05). Conclusion: MPFC in RWIS caused by rats. In gastric mucosal injury, it has an important regulatory effect on gastric function activity. Experiment three: combined with the results of experiment one and experiment two, the effects of two parts of MPFC, PL and IL, on gastric function activities (gastric motility and gastric acid secretion) in rats were further explored. For the sham operation group, bilateral PL damage group, bilateral IL damage group, bilateral PL and IL simultaneously damaged group. The gastric motility in the four groups of rats in awake non stress state and awake RWIS 4 h process (focus on the average amplitude of gastric motility, gastric motility index and contraction fraction), gastric acid secretion change. Results: 1) rats were in the awake non should. The average amplitude of gastric motility, the gastric motility index and the systolic fraction of the rats in the experimental group were significantly increased in the experimental group compared with the sham operation group, and the increase was: bilateral PL and IL simultaneously damaged group bilateral IL damage group bilateral PL damage group; 2) when the rats were in the RWIS 4 h process, the stomach movement of the sham operation group showed significant hyperactivity: various statistics There were significant differences in the indexes compared with those before stress (P? 0.05 or P? 0.01). The average amplitude of gastric motility in the rats with bilateral PL damage group, and the gastric motility index in the whole stress process, had no significant changes compared with that before stress. The average amplitude of gastric motility in rats with bilateral IL damage group and bilateral PL and IL damaged rats, stomach movement Both the dynamic index and the contraction fraction were significantly suppressed (P? 0.05 or P? 0.01). The results were echoed with the results of experiment two, suggesting that the damage of PL and IL was inhibited by RWIS induced gastric hyperactivity, the degree of inhibition was: bilateral PL and IL damage group bilateral IL damage group bilateral PL lesion group; 3) rats in the RWIS 4 h process, and false hands. Compared with the operation group, the gastric juice secretion and H+ secretion of both bilateral PL and IL rats were significantly increased (P? 0.05), and the content of bicarbonate decreased significantly (P? 0.05). 1) in the sober non stress state, the efferent information of MPFC was inhibited by the MPFC, and the inhibitory effect of IL was stronger than that of PL, and the effect of the two combined effect was stronger; 2 In the course of conscious RWIS stress, the efferent information of MPFC promoted the motion of the stomach, and the effect of IL was stronger than that of PL, and the combination of the two had the strongest effect. At the same time, the efferent information of MPFC also had a certain influence on the secretion of gastric acid. Experiment four: combined with the result of experiment three, the different types of neurons in PL and IL were further explored in the rat clear. The roles played during the wake of RWIS, as well as the interaction of neuronal activity between PL and IL two, are used in this study to monitor the neural signal front potential (Spike) and local field potential (Local field potentials, LFPs) of the two different modes of neural signal acquisition. The Spike electrical activity and LFPs activity of pyramidal neurons and intermediate neurons in PL and IL in stimulated state and awake RWIS process. Results: 1) the explosive discharge rate of pyramidal neurons in PL was significantly higher than that of IL (P? 0.05) in the sober non stress state, while the explosive rate of the intermediate deity in IL was significantly higher than that of PL (P? 0.05); 2) In the RWIS 4 h process, most pyramidal neurons and all the intermediate neurons in PL showed inhibitory effects (P? 0.05 or P? 0.01), and with the prolonged stress time, the discharge rate, explosive discharge rate and the percentage of explosive discharge were significantly decreased (P? 0.05 or P? 0.01). At the same time, RWIS led to the low frequency component of LFPs in PL. The number (0.5-3 Hz, 3-7 Hz) was significantly increased while the medium and high frequency component signals (7-12 Hz, 12-30 Hz, 30-100 Hz) were significantly reduced (P? 0.05 or P? 0.01), and the power spectral density analysis and time domain analysis of LFPs were all consistent with the trend of this result. During IS 4 h, all the intermediate and few pyramidal neurons in IL showed inhibitory effects (P? 0.05 or P? 0.01). Most of the rest of the pyramidal neurons were excited by RWIS, but the excitement was only under stress first h, second h was obvious, and there was a significant difference compared with pre stress (P? 0.05 or P? 0.01). And PL LFPs. On the contrary, RWIS leads to a significant decrease in the low frequency component signal (0.5-3 Hz, 3-7 Hz) in the IL LFPs and a significant increase in the high frequency component signal (12-30 Hz, 30-100 Hz) (P? 0.05 or P? 0.01), and the LFPs power spectral density analysis and time domain analysis results are in accordance with the trend of the result. There is a tendency to increase the degree of excitement. Summary: 1) in the non stress state of the rat, the excitatory degree of the PL pyramidal neurons is relatively high in the conscious and non stress state, making the whole PL relatively excited state, while the degree of excitement in the IL intermediate neurons is relatively high, and most of them may be inhibitory neurons, so that the whole IL is relative. Inhibition state; 2) RWIS can inhibit the pyramidal neurons in the rat PL, and produce excitatory effect on the pyramidal neurons in IL,.3) in the awake non stress state, or in the RWIS process, the neuronal activity rules in PL and IL are different, and the relative active state is shown. Experiment five: PL and IL are considered. The important role in cognition, emotion and executive control combined with the relationship between psychological stress factors and MPFC in RWIS. This study designed a open field experiment to observe the changes in emotional, emotional and cognitive abilities of rats after damaging MPFC. Results: compared with the sham operation group, the damage of MPFC can lead to anxiety and conditioned fear in rats. It is more sensitive to external stimuli, more timid and cowardly, accompanied by a decrease in cognitive ability. Conclusion: MPFC mainly performs inhibition in emotional and emotional expression in rats.

【学位授予单位】：山东师范大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R338

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