STG中幽门三相节律的观察和阻断I_k（ca）离子通道后放电节律变化的研究

【作者】 董珊；

【导师】 古华光；

【作者基本信息】 陕西师范大学 ， 生物物理学， 2011， 硕士

【摘要】 中枢模式发生器(Central pattern generator, CPG)是由一系列神经元以及其相互间突触连接集合而成,在缺少外界输入的情况下(如感觉输入和来自外周的伴随反射),其控制效应器产生节律性模式输出,调节完成相应的节律性运动。CPG产生节律模式的输出依赖于网络自身的特性以及网络受外部条件调控的影响。无脊椎动物(龙虾,蝗虫)标本在此研究中具有重要作用。它的神经系统解剖结构清楚,胞体数量和突触间联系固定并且易于制备。在离体情况下网络节律性仍然保持不变,胞外神经纤维记录有明显的节律性而且易于区分各类神经元放电。甲壳类动物的口胃神经节(stomatogastric ganglion, STG)是一种CPG网络,它位于此类动物的胃部,并且控制其幽门和胃磨的运动。虾类STG是由最少30个神经元组成的一个小网络,包括幽门胃CPG和胃磨体CPG。其中幽门胃网络是由6类、14个STG神经元组成,分别是AB、PD (2)、LP、PY (8)、IC和VD,其中AB为唯一的中间神经元。幽门网络中各神经元的性质,在网络中的作用和相互间的突触联系对于整个生命活动完成尤为重要。由于上端STG中各神经元通过神经纤维进行信息传递,胞外记录lvn神经纤维可观察到PD、LP、PY神经元依次排列形成的典型三相节律,这是由解剖结构决定的。lvn神经纤维一般用于记录幽门神经网络放电。在幽门神经系统中,三相节律输出作为一种胃部活动反映其消化、吸收食物的快慢程度。在网络中,AB神经元是最先开始放电的中间神经元,通过电耦联与2个PD神经元协同放电,这就使得AB和2个PD组成起搏神经元群,它们通过突触抑制其他的5类运动神经元,所以在AB/PD放电静息期这些运动神经元基本上开始放电。同步胞内记录表明,AB和PD由于电偶联作用时相基本相同；LP与IC时相大部分重合；PY与VD时相大体相同。当前国内对于感觉神经系统的研究较多,但运动神经系统较少。神经网络的行为在多个学科被当作热点研究。基于前人的研究进展,本研究主要采用胞外实验记录方式观察了克氏鳌虾(Procambarus clarkia(Girard)) STG中幽门三相节律的稳定放电,利用钙依赖钾离子通道阻断剂TEA进行作用,观察三相节律性电活动的变化,从而加强对鳌虾STG网络活动的认识。主要的研究结果：1.鳌虾STG幽门三相节律的稳定记录实验中,在正常胞外液下鳌虾STG幽门神经元网络表现出特定的三相放电节律模式,即LP、PY、PD神经元形成包含三个时相的循环放电模式。此节律中各神经元放电表现为：PD神经元高频、低幅值放电,LP神经元较低频率、高幅值放电,以及PY神经元的低频率、较低幅值放电。在PD神经元放电结束后,会产生一段静息期,而后LP继续开始放电。幽门网络三相节律以这种特定放电形式和放电顺序循环产生。2.TEA影响STG神经元三相放电节律的变化实验表明在STG幽门网络中的神经元自发放电活动中,钙依赖性钾通道参与活动。在TEA作用下三相节律中神经元放电节律模式发生改变,从较均匀的形式转为有休止期交替的簇放电,但是同时对于三相节律的顺序关系没有影响,神经元网络基本状态没有改变。其次,30mmol/L TEA使得处于正常稳定三相放电节律周期呈现变短的过程,三相节律中三类神经元对应的放电持续期以及各自放电中的峰峰间期(Interspike Interval, ISI)也有一定的变化。三相节律周期和PY放电持续期减少有极显著意义(P<0.01)；LP神经元放电持续期增加有显著意义(P<0.05); LP、PD放电周期中ISI减少有显著意义(P<0.05)。3.TEA影响STG神经元三相放电节律时相的变化实验表明,随着正常胞外溶液被30mmol/L TEA溶液所替换,整体三相放电节律周期时相有一定的变化。在一个时相(一个幽门网络的三相节律周期)中,三种神经元PY所占时相明显减小,相反LP、PD所占时相增加。经过统计学分析,TEA加入后LP神经元和静息期时相增加、PY神经元时相减小都有极显著意义(P<0.01)。更多还原

【Abstract】 CPG-neural networks composed of a series of neurons and synapses, has ability of spontaneously producing rhythmic, patterned neural outputs in the absence of sensory feedback or patterned central input-exist. The rhythmic motor patterns of CPG depend on the nature of the network and external conditions on network regulation. Invertebrate preparations (lobster and locust) have always played a important role in the studies because of easily maintaining activities in vitro of these preparations, clearly anatomically distributed nervous systems, large neurons which are easily identified from different animals of the same species, and fixed neural synaptic connections and the populations. In vitro rhythmic motor patterns remain unchanged, and extracellular recordings from nerves which is distinguished firing of the various types of neurons has the fundamental rhythmicity.The crustacean stomatogastric ganglion (STG) is a central pattern generator network that sits on top of the crustacean stomach and controls the movements of the pylorus and gastric mill. The STG of lobster contains about 30 neurons, and controls the movements of the lobster’s stomach. The stomatogastric ganglion(STG) contains two networks, one controlling the teeth of the gastric mill (the gastric CPG) and one controlling the pyloric filter apparatus (the pyloric CPG). The complete pyloric circuit consists of 14 neurons, one of which is an interneuron (AB) and the rest of which are motor neurons (PD, PY, LP, IC, VD). The roles and importance of cellular properties in the network and the synaptic connectivity are important in lives. Some of the inputs to the STG normally fire in bursts. However, their potentiation and acceleration of the output pattern are also produced by tonic stimulation of the nerve. The primary projection from the STG is the dorsal ventricular nerve (dvn). The dvn quickly splits into two medial ventricular nerve (mvn) and further down into lateral ventricular nerve (lvn). The lvn that consists of the LP, PY, and PD from the pyloric circuit is used to recording in vitro.Food has been macerated by the gastric millpasses into the pyloric region where it is filtered, kneaded further, and then passed on into the gut. Recording from the pyloric muscles in intact show a three-phase activity pattern which reflects stomach activities. In the pyloric system the driver neurons (PD/AB) put strong inhibitory synapses on the remaining "follower" neurons so that during driver activity, followers are shut off. After driver bursts terminate spontaneously, followers are free to fire in a sequence which is determined partly by their synaptic connections and partly by their cellular properties. The synchronous recording in vivo shows the same phase between AB and PD neurons. The identical result was recorded between IC and LP neurons, VD and PY neurons.At present, the research in sensory neural system is more than in motor neural system. The purpose of this work is to reinforce the research of STG in crayfish. Three-phase rhythms were recorded in normal condition and in condition of TEA being as blocker of the Ca2+-dependent potassium currents (IK(ca)) in STG of crayfish, chosen as experimental model in this paper.The main results are as follows:The recording of three-phase rhythm in normal condition of STG neuronsThree-phase rhythm composed of the firings generated by PD, LP and PY pyloric neurons in sequence, recorded in the stomatogastric ganglion (STG) of spiny lobster in normal condition. The rhythm pattern forms low frequency and high amplitude of the LP neurons, low frequency and low amplitude of the PY neurons, high frequency and lower amplitude of the PD neurons discharge. LP neuron does not start to discharge until PD neurons being resting. The three-phase rhythm shows the specfic forms and sequence.The change of three-phase rhythm under the influence of TEAThe Ca2+-dependent potassium currents (IK(ca)) plays a part in STG during spontaneous firing of neurons. The firing rhythm pattern was changed in condition of TEA, that bursting pattern was altered from homogeneous to being resting period. The firing patterns of LP and PY neurons were changed under the influence of 30 mmol/L TEA, while the firing sequence corresponding to each neuron of the three-phase rhythm did not change. The period of three-phase rhythm and the firing duration of PY neuron were decreased significantly (P<0.01). The firing duration of LP neuron was increased significantly (P<0.05). The interspike intervals of LP and PD neurons were decreased significantly (P<0.05).The change of mean phases corresponding to the three-phase rhythm under the influence of TEAThe mean phases corresponding to the three-phase rhythm were changed in condition of 30 mmol/L TEA. The phase of PY neuron was decreased significantly (P<0.01), the phase of LP neuron and resting were increased significantly (P<0.01) in the three-phase rhythm under the influence of TEA.更多还原