内吗啡肽在导水管周围灰质发挥镇痛效应的机制
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摘要
导水管周围灰质(periaqueductal gray,PAG)位于中脑,是哺乳动物脑干内下行痛抑制系统(descending pain inhibitory system,DPIS)内的重要组成。PAG的镇痛作用主要是通过激活其腹外侧区/柱(ventrolateral column of the PAG,vlPAG)内的5-HT能神经元的活性而实现的。vlPAG内的5-HT能神经元兴奋后,可以通过直接或间接的方式抑制脊髓的伤害性感受神经元,从而产生镇痛效应。
在PAG内同时存在一个调控5-HT能神经元活性的局部环路。其基本构成如下:PAG内大量存在的γ-氨基丁酸(γ-aminobutyric acid,GABA)能中间神经元可以对5-HT能神经元产生持续性的抑制效应,而阿片类物质通过与表达在GABA能神经元上的μ型阿片受体(MOR)结合,能够抑制GABA能神经元的活性,从而使5-HT能神经元间接激活(脱抑制,disinhibition),最终产生镇痛效应。
内吗啡肽(endomorphin,EM)是新近发现的一种内源性阿片肽,也是MOR高选择性的内源性配体。其镇痛效应与吗啡相似,但其副作用却远小于吗啡,在临床镇痛治疗方面有广泛的应用前景。内吗啡肽可以分为内吗啡肽1( endomorphin 1, EM1)和内吗啡肽2(endomorphin 2, EM2)两种亚型。内吗啡肽的胞体在脊髓上位脑区主要分布于下丘脑和孤束核,而纤维则遍布全脑,特别是在PAG的不同亚区内都分布有比较密集的EM能纤维和终末,提示EM可能参与了PAG内功能的调控。但是迄今为止,关于EM参与PAG内镇痛效应机制的研究还未见报道。因此,本论文综合应用当代神经科学研究方法,对以下几个问题进行了探讨:
1. PAG内大量存在的EM能纤维和终末的来源部位在哪里,其来源部位和PAG的不同亚区之间是否存在一定的对应关系?
2. vlPAG是5-HT能DPIS的起源部位,而该部位的EM是否参与了对DPIS局部调控的环路?
3. EM参与DPIS调控的机制是什么?
主要结果:
1.PAG内EM阳性纤维和终末的来源
将荧光逆行追踪剂荧光金(Fluoro-Gold,FG)分别电泳入大鼠PAG的不同亚区,通过结合EM1或EM2的免疫荧光组织化学染色技术,我们观察到EM1/FG和EM2/FG双重标记的神经元主要位于下丘脑的不同核团/区域内,孤束核内未见到双标神经元分布。其中,下丘脑结节乳头体区内侧的背内侧核( dorsomedial hypothalamic nucleus , DMH )、DMH及腹内侧核( ventromedial hypothalamic nucleus)之间的中央内侧区(centromedial hypothalamic region,CMH)以及弓状核(arcuate nucleus of the hypothalamus,Arc)内包含了绝大部分双标神经元。它们的分布特点如下:
1)将FG注入vlPAG后,DMH内18.0%的EM1(15.7±6.0)和14.0%的EM2(8.2±2.6)阳性神经元,CMH内15%的EM1(30.7±5.9)和18.6%的EM2(17.2±4.7)阳性神经元以及Arc内10.5%的EM1(6.3±1.9)和12.1%的EM2(5.7±2.0)阳性神经元同时被FG逆行标记;
2)将FG注入PAG外侧区(lateral column of the PAG, lPAG)后,DMH内14.8%的EM1(12.2±4.3)和10.8%的EM2(6.7±2.7)阳性神经元,CMH内11.1%的EM1(23.7±7.9)和10.3%的EM2(9.0±3.2)阳性神经元以及Arc内9.2%的EM(15.2±2.6)和5.6%的EM(22.8±1.2)阳性神经元同时被FG逆行标记;
3)将FG注入PAG背外侧区(dorsolateral column of the PAG, dlPAG)后,DMH内7.0%的EM1(6.2±3.3)和6.2%的EM2(3.8±2.3)阳性神经元,CMH内5.3%的EM(111.6±3.6)和3.4%的EM(23.0±1.6)阳性神经元以及Arc内2.0%的EM(11.2±0.8)和2.4%的EM(21.0±0.7)阳性神经元同时被FG逆行标记;
4)将FG注入PAG背内侧区(dorsomedial column of the PAG,dmPAG)后,DMH内8.5%的EM1(7.8±2.9)和7.0%的EM2(4.6±1.8)阳性神经元,CMH内9.5%的EM(119.2±8.1)和5.7%的EM(24.8±2.4)阳性神经元以及Arc内3.5%的EM1(2.0±1.2)和3.9%的EM(21.8±1.8)阳性神经元同时被FG逆行标记。
综合以上结果,可以看出:①下丘脑内EM1/FG双标神经元的数量远多于EM2/FG双标神经元的数量(802 vs. 392);②在下丘脑的不同核团和区域中,CMH内的EM/FG双标神经元数目最多(480 EM1/FG;196 EM2/FG),随后是DMH(237 EM1/FG;131 EM2/FG)及Arc(85 EM1/FG;65 EM2/FG);③将FG注入vlPAG后,在下丘脑内的EM/FG双标神经元数目最多,说明下丘脑内的EM能神经元向vlPAG发出最多的投射,随后是lPAG及dmPAG,最少的是dlPAG。
以上结果说明:①PAG的不同亚区内的EM能纤维和终末主要来源于下丘脑而不是孤束核;②下丘脑的不同核团和区域与PAG内的不同亚区之间存在明确的对应关系;③来源于下丘脑的EM能纤维和终末可能参与了PAG的多种功能活动,特别是痛觉的调制。
2.对于EM参与vlPAG内痛觉调控环路的机制问题,我们分别从形态学和行为药理学角度进行了论证。
2.1 EM参与vlPAG内痛觉调控环路的电镜观察
通过使用电镜双重标记技术,我们观察了大鼠vlPAG内EM1或EM2阳性的轴突终末与谷氨酸脱羧酶( glutamate decarboxylase,GAD)、MOR以及5-HT阳性的神经元胞体及树突之间的突触联系。GAD是GABA合成中的限速酶,也是GABA能神经元的标志,其分布与GABA相一致,GAD阳性结构即可认为是GABA阳性结构。结果显示:
1) vlPAG内含有大量的EM1和EM2阳性的轴突以及轴突终末以及GAD、MOR以及5-HT阳性的胞体和树突存在;
2) EM1以及EM2阳性的轴突终末能够与MOR、GAD及5-HT阳性的胞体和树突形成突触。其中:
EM1及EM2阳性轴突终末与MOR阳性胞体和树突主要形成对称性/抑制性突触(EM1:非对称/对称=37.8/62.2×100%;EM2:非对称/对称=41.4/58.6×100%)。
EM1及EM2阳性轴突终末与GAD阳性胞体和树突也主要形成对称性突触(EM1:非对称/对称=17.5/82.5×100%;EM2:非对称/对称=13.6/86.4×100%)。
EM1及EM2阳性轴突终末与5-HT阳性胞体和树突则主要形成非对称性/兴奋性突触(EM1:非对称/对称=58.3/41.7×100%;EM2:非对称/对称=56.7/43.3×100%)。
3) EM2阳性轴突终末间也可形成以非对称性突触(92%)为主的突触联系。
以上结果从电镜水平证实了vlPAG内EM能够对GABA能神经元产生抑制效应,且对5-HT能神经元存在直接兴奋效应,而以上的效应可能是通过MOR介导。
2.2 EM参与vlPAG内痛觉调控环路的共聚焦显微镜观察
通过使用谷氨酸脱羧酶67-绿色荧光蛋白(GAD67-GFP)基因敲入小鼠,我们进一步观察了vlPAG内EM1及EM2阳性纤维和终末以及表达GFP的GAD阳性胞体与MOR或5-HT阳性胞体之间的共存情况。结果显示:
1) GFP阳性胞体与MOR存在广泛的共存关系,几乎所有的GFP阳性神经元(98%)都表达MOR。而EM1或EM2阳性纤维和终末能够与GFP/MOR双标神经元形成密切接触;
2)部分GFP阳性胞体发出纤维与5-HT能神经元形成密切接触,而EM1或EM2阳性纤维和终末又能够与该GFP阳性胞体发生密切接触。
以上结果从形态学光镜水平为vlPAG内EM抑制GABA能神经元(通过MOR介导),从而间接兴奋5-HT能神经元的局部环路的存在提供了形态学依据。
2.3 EM参与vlPAG内痛觉调控环路的行为学观察
大鼠vlPAG区埋管制备动物模型,经管给予EM、GABAA受体的激动剂和拮抗剂以及MOR受体的拮抗剂,观察了大鼠给药前后对于热刺激反应潜伏期以及机械刺激反应阈值的变化,探讨EM在vlPAG内作用的机制。结果如下:
1)不同剂量的EM1(4、8、16、32 nmol/0.5μl)以及EM2(2、4、8、16 nmol/0.5μl)可以引发实验动物产生明显的镇痛效应。而该效应可以被MOR拮抗剂完全翻转;
2) GABAA受体的拮抗剂与EM能够产生强力的协同镇痛效应,而GABAA受体的激动剂则可以完全抑制EM的镇痛效应。
以上结果从行为学角度说明了EM的作用通过抑制GABA能神经元活性实现,而该效应通过MOR介导。
总之,上述形态学和行为学实验说明vlPAG内EM可以产生明确的镇痛效应,而该效应主要是通过抑制表达MOR的GABA能神经元的活性,进而使表达GABAA受体的5-HT能神经元摆脱GABA抑制而间接实现的。
The periaqueductal gray (PAG), which is located in the midbrain, is an important structure in consisting of the descending pain inhibitory system (DPIS) in mammalian animals. The function of the PAG is mainly carried out by exciting the 5-HTergic neurons in the ventrolateral column/part of the PAG (vlPAG). After excitation, the 5-HTergic neurons could inhibit directly or indirectly the nociceptive neurons in the spinal dorsal horn and exert antinociceptive effect.
There is a sprcial kind local circuit in regulating the activity of the 5-HTergic neurons in the PAG. That is, the widely distributedγ-aminobutyric acid (GABA) like immunoreactivity (LI) neurons can cause a tonic inhibitory effect on the 5-HTergic neurons. The opioid like substances (OLS) can inhibit the activities of the GABA-LI neurons via theμ-opioid receptor (MOR), and finally, to excite indirectly (disinhibit) the activities of the 5-HT-LI neurons.
Endomorphin (EM) is a new found member of the endogenous opioid peptide family and also the endogenous ligand for the MOR. With the similar effect in pain inhibition as morphine, EM has much less side effect, indicating their significant clinical potential. EM is consisted of two subtypes—endomorphin 1 (EM1) and endomorphin 2 (EM2). In the brain, EMergic neuronal cell bodies are located mainly in the hypothalamus and the nucleus tractus solitary (NTS), while the EMergic fibers being widely distributed across many structures. There are moderate to dense density of EM1- and EM2-LI fibers and terminals found in different parts the PAG, suggesting EM should be involved in many functions of the PAG. Since the functional role of EM in the PAG has not been reported yet, we studied the possible regulatory mechanisms of EM on the nociceptive transmission in the PAG by modern neuroscience methods. The main problems we met prior to the experiment were as follows:
1. Where are the possible origins of the EMergic fibers and terminals in the PAG? Is there any topographical correspondence of the origin areas and the different parts of the PAG?
2. Whether EM is involved in the modulation of local pain regulatory circuit in the vlPAG, which is the originating area of the 5-HTegic DPIS?
3. What is the possible modulation mechanism of EM on the nociceptive transmission in the vlPAG?
Methods and results:
1. The origins of the EM-LI fibers and terminals in the PAG
By combining the injection of the retrograde tracer, Fluoro-Gold (FG), into the different columns of the PAG with the EM1 or EM2 immunofluorescent staining, it’s found that the EM1/FG and EM2/FG double-labeled neurons were mainly distributed in different nuclei and areas of the hypothalamus, especially in the dorsomedial hypothalamic nucleus (DMH), areas between the ventromedial hypothalamic nucleus and DMH (centromedial hypothalamic region, CMH) and the arcuate nucleus (Arc). The distribution characteristics are as follows:
1) For FG injection into the vlPAG, 18% EM1-LI (15.7±6.0) and 14.0% EM2-LI (8.2±2.6) neurons in the DMH, 15% EM1-LI (30.7±5.9) and 18.6% EM2-LI (17.2±4.7) in the CMH and 10.5% EM1-LI (6.3±1.9) and 12.1% EM2-LI (5.7±2.0) in the Arc were labeled with FG;
2) For FG injection into the vlPAG, 14.8% EM1-LI (12.2±4.3) and 10.8% EM2-LI (6.7±2.7) neurons in the DMH, 11.1%的EM1-LI (23.7±7.9) and 10.3% EM2-LI (9.0±3.2) in the CMH and 9.2% EM1-LI (5.2±2.6) and 5.6% EM2-LI (2.8±1.2) in the Arc were labeled with FG;
3) For FG injection into the vlPAG, 7.0% EM1-LI (6.2±3.3) and 6.2% EM2-LI (3.8±2.3) neurons in the DMH, 5.3% EM1-LI (11.6±3.6) and 3.4% EM2-LI (3.0±1.6) neurons in the CMH and 2.0% EM1-LI (1.2±0.8) and 2.4% EM2-LI (1.0±0.7) neurons in the Arc were labeled with FG;
4) For FG injection into the vlPAG, 8.5% EM1-LI (7.8±2.9) and 7.0% EM2-LI (4.6±1.8) neurons in the DMH, 9.5% EM1-LI (19.2±8.1) and 5.7% EM2-LI (4.8±2.4) in the CMH and 3.5% EM1-LI (2.0±1.2) and 3.9% EM2-LI (1.8±1.8) in the Arc were labeled with FG;
Summery:①The number of EM1/FG double-labeled neurons is much larger than that of the EM2/FG double-labeled neurons in the hypothalamus (802 vs. 392);②The number of EM/FG double-labeled neurons in the CMH (480 EM1/FG; 196 EM2/FG) is larger that those in the DMH (237 EM1/FG; 131 EM2/FG) and Arc (85 EM1/FG; 65 EM2/FG);③The greatest number of EM/FG double-labeled neurons appears for FG injection into the vlPAG, indicating that EMergic neurons in the hypothalamus have the most close connection with the vlPAG, followed with the lPAG, dmPAG and dlPAG.
The present results indicate that:①EM-LI fiber and terminals in the different parts of the PAG mainly come from the hypothalamus but not from the NTS;②There exists an obvious topographical correspondence between the different nuclei/areas in hypothalamus and different parts of the PAG;③The hypothalamus-PAG projecting EMergic fibers and terminals may be involved in the modulation of the PAG’s function.
2. By using morphological and behavioral research works, we studied the mechanism of EM in the modulation of pain regulatory circuit in the vlPAG.
2.1 The regulatory circuit constituted by EM-LI structures which might be involved in the vlPAG
Under the electron microscope, the synaptic connections between the EM1- or EM2-LI terminals and the glutamate decarboxylase (GAD), MOR or 5-HT-LI cell bodies and dendrites were observed in the rat vlPAG. The results were as follows:
1) There existed many EM1- and EM2-LI axons/terminals and GAD-, MOR- and 5-HT-LI cell bodies/dendrites in the vlPAG;
2) EM1- and EM2-LI terminals could make synaptic connections with the MOR-, GAD- or 5-HT-LI cell bodies and dendrites. That is:
EM1- and EM2-LI terminals mainly made symmetric/inhibitory synaptic connections with the MOR-LI cell bodies and dendrites (EM1: asymmetric/symmetric=37.8/62.2×100%; EM2: asymmetric/symmetric=41.4/58.6×100%).
EM1- and EM2-LI terminals mainly made symmetric synaptic connections with the GAD-LI cell bodies and dendrites (EM1: asymmetric/symmetric=27.5/72.5×100%; EM2: asymmetric/symmetric =23.6/76.4×100%).
EM1- and EM2-LI terminals mainly made asymmetric synaptic connections with the 5-HT-LI cell bodies and dendrites (EM1: asymmetric/symmetric=58.3/41.7×100%; EM2: asymmetric/symmetric=56.7/43.3×100%).
3) EM2-LI terminals also made synapses with EM2-LI axons and terminals, most of which (92%) were asymmetric.
The present results suggest that EM can inhibit the activities of GABA-LI neurons and excite the 5-HT-LI neurons.
2.2 The confocal-microscopic observation of the regulatory circuit involved with EM in the vlPAG
By introducing the glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) gene knock-in mouse, we observed the distribution of EM1- or EM2-LI fibers and terminals, GFP-expressing GAD-LI neurons and MOR- or 5-HT-LI neurons. The results were as follows:
1) There were extensive co-existences between GFP- and MOR-LI neuronal cell bodies. Almost all of the GFP-LI cell bodies (98%) were labeled with MOR immunoreactivities. EM1- or EM2-LI fibers and terminals could make close contacts with the GFP/MOR double-labeled neurons.
2) Fibers from some GFP-LI neuronal cell bodies could make close contacts with the 5-HT-LI cell bodies, while the EM1- or EM2-LI fibers and terminals connected closely with these GFP-LI cell bodies.
The present results suggest that there should exist a special kind of regulatory circuit, in which EM might inhibit the GABA (via the MOR) and excite indirectly the 5-HTergic neurons.
2.3 The behavioral study of the regulatory circuit involved with EM in the vlPAG
After EM, antagonist of MOR and antagonist and agonist of the GABAA receptor were injected into the vlPAG via a guide cannula respectively, the rat’s paw withdrawal threshold for mechanical stimuli and latency for heat stimuli were measured. The possible mechanisms of EM in the pain regulatory circuit were discussed. The results were as follows:
1) Different doses of EM1 (4, 8, 16 and 32 nmol/0.5μl) and EM2 (2, 4, 8, 16 nmol/0.5μl) can cause obvious analgesia effect in animals, which can be blocked completely by the MOR antagonist;
2) These GABAA receptor antagonist can potentiate the analgesia effect of EM, while the GABAA receptor agonist reversed the EM caused analgesia effect.
The results indicate that the EM can cause analgesia effect by inhibiting the activity of the GABAergic neurons via the MOR.
In summary, the morphological and behavioral studies have demonstrated that the EM can induce obvious analgesia effect in the vlPAG, which is carried out by inhibiting the activity of the GABAergic neurons (via the MOR) and exciting indirectly the activity of the 5-HTergic neurons.
在PAG内同时存在一个调控5-HT能神经元活性的局部环路。其基本构成如下:PAG内大量存在的γ-氨基丁酸(γ-aminobutyric acid,GABA)能中间神经元可以对5-HT能神经元产生持续性的抑制效应,而阿片类物质通过与表达在GABA能神经元上的μ型阿片受体(MOR)结合,能够抑制GABA能神经元的活性,从而使5-HT能神经元间接激活(脱抑制,disinhibition),最终产生镇痛效应。
内吗啡肽(endomorphin,EM)是新近发现的一种内源性阿片肽,也是MOR高选择性的内源性配体。其镇痛效应与吗啡相似,但其副作用却远小于吗啡,在临床镇痛治疗方面有广泛的应用前景。内吗啡肽可以分为内吗啡肽1( endomorphin 1, EM1)和内吗啡肽2(endomorphin 2, EM2)两种亚型。内吗啡肽的胞体在脊髓上位脑区主要分布于下丘脑和孤束核,而纤维则遍布全脑,特别是在PAG的不同亚区内都分布有比较密集的EM能纤维和终末,提示EM可能参与了PAG内功能的调控。但是迄今为止,关于EM参与PAG内镇痛效应机制的研究还未见报道。因此,本论文综合应用当代神经科学研究方法,对以下几个问题进行了探讨:
1. PAG内大量存在的EM能纤维和终末的来源部位在哪里,其来源部位和PAG的不同亚区之间是否存在一定的对应关系?
2. vlPAG是5-HT能DPIS的起源部位,而该部位的EM是否参与了对DPIS局部调控的环路?
3. EM参与DPIS调控的机制是什么?
主要结果:
1.PAG内EM阳性纤维和终末的来源
将荧光逆行追踪剂荧光金(Fluoro-Gold,FG)分别电泳入大鼠PAG的不同亚区,通过结合EM1或EM2的免疫荧光组织化学染色技术,我们观察到EM1/FG和EM2/FG双重标记的神经元主要位于下丘脑的不同核团/区域内,孤束核内未见到双标神经元分布。其中,下丘脑结节乳头体区内侧的背内侧核( dorsomedial hypothalamic nucleus , DMH )、DMH及腹内侧核( ventromedial hypothalamic nucleus)之间的中央内侧区(centromedial hypothalamic region,CMH)以及弓状核(arcuate nucleus of the hypothalamus,Arc)内包含了绝大部分双标神经元。它们的分布特点如下:
1)将FG注入vlPAG后,DMH内18.0%的EM1(15.7±6.0)和14.0%的EM2(8.2±2.6)阳性神经元,CMH内15%的EM1(30.7±5.9)和18.6%的EM2(17.2±4.7)阳性神经元以及Arc内10.5%的EM1(6.3±1.9)和12.1%的EM2(5.7±2.0)阳性神经元同时被FG逆行标记;
2)将FG注入PAG外侧区(lateral column of the PAG, lPAG)后,DMH内14.8%的EM1(12.2±4.3)和10.8%的EM2(6.7±2.7)阳性神经元,CMH内11.1%的EM1(23.7±7.9)和10.3%的EM2(9.0±3.2)阳性神经元以及Arc内9.2%的EM(15.2±2.6)和5.6%的EM(22.8±1.2)阳性神经元同时被FG逆行标记;
3)将FG注入PAG背外侧区(dorsolateral column of the PAG, dlPAG)后,DMH内7.0%的EM1(6.2±3.3)和6.2%的EM2(3.8±2.3)阳性神经元,CMH内5.3%的EM(111.6±3.6)和3.4%的EM(23.0±1.6)阳性神经元以及Arc内2.0%的EM(11.2±0.8)和2.4%的EM(21.0±0.7)阳性神经元同时被FG逆行标记;
4)将FG注入PAG背内侧区(dorsomedial column of the PAG,dmPAG)后,DMH内8.5%的EM1(7.8±2.9)和7.0%的EM2(4.6±1.8)阳性神经元,CMH内9.5%的EM(119.2±8.1)和5.7%的EM(24.8±2.4)阳性神经元以及Arc内3.5%的EM1(2.0±1.2)和3.9%的EM(21.8±1.8)阳性神经元同时被FG逆行标记。
综合以上结果,可以看出:①下丘脑内EM1/FG双标神经元的数量远多于EM2/FG双标神经元的数量(802 vs. 392);②在下丘脑的不同核团和区域中,CMH内的EM/FG双标神经元数目最多(480 EM1/FG;196 EM2/FG),随后是DMH(237 EM1/FG;131 EM2/FG)及Arc(85 EM1/FG;65 EM2/FG);③将FG注入vlPAG后,在下丘脑内的EM/FG双标神经元数目最多,说明下丘脑内的EM能神经元向vlPAG发出最多的投射,随后是lPAG及dmPAG,最少的是dlPAG。
以上结果说明:①PAG的不同亚区内的EM能纤维和终末主要来源于下丘脑而不是孤束核;②下丘脑的不同核团和区域与PAG内的不同亚区之间存在明确的对应关系;③来源于下丘脑的EM能纤维和终末可能参与了PAG的多种功能活动,特别是痛觉的调制。
2.对于EM参与vlPAG内痛觉调控环路的机制问题,我们分别从形态学和行为药理学角度进行了论证。
2.1 EM参与vlPAG内痛觉调控环路的电镜观察
通过使用电镜双重标记技术,我们观察了大鼠vlPAG内EM1或EM2阳性的轴突终末与谷氨酸脱羧酶( glutamate decarboxylase,GAD)、MOR以及5-HT阳性的神经元胞体及树突之间的突触联系。GAD是GABA合成中的限速酶,也是GABA能神经元的标志,其分布与GABA相一致,GAD阳性结构即可认为是GABA阳性结构。结果显示:
1) vlPAG内含有大量的EM1和EM2阳性的轴突以及轴突终末以及GAD、MOR以及5-HT阳性的胞体和树突存在;
2) EM1以及EM2阳性的轴突终末能够与MOR、GAD及5-HT阳性的胞体和树突形成突触。其中:
EM1及EM2阳性轴突终末与MOR阳性胞体和树突主要形成对称性/抑制性突触(EM1:非对称/对称=37.8/62.2×100%;EM2:非对称/对称=41.4/58.6×100%)。
EM1及EM2阳性轴突终末与GAD阳性胞体和树突也主要形成对称性突触(EM1:非对称/对称=17.5/82.5×100%;EM2:非对称/对称=13.6/86.4×100%)。
EM1及EM2阳性轴突终末与5-HT阳性胞体和树突则主要形成非对称性/兴奋性突触(EM1:非对称/对称=58.3/41.7×100%;EM2:非对称/对称=56.7/43.3×100%)。
3) EM2阳性轴突终末间也可形成以非对称性突触(92%)为主的突触联系。
以上结果从电镜水平证实了vlPAG内EM能够对GABA能神经元产生抑制效应,且对5-HT能神经元存在直接兴奋效应,而以上的效应可能是通过MOR介导。
2.2 EM参与vlPAG内痛觉调控环路的共聚焦显微镜观察
通过使用谷氨酸脱羧酶67-绿色荧光蛋白(GAD67-GFP)基因敲入小鼠,我们进一步观察了vlPAG内EM1及EM2阳性纤维和终末以及表达GFP的GAD阳性胞体与MOR或5-HT阳性胞体之间的共存情况。结果显示:
1) GFP阳性胞体与MOR存在广泛的共存关系,几乎所有的GFP阳性神经元(98%)都表达MOR。而EM1或EM2阳性纤维和终末能够与GFP/MOR双标神经元形成密切接触;
2)部分GFP阳性胞体发出纤维与5-HT能神经元形成密切接触,而EM1或EM2阳性纤维和终末又能够与该GFP阳性胞体发生密切接触。
以上结果从形态学光镜水平为vlPAG内EM抑制GABA能神经元(通过MOR介导),从而间接兴奋5-HT能神经元的局部环路的存在提供了形态学依据。
2.3 EM参与vlPAG内痛觉调控环路的行为学观察
大鼠vlPAG区埋管制备动物模型,经管给予EM、GABAA受体的激动剂和拮抗剂以及MOR受体的拮抗剂,观察了大鼠给药前后对于热刺激反应潜伏期以及机械刺激反应阈值的变化,探讨EM在vlPAG内作用的机制。结果如下:
1)不同剂量的EM1(4、8、16、32 nmol/0.5μl)以及EM2(2、4、8、16 nmol/0.5μl)可以引发实验动物产生明显的镇痛效应。而该效应可以被MOR拮抗剂完全翻转;
2) GABAA受体的拮抗剂与EM能够产生强力的协同镇痛效应,而GABAA受体的激动剂则可以完全抑制EM的镇痛效应。
以上结果从行为学角度说明了EM的作用通过抑制GABA能神经元活性实现,而该效应通过MOR介导。
总之,上述形态学和行为学实验说明vlPAG内EM可以产生明确的镇痛效应,而该效应主要是通过抑制表达MOR的GABA能神经元的活性,进而使表达GABAA受体的5-HT能神经元摆脱GABA抑制而间接实现的。
The periaqueductal gray (PAG), which is located in the midbrain, is an important structure in consisting of the descending pain inhibitory system (DPIS) in mammalian animals. The function of the PAG is mainly carried out by exciting the 5-HTergic neurons in the ventrolateral column/part of the PAG (vlPAG). After excitation, the 5-HTergic neurons could inhibit directly or indirectly the nociceptive neurons in the spinal dorsal horn and exert antinociceptive effect.
There is a sprcial kind local circuit in regulating the activity of the 5-HTergic neurons in the PAG. That is, the widely distributedγ-aminobutyric acid (GABA) like immunoreactivity (LI) neurons can cause a tonic inhibitory effect on the 5-HTergic neurons. The opioid like substances (OLS) can inhibit the activities of the GABA-LI neurons via theμ-opioid receptor (MOR), and finally, to excite indirectly (disinhibit) the activities of the 5-HT-LI neurons.
Endomorphin (EM) is a new found member of the endogenous opioid peptide family and also the endogenous ligand for the MOR. With the similar effect in pain inhibition as morphine, EM has much less side effect, indicating their significant clinical potential. EM is consisted of two subtypes—endomorphin 1 (EM1) and endomorphin 2 (EM2). In the brain, EMergic neuronal cell bodies are located mainly in the hypothalamus and the nucleus tractus solitary (NTS), while the EMergic fibers being widely distributed across many structures. There are moderate to dense density of EM1- and EM2-LI fibers and terminals found in different parts the PAG, suggesting EM should be involved in many functions of the PAG. Since the functional role of EM in the PAG has not been reported yet, we studied the possible regulatory mechanisms of EM on the nociceptive transmission in the PAG by modern neuroscience methods. The main problems we met prior to the experiment were as follows:
1. Where are the possible origins of the EMergic fibers and terminals in the PAG? Is there any topographical correspondence of the origin areas and the different parts of the PAG?
2. Whether EM is involved in the modulation of local pain regulatory circuit in the vlPAG, which is the originating area of the 5-HTegic DPIS?
3. What is the possible modulation mechanism of EM on the nociceptive transmission in the vlPAG?
Methods and results:
1. The origins of the EM-LI fibers and terminals in the PAG
By combining the injection of the retrograde tracer, Fluoro-Gold (FG), into the different columns of the PAG with the EM1 or EM2 immunofluorescent staining, it’s found that the EM1/FG and EM2/FG double-labeled neurons were mainly distributed in different nuclei and areas of the hypothalamus, especially in the dorsomedial hypothalamic nucleus (DMH), areas between the ventromedial hypothalamic nucleus and DMH (centromedial hypothalamic region, CMH) and the arcuate nucleus (Arc). The distribution characteristics are as follows:
1) For FG injection into the vlPAG, 18% EM1-LI (15.7±6.0) and 14.0% EM2-LI (8.2±2.6) neurons in the DMH, 15% EM1-LI (30.7±5.9) and 18.6% EM2-LI (17.2±4.7) in the CMH and 10.5% EM1-LI (6.3±1.9) and 12.1% EM2-LI (5.7±2.0) in the Arc were labeled with FG;
2) For FG injection into the vlPAG, 14.8% EM1-LI (12.2±4.3) and 10.8% EM2-LI (6.7±2.7) neurons in the DMH, 11.1%的EM1-LI (23.7±7.9) and 10.3% EM2-LI (9.0±3.2) in the CMH and 9.2% EM1-LI (5.2±2.6) and 5.6% EM2-LI (2.8±1.2) in the Arc were labeled with FG;
3) For FG injection into the vlPAG, 7.0% EM1-LI (6.2±3.3) and 6.2% EM2-LI (3.8±2.3) neurons in the DMH, 5.3% EM1-LI (11.6±3.6) and 3.4% EM2-LI (3.0±1.6) neurons in the CMH and 2.0% EM1-LI (1.2±0.8) and 2.4% EM2-LI (1.0±0.7) neurons in the Arc were labeled with FG;
4) For FG injection into the vlPAG, 8.5% EM1-LI (7.8±2.9) and 7.0% EM2-LI (4.6±1.8) neurons in the DMH, 9.5% EM1-LI (19.2±8.1) and 5.7% EM2-LI (4.8±2.4) in the CMH and 3.5% EM1-LI (2.0±1.2) and 3.9% EM2-LI (1.8±1.8) in the Arc were labeled with FG;
Summery:①The number of EM1/FG double-labeled neurons is much larger than that of the EM2/FG double-labeled neurons in the hypothalamus (802 vs. 392);②The number of EM/FG double-labeled neurons in the CMH (480 EM1/FG; 196 EM2/FG) is larger that those in the DMH (237 EM1/FG; 131 EM2/FG) and Arc (85 EM1/FG; 65 EM2/FG);③The greatest number of EM/FG double-labeled neurons appears for FG injection into the vlPAG, indicating that EMergic neurons in the hypothalamus have the most close connection with the vlPAG, followed with the lPAG, dmPAG and dlPAG.
The present results indicate that:①EM-LI fiber and terminals in the different parts of the PAG mainly come from the hypothalamus but not from the NTS;②There exists an obvious topographical correspondence between the different nuclei/areas in hypothalamus and different parts of the PAG;③The hypothalamus-PAG projecting EMergic fibers and terminals may be involved in the modulation of the PAG’s function.
2. By using morphological and behavioral research works, we studied the mechanism of EM in the modulation of pain regulatory circuit in the vlPAG.
2.1 The regulatory circuit constituted by EM-LI structures which might be involved in the vlPAG
Under the electron microscope, the synaptic connections between the EM1- or EM2-LI terminals and the glutamate decarboxylase (GAD), MOR or 5-HT-LI cell bodies and dendrites were observed in the rat vlPAG. The results were as follows:
1) There existed many EM1- and EM2-LI axons/terminals and GAD-, MOR- and 5-HT-LI cell bodies/dendrites in the vlPAG;
2) EM1- and EM2-LI terminals could make synaptic connections with the MOR-, GAD- or 5-HT-LI cell bodies and dendrites. That is:
EM1- and EM2-LI terminals mainly made symmetric/inhibitory synaptic connections with the MOR-LI cell bodies and dendrites (EM1: asymmetric/symmetric=37.8/62.2×100%; EM2: asymmetric/symmetric=41.4/58.6×100%).
EM1- and EM2-LI terminals mainly made symmetric synaptic connections with the GAD-LI cell bodies and dendrites (EM1: asymmetric/symmetric=27.5/72.5×100%; EM2: asymmetric/symmetric =23.6/76.4×100%).
EM1- and EM2-LI terminals mainly made asymmetric synaptic connections with the 5-HT-LI cell bodies and dendrites (EM1: asymmetric/symmetric=58.3/41.7×100%; EM2: asymmetric/symmetric=56.7/43.3×100%).
3) EM2-LI terminals also made synapses with EM2-LI axons and terminals, most of which (92%) were asymmetric.
The present results suggest that EM can inhibit the activities of GABA-LI neurons and excite the 5-HT-LI neurons.
2.2 The confocal-microscopic observation of the regulatory circuit involved with EM in the vlPAG
By introducing the glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) gene knock-in mouse, we observed the distribution of EM1- or EM2-LI fibers and terminals, GFP-expressing GAD-LI neurons and MOR- or 5-HT-LI neurons. The results were as follows:
1) There were extensive co-existences between GFP- and MOR-LI neuronal cell bodies. Almost all of the GFP-LI cell bodies (98%) were labeled with MOR immunoreactivities. EM1- or EM2-LI fibers and terminals could make close contacts with the GFP/MOR double-labeled neurons.
2) Fibers from some GFP-LI neuronal cell bodies could make close contacts with the 5-HT-LI cell bodies, while the EM1- or EM2-LI fibers and terminals connected closely with these GFP-LI cell bodies.
The present results suggest that there should exist a special kind of regulatory circuit, in which EM might inhibit the GABA (via the MOR) and excite indirectly the 5-HTergic neurons.
2.3 The behavioral study of the regulatory circuit involved with EM in the vlPAG
After EM, antagonist of MOR and antagonist and agonist of the GABAA receptor were injected into the vlPAG via a guide cannula respectively, the rat’s paw withdrawal threshold for mechanical stimuli and latency for heat stimuli were measured. The possible mechanisms of EM in the pain regulatory circuit were discussed. The results were as follows:
1) Different doses of EM1 (4, 8, 16 and 32 nmol/0.5μl) and EM2 (2, 4, 8, 16 nmol/0.5μl) can cause obvious analgesia effect in animals, which can be blocked completely by the MOR antagonist;
2) These GABAA receptor antagonist can potentiate the analgesia effect of EM, while the GABAA receptor agonist reversed the EM caused analgesia effect.
The results indicate that the EM can cause analgesia effect by inhibiting the activity of the GABAergic neurons via the MOR.
In summary, the morphological and behavioral studies have demonstrated that the EM can induce obvious analgesia effect in the vlPAG, which is carried out by inhibiting the activity of the GABAergic neurons (via the MOR) and exciting indirectly the activity of the 5-HTergic neurons.
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