【作者】 夏建霞；

【导师】 胡志安；

【作者基本信息】 第三军医大学 ， 生理学， 2008， 博士

【摘要】 Oreixns,即hypocretins,是1998年分别由两个研究组同时发现的一种神经肽,orexin神经元主要位于穹隆周围的外侧下丘脑区。Orexin系统包括来自同一前体物质的二个单体orexin A (hypocretin 1)和orexin B (hypocretin 2),主要是通过活化两个G蛋白偶联受体orexin受体1和orexin受体2发挥作用。大量研究已经表明,orexins在促觉醒中发挥着关键性作用,这种促觉醒作用主要是通过兴奋多个皮层下觉醒系统及大脑皮层实现的。电生理研究显示,orexin神经元的活动受许多神经递质、神经调质和代谢信号的影响。研究证实,这些神经递质、调质及代谢信号对orexin神经元活动的调节在睡眠觉醒周期的调控中具有关键性作用。其中,神经调质――腺苷,由机体组织代谢活动产生的一种小分子物质,是目前研究最多、最重要的睡眠稳态调控因子,既可以直接作用于突触后腺苷受体、又可以改变突触前兴奋性或抑制性突触传递活动,影响神经元放电活动。文献报道,orexin神经元表达A1受体,但腺苷不能直接通过激活突触后A1受体改变神经元膜电位水平,而是通过结合突触前A1受体减少兴奋性谷氨酸能突触传入,调节orexin神经元的放电活动。除了谷氨酸能突触传递外,orexin神经元还接受大量的抑制性GABA能突触传递,这些抑制性纤维传入主要来自睡眠中心腹外侧视前区和外侧下丘脑区的局部中间神经元。然而,腺苷是否能调节orexin神经元上抑制性突触传递活动,是否存在内源性腺苷的释放调节orexin神经元上的突触传递活动,目前仍不清楚。此外,本室的前期钙成像实验显示,orexin A可以使少量培养的皮层神经元的胞内游离钙增加。同时,在电生理实验中,我们还发现orexin A能增加急性分离的前额叶皮层深层神经元的放电频率,但是,orexin A是否能影响前额叶皮层深层神经元的胞内钙水平及其作用机制,还需进一步的研究。本研究旨在通过膜片钳技术,深入研究腺苷对orexin神经元外侧下丘脑神经元上的抑制性突触传递活动的影响。首先,以orexin-EGFP转基因小鼠中的外侧下丘脑orexin神经元为研究对象,采用穿孔膜片钳技术,观察腺苷对诱发的抑制性突触后电流的影响及其受体和突触前机制。并进一步观察了不同刺激频率下内源性腺苷的释放。同时,在钙成像实验中,采用钙荧光指示剂fluo-4/AM负载急性分离的前额叶皮层深层神经元,结合药理学方法,观察orexin A对急性分离的前额叶皮层神经元的胞内钙影响及其作用机制。1.腺苷抑制诱发的orexin神经元上的抑制性突触后电流穿孔膜片钳记录显示,胞外灌流液中加入腺苷(100μM),能显著降低orexin神经元上诱发的抑制性突触后电流(evoked inhibitory postsynaptic currents, evIPSCs)幅度降至给药前的51.4241% (n=14,P<0.05)。这种抑制效应具有剂量依赖性,50μM、10μM、1μM腺苷加入后,幅度为给药前的56.8569% (n=7,P<0.05)、75.1114% (n=9,P<0.05)、93.0183% (n=6,P>0.05)。2.突触前A1受体介导腺苷对抑制性突触后电流的影响胞外液中预先加入腺苷A1受体拮抗剂CPT(200nM),evEPSCs幅度无明显变化(98.408% of control;n=9,P>0.05),加入腺苷50μM后,电流幅度未发生显著改变。同样,预先加入腺苷A2受体拮抗剂DMPX(10μM)后,并不影响evEPSCs幅度(99.351 % of control;P>0.05,n=8),但加入50μM腺苷后,电流幅度降低(59.139 % of control;P<0.05)。提示A1受体介导腺苷的抑制效应。分析配对脉冲比(paired-pulse ratio, PPR),加入腺苷50μM后,比值从1.095891增加到1.75679 (n=9,P<0.05)。实验进一步显示,腺苷50μM显著降低微小抑制性突触后电流(mini inhibitory postsynaptic currents, mIPSC)的频率,而不影响幅度。此外,腺苷并不能改变外源性给GABAA受体激动剂muscimol(50μM)产生的抑制性电流的幅度。提示腺苷主要是通过激活位于突触前的A1受体降低抑制性突触传递。3.突触活动依赖性释放的内源性腺苷抑制orexin神经元突触后兴奋性电流腺苷50μM显著降低orexin神经元上诱发的兴奋性突触后电流(evoked excitatory postsynaptic currents, evEPSCs)幅度。而在较低刺激频率下(0.033Hz,1Hz),CPT(200nM)加入前后,evEPSCs幅度无任何改变(n=9,P>0.05)。提高刺激频率,2Hz、3Hz、5Hz时,evEPSCs的幅度随着刺激时间(持续600s)而逐渐降低,加入CPT后,这种幅度的降低趋势无显著变化。10Hz,CPT在刺激后期能部分阻断电流幅度的降低(n=10,P<0.05)。而受体2拮抗剂DMPX(10μM),不能阻断电流幅度的降低(n=8,P>0.05)。100Hz刺激1s,也能部分拮抗刺激引起的幅度降低(n=11,P<0.05)。4.突触活动依赖性释放的内源性腺苷抑制orexin神经元突触后抑制性电流在较低刺激频率下(0.033Hz,1Hz),CPT加入前后,evIPSCs幅度无任何改变。提高刺激频率,2Hz、3Hz、5Hz时,evIPSCs的幅度随着刺激时间(持续600s)而逐渐降低,加入CPT后,这种幅度的降低趋势无显著变化。10Hz,CPT在刺激后期能部分阻断电流幅度的降低(n=9,P<0.05)。100Hz刺激1s,CPT并不能影响刺激引起的幅度降低(n=6,P>0.05)。5.腺苷A1受体拮抗剂增强orexin神经元长时程增强的诱导穿孔膜片钳下,100Hz高频刺激和theta频率刺激诱导orexin神经元上的兴奋性突触后电流(EPSCs)活动出现长时程增加(long term potentiation, LTP)的可塑性,分别增加为119.836% (n=12)和128% (n=12)。高频刺激诱导时加入腺苷A1受体拮抗剂CPT,诱导后洗脱,记录到的EPSCs增加为诱导前的165.9375%(n=12,P<0.05).。提示内源性腺苷可能参与了orexin神经元上突触可塑性的形成。此外,我们还发现,ATP代谢中间产物ADP加至胞外灌流液中,能显著降低evEPSCs的幅度大小。6.PKC、PLC介导的L型钙通道引起的钙内流参与orexin A的升钙效应钙成像研究显示orexin A可引起20%(n=8,8/40,P<0.05)急性分离的前额叶皮层神经元胞内钙增加,且呈剂量依赖性。orexin受体1拮抗剂SB 334867存在情况下,orexin A不能引起胞内钙增加。分别预孵PLC、PKC抑制剂D609和BIS II 1h后,未观察到orexin A的升钙效应。胞外无钙情况下,orexin A不能引起胞内钙增加。预孵钙库抑制剂thapsigargin1h后,orexin A仍能引起胞内钙增加。加入L型钙通道阻断剂nifedipine后,orexin A未能引起钙增加。结果说明,orexn A主要是通过orexin受体1,激活PLC、PKC信号,引起L型钙通道开放,引起胞内钙增加。综上所述,本研究结果表明,腺苷可以突触活动依赖的通过突触前A1受体,抑制orexin神经元上的兴奋性和抑制性突触后活动;内源性腺苷还参与了orexin神经元兴奋性突触传递活动可塑性的形成。Orexin A可以结合orexin受体1,通过PLC、PKC胞内信号,开放L型钙通道,引起前额叶皮层神经元胞内钙增加。更多还原

【Abstract】 Orexins localized specifically in neurons within the lateral hypothalamus and perifornical area are the novel neuropeptides discovered in 1998 by two groups respectively. Orexin system includes two separate peptides orexin A (hypocretin 1) and B (hypocretin 2) proteolytically derived from the same precursor protein. The actions of orexins are mainly transduced by orexin receptor 1 and orexin receptor 2 belonging to G-protein coupled receptors superfamily. Many studies have shown that these peptides are importantly implicated in the regulation and promotion of wakefulness. And this role may be mainly fulfilled by the excitatory actions of orexins on multiple subcortical arousal systems and cerebral cortex. As shown by recent electrophysiological studies, the activities of orexin neurons are influenced by several neurontransmitters and neuromodulators, as well as metabolic signals. It is implied that modulation of the activity of orexin neurons by these neurotransmitters or metabolic cues are critical for the regulation of sleep and wakefulness.Among these neuromodulators, adenosine, widely distributed in the mammalian central nervous system, has been proposed as an endogenous homeostatic sleep-promoting factor that accumulates during waking. A growing body of evidence has shown that adenosine may modulate excitatory glutamatergic synaptic transmission, inhibitory GABAergic synaptic transmission or both. An earlier study has demonstrated that adenosine exerts an inhibitory effect on glutamatergic transmission on orexin neurons, which is mediated by A1 receptors located on presynaptic terminals. It is well known that GABAergic synaptic transmission is the major inhibitory input to orexin neurons; however, whether adenosine modulates the inhibitory GABAergic transmission on orexin neurons as well has not been investigated. Specially, whether endogenous adenosine released in lateral hypothalamus is involved in the regulation of synaptic transmission on orexin neurons is still unclear.In addition, our previous study has shown that a few of cultured cortical cells tested show intracellular calcium elevation in response to orexin A, which depend on extracellular Ca2+ entry. Also, our electrophysiological results have illuminated that orexin A can excite the freshly dissociated neurons from layers V and VI of prefrontal cortex (PFC). It is intriguing to test whether neurons in PFC show the same intracellular calcium increase in response to orexin A.In the present study, we have focused on a possible role for adenosine in regulating inhibitory synaptic transmission on orexin neurons by performing perforated patch clamp recording in hypothalamic slices from orexin-EGFP transgenic mouse under voltage-clamp conditions. And we also examined the activity-dependent release of endogenous adenosine in regulating the activity of synaptic transmission as well. The effect and mechanisms underlying cytosolic Ca2+ changes induced by orexin A was investigated by using calcium imaging in acutely dissociated neurons from layers V and VI of PFC.1. Adenosine suppresses evoked inhibitory synaptic transmission on orexin neuronsPerforated path clamp recordings of orexin neurons in hypothalamic slices from orexin-EGFP transgenic mice showed that evoked inhibitory postsynaptic currents (evIPSCs) was 51.4241% of control after superfusion with adenosine (100μM; n=14, P<0.05) and 87.1222% of control after its withdrawal. Moreover, adenosine reduced the evIPSCs amplitude in a concentration-dependent manner. The amplitude of evIPSCs was reduced, on average, to 93.0183% (n=6, P>0.05), 75.1114% (n=9, P<0.05) and 56.8569% (n=7, P<0.05) of control for 1, 10, 50μM adenosine, respectively.2. Presynaptic A1 adenosine receptor is responsible for the effect of adenosine on GABAergic synaptic transmissionApplication of A1 receptor antagonist CPT (200 nM) completely blocked the inhibitory effects of adenosine on evIPSCs (98.408% of control; n=9, P>0.05). During the course of the experiments it was noted that bath application of CPT alone did not affect evIPSC amplitude compared to control condition. Application of DMPX (10μM) did not alter the amplitude or the duration of evIPSCs (99.351 % of control; n=8, P>0.05), nor did it affect the inhibitory action of adenosine on the evIPSCs of orexin neurons (59.139 % of control; P<0.05). These results suggest that adenosine inhibits evoked inhibitory transmission in orexin neurons through activation of A1 receptors.In the presence of 50μM adenosine, the average of paired-pulse ratio (PPR) was increasing from 1.095891 to 1.75679 (n=9, P<0.05). Application of adenosine did affect the cumulative inter-event interval distribution of the mIPSC, producing a rightward shift of the curve. The cumulative amplitude distribution of the mIPSC was not affected after application of adenosine. These observations indicate that adenosine inhibits evIPSCs in orexin neurons by a predominantly presynaptic mechanism (n=10, P<0.05).3. Activity-dependent release of adenosine inhibits excitatory synaptic transmission on orexin neuronsBath application of adenosine (100μM) induced a decline in the amplitude of evEPSCs to 56.413% of controls (n=11, P<0.05). Glutamatergic synaptic transmission after the application of CPT (200 nM) did not differ from the control condition when the stimulation rate was 0.033Hz and 1 Hz (n=7, P>0.05). The depression of evEPSCs elicited at 2Hz~5Hz was not abolished (P>0.05) by CPT (200nM), whereas when the frequency of stimuli was raised to 10 Hz, the depression of evEPSCs began to be partially blocked by CPT after 360s of stimuli (n=10, P<0.05). Application of DMPX (10μM), a A2 receptor antagonist, did not block the depression induced by 10 Hz stimulation (n=8, P>0.05). In the presence of CPT (200 nM), the depression of evEPSCs induced by 100Hz for 1s was also significantly partially inhibited during the course of stimulation (n=11, P<0.05).4. Activity-dependent release of adenosine inhibits inhibitory synaptic transmission on orexin neuronsNo change of the amplitude of evIPSCs was observed after application of CPT (200 nM) (n=9, P>0.05) in 1Hz stimulation. In the presence of CPT, the depression of evIPSCs elicited at 2Hz~5Hz was not abolished. When the frequency of stimuli reached to 10 Hz, the depression was then partially inhibited by CPT (200 nM) (n=9, P<0.05), while superfusion of DMPX (10μM) did not block the depression (n=6, P>0.05). When the frequency of stimulation increased up to 100Hz for 1s, the depression of evIPSCs was not inhibited by the CPT (n=6, P>0.05).5. A1 receptor antagonist enhances the induction of long term potentiation on excitatory synaptic transmission of orexin neuronsIn the perforated path clamp recordings, high frequency stimulation (HFS) or theta burst stimulation (TBS) can induce the long term potentiation (LTP) of excitatory synaptic transmission in orexin neurons, and the amplitude after induction was 119.836% (n=12) and 128% (n=12) of control, respectively. While in the presence of CPT during the induction by HFS, the amplitude of currents will increase up to 165.9375% after induction (n=12, P<0.05). The results indicate that endogenous adenosine may be involved in the induction of LTP in orexin neurons. 6. Extracellular calcium through L-type Ca2+ channels by activation of PLC-PKC pathway contribute to elevation of intracellular calcium of acutely dissociated neurons from PFCThe calcium imaging studies demonstrated that intracellular calcium in about of 20% (n=8, 8 of 40) of dissociated acutely PFC neurons was increased in a dose-dependent manner in response to orexin A (1μM). The increase in peak of calcium fluorescence intensity induced by orexin A was abolished in the presence of orexin receptor 1 antagonist SB 334867. Preincubation of PLC inhibitor D609 or PKC inhibitor BIS II, no elevation of intracellular calcium was observed in the presence of orexin A. Also, in extracellular calcium free condition, orexin A failed to increase the intracellular calcium, but the elevation was still observed when pretreatment of thapsigargin, an inhibitor of calcium store, was applied. Furthermore, the elevation induced by orexin A was not examined in the presence of L-type calcium channels inhibitor nifedipine. These findings illuminate that orexin A-induced increase of intracellular calcium in PFC neurons is mainly from extracelluar calcium influx through L-type calcium channels by activation of orexin receptor 1 and PLC-PKC signaling pathway.In conclusion, these observations provide evidence that the inhibitory effects of adenosine on inhibitory synaptic transmission in orexin neurons are mediated by presynaptic A1 receptors in a dose-dependent manner. Under strong and sustained synaptic transmission activities, endogenous adenosine will be released to extracellular space in hypothalamus, modulating the activity of synaptic transmission as well as the synaptic plasticity. Moreover, the increase in intracellular calcium induced by orexin A in freshly dissociated PFC neurons may be attributed to the extracellullar calcium influx through L-type calcium channels mediated by activation of orexin receptor 1 and PLC-PKC signaling pathway.更多还原