多巴胺D1受体经cAMP-PKA途径对延髓面神经后核内侧区吸气神经元/双相呼气神经元放电活动调控机制的研究
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摘要
基本节律性呼吸是维持哺乳动物生命活动的基本条件,基本呼吸节律产生的部位位于低位脑干的延髓。但迄今基本节律性呼吸发生及其调控机制仍未清楚;临床日常用药如运用吗啡类药物镇痛时会引起呼吸功能的异常;另一方面在临床工作中很多疾病如各种中枢性呼吸衰竭、中枢性呼吸节律紊乱等很常见,也是引起病人死亡的常见原因之一。因此阐明呼吸节律发生的确切部位揭示其发生与调控机制无疑是神经科学领域重要课题之一;研究呼吸节律阐明基本生命现象的本质不仅在理论上有着重要的意义,而且对于指导临床工作也具有重要的应用价值。
为更好地研究呼吸节律,前人建立了新生大鼠离体延髓-脊髓标本模型、延髓脑片标本、pre-B(o|¨)tC“岛”,它们是研究呼吸节律的产生和调节机制良好的体外标本。正是借助这些标本,国内外的学者对呼吸中枢的确切发生部位又做了大量的研究工作。目前主要有以下几种观点:1.延髓面神经后核内侧区(the medialarea of nucleus retrofacialis,mNRF)1986年我室吴中海等提出mNRF是节律性呼吸产生的部位。mNRF包括面神经后核内侧、网状小细胞核腹外侧、网状巨细胞核背外侧和外侧网状核内侧部分。2.pre-B(o|¨)tzinger复合体(pre-B(o|¨)tzingercomplex,pre-B(o|¨)tC)1991年Smith等人的实验表明,在延髓吻端的一个部位对呼吸节律的发生极为重要,这个部位称为pre-B(o|¨)tC;在该Pre-B(o|¨)tzinger复合体内存在着一类电压依赖性的、能够产生节律性发放活动的神经元,称之为起步神经元(Pace-maker neurons);它对于产生节律性的呼吸活动可能具有重要的意义。3.延髓头腹外侧区(the rostral ventrolateral medulla,RVLM)Onimaru等学者认为可能RVLM是产生基本呼吸节律的关键部位。4.面神经核周围区(thevicinities of nucleus facialis,VFN)VFN包括面神经后核、斜方体后核、pre-B(o|¨)tC。这四种定位的区域全部位于延髓头腹外侧区,相互之间不完全相同,但有重叠。对于呼吸节律产生机制的认识上目前主要存在两种学说,即起步细胞学说(pacemaker neuron hypothesis)、神经网络学说(neurons network hypothesis)起步细胞学说认为在延髓存在具有“起步”性质的呼吸神经元,它们表现有内在的节律性活动,这种活动影响和决定了其它呼吸相关神经元的活动。我室在整体及新生鼠离体延髓脑薄片标本的mNRF记录到具有“起步”特征的呼气-吸气跨时相神经元(Expiratory-Inspiratory phase spanning neuron,E-I PS),这类神经元放电开始于吸气前(呼气相的中、晚期),以频率递增的形式发放至吸气开始,然后以恒频的形式持续,最后与吸气性放电同步结束。这类放电总是先于吸气放电的神经元可能就是“起步神经元”(Pacemaker neuron)。神经网络学说是以Richter等为代表,该学说认为由呼吸节律发生器(RRG)和吸气形式发生器(IPG)组成呼吸神经元网络,呼吸节律的产生依赖于延髓内呼吸神经元之间复杂的相互联系和相互作用。
诸多的神经递质、调质控制呼吸网络的活动。多巴胺(dopamine,DA)是一种小分子神经递质,属于儿茶酚胺类。在纹状体、延髓、大脑皮层等部位有大量的多巴胺受体分布。多巴胺参与呼吸活动、药物成瘾、痛觉以及缺血再灌注损伤等的调节。依据细胞内信号转导过程的差异和氨基酸序列的差异,DA受体可以分为多巴胺D1、D2、D3、D4、D5五种受体,它们都是G-蛋白耦联受体。D1、D5受体通过Gs蛋白与腺苷酸环化酶(adenylate cyclase,AC)正偶联,使AC的活力增加,使细胞内的cAMP水平升高,进而磷酸化效应蛋白,产生各种生理病理效应;多巴胺D2、D3、D4与Gi蛋白与AC负相关,从而抑制AC的活力,降低cAMP水平,以及提高磷脂酰肌醇代谢等,产生一系列的生理学效应。
多巴胺作为一种神经递质,参与呼吸的调节。在体研究表明,静脉途径给予多巴胺D1受体激动剂,可以增强膈神经和吸气神经元的放电活动;多巴胺D1受体阻断剂能够抑制膈神经放电、增强呼气神经元的兴奋性;多巴胺D1受体激动剂可以阻逆阿片类物质所引起的呼吸抑制,而不影响阿片类物质的镇痛效果,这些在体的研究结果提示多巴胺D1受体可能与呼吸的调节有关。
大量的研究表明激活或抑制多巴胺及其D1受体会影响cAMP的水平。ChengL等人在负鼠肾细胞的研究表明激活的多巴胺D1受体可激活细胞内腺苷酸环化酶使cAMP出现剂量依赖性的增加,并且在磷酸二酯酶抑制剂3-异丁基-1-甲基黄嘌呤存在的条件下,还会引起cAMP的显著增加;且这种作用可被D1受体阻断剂阻断。利用逆转录聚合酶链反应技术Smith DO等发现小鸡胚胎脊髓运动神经元表达多巴胺D1受体,将神经元用100 microM多巴胺抚育后,神经元内的cAMP水平将会增加33%。对家兔动脉血中cAMP含量进行测试的结果表明,多巴胺D1受体激动剂非诺多巴可以引起。肾、肺脏、肠系膜、股动脉cAMP出现剂量依赖性增加,多巴胺D1受体特异性拮抗剂SCH-23390可以显著地阻断这种效应。
cAMP作为一种第二信使,在细胞信号转导中起着重要的作用。研究表明cAMP在中枢呼吸节律产生的调节中同样起着重要的作用:在离体延髓脑片上clonidine通过α_2-肾上腺素能受体降低细胞内cAMP的水平,从而抑制C4和前吸气神经元的放电活动;甲基黄嘌呤、Db-cAMP与forskolin有相似的作用,都可以增加前吸气神经元的发放频率;cAMP可以通过调节前吸气神经元的内在发放特性来调控呼吸节律的产生。激活AC、增加细胞内cAMP的水平或者是激活多巴胺D1受体可以逆转阿片和PGE1所引起呼吸抑制。2003年Shao在离体延髓脑片研究了AMPA受体对吸气神经元放电活动的调节机制后指出,提高细胞内cAMP水平或者用蛋白激酶A抑制剂Rp-cAMPS阻断呼吸细胞内cAMP的作用可影响呼吸细胞节律性放电活动,cAMP-PKA信号转导途径可调控pre-B(o|¨)tC区吸气神经元的兴奋性,且内源性PKA的水平是影响静息呼吸频率的一个重要因素。
在离体脑片上多巴胺D1受体对呼吸基本节律性发生和调节有什么样的作用,通过什么样的途径起作用的,这些问题还不是很清楚,为了解决这些问题设计了本课题。旨在利用包含舌下神经根和mNRF的离体延髓脑薄片:①探讨多巴胺D1受体对舌下神经根呼吸节律性放电活动的影响;②观察多巴胺D1对舌下神经根及吸气神经元/双相呼气神经元放电活动的调制作用;③研究cAMP变化对呼吸神经元基本放电活动的影响。④观察pre-B(o|¨)C“岛”多巴胺D1受体的表达情况;⑤探讨多巴胺D1受体调控基本呼吸节律的可能机制。
1、多巴胺D1受体对舌下神经根呼吸节律性放电活动(the respiratoryrhythmic discharge activity,RRDA)的影响
1.1不同浓度A68930组对RRDA影响
用不同浓度A68930灌流脑片后,呼吸周期(respiratory cycle,RC)和呼气时间(expiratory time,TE)随浓度的增加逐渐缩短、放电积分幅度(integral amplitude,IA)逐渐增大;不同浓度组间RRDA的RC比较总体上有差别(F=48.663,P＜0.001),TE总体上有差别(F=53.655,P＜0.001),IA总体上有差别(F=7.760,P=0.002)。其中5μmol/L组的作用效果最明显;5μmol/L组3 min三相指标RC、TE、IA与5 min时的三项相应指标比较没有统计学差异,P值分别为0.797、0.809、0.226,即3-5分钟可以取得药物的稳定作用,选择5μmol/L作为实验的合适浓度。
1.2不同浓度SCH-23390组对RRDA影响
给予浓度SCH-23390灌流脑片后,RC(F=70.561,P＜0.001)和TE(F=79.659,P=0.000)随浓度的增加逐渐延长、吸气时间(inspiratory time,TI)(F=21.800,P＜0.001)逐渐缩短、IA(F=6.617,P=0.004)逐渐减小;不同浓度组间RRDA的RC总体上有差别(F=70.561,P＜0.001)、TE总体上有差别(F=79.659,P=0.000)、TI总体上有差别(F=21.800,P＜0.001),IA总体上有差别(F=6.617,P=0.004);其中2μmol/L组的作用效果最明显;2μmol/L组3 min时的RC、TI、TE、IA与5min相应指标比较没有统计学差异,P分别为0.874、0.854、0.865、0.238,即3-5分钟可以取得药物的稳定作用,选择2μmol/L作为实验的合适浓度。
1.3 A68930组与A68930+SCH-23390组
给予5μmol/LA68930灌流脑片,RC、TE显著缩短(P＜0.001)、IA显著增大(P＜0.001);而且A68930的作用可以被2μmol/L SCH-23390部分逆转。
2、多巴胺D1受体对mNRF区吸气神经元/双相呼气神经元放电活动的调控
2.1多巴胺D1受体对吸气神经元的影响
使用A68930灌流脑片5min时与正常对照比较,吸气神经元的RC缩短20.90%(P=0.000)、TE缩短21.91%(P=0.000)、Fn增加14.84%(P=0.001)、IA增加14.27%(P=0.001),但是TI的变化不显著(P=0.825);在A68930灌流条件下,用A68930+SCH-23390灌流脑片,A68930对吸气神经元的作用可以被SCH-23390逆转。
2.2多巴胺D1受体对双相呼气神经元的作用
A68930灌流脑片5min时与正常对照比较,双向呼气神经元的RC和TE均显著缩短(P=0.000)、IA和PFn均显著增加(P=0.000),而TI的变化不显著(P=0.844);用A68930灌流脑片条件下使用A68930+SCH-23390灌流脑片,A68930对双相呼气神经元的作用被SCH-23390所逆转。
3、cAMP在多巴胺D1受体对面神经后核内侧区吸气神经元/双相呼气神经元放电活动调控中的作用
3.1 IBMX、forskolin与clonidine、Rp-cAMPS对面神经后核内侧区吸气神经元/双相呼气神经元放电活动调控中的作用
3.1.1 IBMX与IBMX+clonidine、forskolin与forskolin+Rp-cAMPS对吸气神经元放电活动的作用
结合参考文献及所做的Rp-cAMPS、clonidine对舌下神经根节律放电活动量效曲线,实验确定IBMX、clonidine、forskolin、Rp-cAMPS四种试剂的最适浓度分别为5、10、5、10μmol/L。
3.1.1.1 IBMX与IBMX+clonidine对吸气神经元放电活动的作用
使用IBMX灌流脑片5min时与正常对照比较,吸气神经元的RC和TE分别缩短18.85%(P=0.003)和20.18%(P=0.003)、TI延长8.82%(P=0.000),IA和Fn分别增加17.16%(P=0.001)和11.85%(P=0.001)。在IBMX灌流条件下,用IBMX+可乐定灌流脑片,IBMX对吸气神经元的作用可以被可乐定逆转,各观察指标基本都恢复至对照水平。
3.1.1.2 forskolin与forskolin+Rp-cAMPS对吸气神经元放电活动的作用
使用forskolin灌流脑片5min时与正常对照比较,吸气神经元的RC缩短18.81%(P=0.004)、TE缩短20.18%(P=0.003)、TI延长8.34%(P=0.000),IA增加12.36%(P=0.001)、Fn增加18.96%(P=0.001);在forskolin灌流条件下,用forskolin+Rp-cAMPS灌流脑片,forskolin对吸气神经元的作用可以被Rp-cAMPS逆转,各观察指标基本都恢复至对照水平。
3.1.2 IBMX、clonidine、forskolin、Rp-cAMPS对双相呼气神经元放电活动的作用
3.1.2.1 IBMX与clonidine对双相呼气神经元放电活动的作用
IBMX灌流脑片5min时与正常对照相比较,双向呼气神经元的RC和TE均显著缩短(P=0.000),IA和PFn均增加(P=0.000),TI显著延长(P=0.003)。用IBMX灌流脑片条件下使用IBMX+clonidine灌流脑片,IBMX对双相呼气神经元的作用可以被可乐定逆转,各观察指标基本都恢复至对照水平。
3.1.2.2 forskolin、Rp-cAMPS对双相呼气神经元放电活动的作用
用forskolin灌流脑片5min时与正常对照相比较,双向呼气神经元的RC和TE均显著缩短(P=0.000),分别缩短31.94%、23.53%;IA和PFn显著增加(P=0.000),分别增加19.55%、30.60%;TI延长10.17%(P=0.012)。用forskolin灌流脑片条件下使用forskolin+Rp-cAMPS灌流脑片,与正常对照相比较,双相呼气神经元的RC、TI、TE、IA和PFn恢复至对照水平,P分别为0.746、0.683、0.679、0.815、0.827。
3.2 Clonidine与Rp-cAMPS在A68930调控吸气/双相呼气神经元放电活动中的作用
3.2.1吸气神经元组
3.2.1.1 clonidine与clonidine+A68930对mNRF区吸气神经元放电活动的作用
使用可乐定灌流脑片5min时与正常对照相比,RC和TE分别延长21.68%(P=0.001)和23.45%(P=0.000)、TI缩短12.86%(P=0.000),同时IA和Fn分别减小11.08%(P=0.000)和12.35%(P=0.002);用可乐定持续灌流脑片条件下,用可乐定+A68930灌流脑片,A68930对吸气神经元的兴奋作用可被可乐定阻断,与单独用可乐定灌流脑片相比较,观察指标RC、TE、TI、IA、Fn变化不显著,P值分别为0.651、0.654、0.639、0.404、0.610。
3.2.1.2 Rp-cAMPS与Rp-cAMPS+A68930对吸气神经元放电活动的作用
使用Rp-cAMPS灌流脑片5min时与正常对照比较,RC和TE分别延长22.04%(P=0.000)和23.81%(P=0.000)、TI缩短14.29%(P=0.000),同时IA和Fn分别减小13.86%(P=0.000)和15.85%(P=0.000);用Rp-cAMPS持续灌流脑片条件下,用Rp-cAMPS+A68930灌流脑片,A68930对吸气神经元的兴奋作用可被Rp-cAMPS阻断,与单独用Rp-cAMPS灌流脑片相比较,观察指标RC、TE、TI、IA、Fn变化不显著,P分别为0.716、0.688、0.704、0.605、0.579。
3.2.2双相呼气神经元组
3.2.2.1 clonidine与clonidine+A68930对双相呼气神经元放电活动的作用
使用可乐定灌流脑片5min后与正常对照相比,RC和TE分别延长25.93%和31.99%(P=0.000)、TI缩短14.57%(P=0.005),同时IA和Fn分别减小10.45%(P=0.001)和11.37%(P=0.000);用可乐定持续灌流脑片条件下,使用可乐定+A68930灌流脑片,A68930对吸气神经元的兴奋作用可以被可乐定阻断,与单独使用可乐定灌流脑片相比较,观察指标RC、TE、TI、IA、PFn变化不显著,P值分别为0.389、0.945、0.337、0.774、0.668。
3.2.2.2 Rp-cAMPS与Rp-cAMPS+A68930对双相呼气神经元放电活动的作用
使用Rp-cAMPS灌流脑片5min后与正常对照相比,RC和TE分别延长10.78%(P=0.001)和13.41%(P=0.000)、TI缩短8.33%(P=0.035),同时IA和PFn分别减小15.31%(P=0.000)和17.41%(P=0.000);用Rp-cAMPS持续灌流脑片条件下,使用Rp-cAMPS+A68930灌流脑片,A68930对双相呼气神经元的兴奋作用可以被Rp-cAMPS阻断,与单独使用Rp-cAMPS灌流脑片相比较,观察指标RC、TE、TI、IA、PFn变化不显著,P分别为0.942、0.759、0.881、0.688、0.751。
4、延髓面神经后核内侧区神经细胞cAMP浓度的测定
采用酶联免疫吸附实验测定延髓面神经后核内侧区神经细胞cAMP浓度的测定,将mNRF“岛”在灌流槽中分别用MKS、5μmol/L A68930、2μmol/LSCH-23390灌流20分钟后用酶联免疫试剂盒测定cAMP浓度。结果显示:激动剂A68930组cAMP的浓度升高,与对照组比较有统计学差异(P=0.000);拮抗剂SCH-23390组cAMP的浓度降低;与对照组比较有统计学差异(P=0.000)。
5、多巴胺D1受体在延髓面神经后核内侧区“岛”的表达
采用荧光定量PCR测定检测多巴胺D1受体在延髓面神经后核内侧区“岛”是否表达及表达的量。结果表明:mNRF“岛”区多巴胺D1受体基因有表达;与对照组(大脑前额皮质多巴胺D1受体基因的表达量)比较有统计出差异(t=-24.226,P=0.000),延髓面神经后核内侧区“岛”多巴胺D1受体基因的表达量低于大脑前额皮质的表达量,为大脑前额皮质表达量的2~(-2.09)倍。
结论:①多巴胺D1受体对舌下神经根呼吸节律性放电活动有调节作用。②多巴胺D1调制mNRF区吸气神经元/双相呼气神经元放电活动;特异性受体激动剂增强吸气神经元/双相呼气神经元放电活动,特异性受体抑制剂抑制吸气神经元/双相呼气神经元放电活动。③多巴胺D1受体影响mNRF区神经细胞内cAMP的水平。④pre-B(o|¨)tC“岛”多巴胺D1受体有表达,相对于大脑前额皮层的表达较少。⑤forskolin与IBMX等cAMP激动剂可以兴奋吸气神经元/双相呼气神经元放电活动;Rp-cAMPS与clonidine等cAMP抑制剂可以抑制呼吸节律性放电活动。⑥Rp-cAMPS与clonidine能够阻断多巴胺D1受体对呼吸神经元放电的兴奋作用。⑦呼吸细胞内cAMP的水平调控呼吸节律性放电活动。⑧多巴胺D1受体是通过cAMP-PKA信号转导途径来调控基本呼吸节律的。
Neuronal networks in the medulla oblongata generate basic respiratory rhythm in mammals and it is essential for life to keep normal rhythmic respiration.However, little is known about the precise site for respiratory rhythm generation and the mechanisms underlying respiratory rhythmogenesis.It is common in clinical work for opioid-or prostaglandin-induced respiratory depression and many diseases such as central respiratory failure and central respiratory rhythm disturbance,which induce the patient to die.So elucidating the mechanisms underlying respiratory rhythmogenesis clearly is singificant in not only pathophysiology but also preventing and curing respiratory central diseases.
In order to study mechanisms underlying respiratory rhythmogenesis conveniently,scholars made the model of medulla oblongata,spinal cord brainstem slices and pre-B(o|¨)tC“island”of neonatal rats.The researchers in the neural science field had paid much attention to the precise site for respiratory rhythm generation and up to now there are some points as below about it:Firstly,professor Zhong-Hai Wu has demonstrated that the medial area of nucleus retrofacialis(mNRF)was the site of respiratory rhythmogenesis in 1986,it was vital to respiratory rhythmogenesis. Sencendly,Smith conformed that the pre-B(o|¨)t complex(PBC)was the site of respiratory rhythmogenesis in 1991.Thirdly,the experimental results of Onimaru showed the rostral ventrolateral medulla(RVLM)was the critical site of respiratory rhythm generation.But in other worker ideas the vicinities of nucleus facialis(VFN) is the important site.The four site statemented as above are all located in the medullary lateral region of abdomen.The anatomical position of them has difference, but it overlapped.The site of basic respiratory rhythmogenesis has been ascertained in the medullary head lateral region of abdomen.There are two hypothesis for the mechanism of respiratory rhythmogenesis,pacemaker neuron hypothesis and neurons network hypothesis.Pacemaker neuron hypothesis is supported by majority of the present findings.We had found Expiratory-Inspiratory phase spanning(E-I PS) neuron characteristic of pacemaker on mNRF in vivo and in vitro in neonate rats.The type of E-I PS neurons begin to discharge before inspiration,burst to initiation inspiration by constant frequency,continue and terminate simultaneously with discharge of inspiration.The type of E-I PS neurons may be pacemaker neurons.
There are many neurotransmitters and neuromodulators modulating the respiratory network.As one of the important neuromodulators of catecholamines in the central nervous system(CNS),dopamine(DA)is involved in many physiological and pathophysiological activities such as learning and memory,respiration and blood pressure regulation as well as cocaine craving.The dopamine membrane receptors include five subtypes:D1R-D5R.D1R and D5R activate adenylate cyclase(AC)via Gs protein and increasing the intracellular cAMP level to generate biological effect but D2R-D4R have the opposite effect and decreasing the intracellular cAMP level.
As one of the neuromodulators,DA may be involved in the rhythmic discharges of respiration.D1R agonist increased the discharge activities of phrenic nerve and inspiratory neurons by intravenous administration in vivo,meanwhile D1R antagonist had the opposite influence on them.D1R agonists might be therapeutically useful for the treatment of opioid disturbances of breathing without impeding analgesia.Such results show the receptor may be modulate the respiratory rhythm.
Activation and suppress D1R would influence the intracellular cAMP level.The stimulation of cAMP production in opossum kidney cells by D1R agonist was dose dependent,and markedly higher levels were observed in the presence of dopamine plus a phosphodiesterase inhibitor,3-isobutyl-1-methylxanthine.The effect of D1R agonist on the opossum kidney cells blocked by a specific D1R antagonist, Sch-23390.Smith found that in cultured embryonic chick motoneurons intracellular cAMP levels increased by 33%following exposure to 100 microM dopamine. Another findings suggested the presence of D1R mediating the cAMP generation system in renal,pulmonary and mesenteric arteries,but SCH-23390 reversed the effect of D1R agonist.
As the second messager,cAMP play one important role in cell signal transduction as well as in modulating respiratory rhythmogenesis.Clonidine,which might decrease intracellular cAMP level viaα_2-receptors,induced long-lasting depression depression of the respiratory rhythm(C4 and Pre-I activity)in brainstem-spinal cord preparation from newborn rat.The effects of IBMX and Db-cAMP were similar to those of forskolin,they all enhanced the burst rate of isolated Pre-I neurons.cAMP is important in respiratory rhythm generation;this may be due to its regulation of the intrinsic burst generating properties of Pre-I neurons. Opioid-and PGE-evoked respiratory depression was reversed upon elevation of endogenous cAMP levels by stimulating adenylyl cyclase with forskolin,activating dopamine D1R or preventing cAMP breakdown with isobutylmethylxanthine. Increasing the intracellular cAMP level or supressing PKA by a PKA inhibitor affect the respiratory rhythm discharge activities,the cAMP-PKA pathway modulates excitability of inspiratory neurons in the preBotC and,therefore,regulates respiratory rhythm.Moreover,the basal level of endogenous PKA activity appears to be a determinant of resting respiratory frequency.
D1R plays an role in regulating respiratory activity in mammals,however,little is known about how this receptor acts to modulate the basic respiratory rhythmgenesis in brainstem-spinal cord preparation from neonatal rat in vitro.Here, by simultaneously recording the activities of biphasic expiratory(BE) neurons/inspiratory(Ⅰ)neurons and theⅫnerve rootlets from brainstem slices in order to solve the questions as below:To explore how D1R affect the respiratory rhythmic discharge activity(RRDA)ofⅫnerve rootlets;to research the modulating of D1R on BE neurons/I neurons;to study the effect of cAMP on respiratory neurons; to survey how D1R expresses in mNRF”island”,the last but important purpose is to explore the underlying mechanisms of D1R regulating the basic respiratory rhythmgenesis.
1.The influences of D1R on RRDA ofⅫnerve rootlets
1.1 The effect of different concentration of A68930 on RRDA
Perfusing the slices with different concentration A68930,a D1R agonist,the respiratory cycle(RC)and the expiratory time(TE)shortened gradually,Concurrently, integral amplitude(IA)increased gradually.Among the different concentration group of A68930;RC(F=48.663,P＜0.001),TE(F=53.655,P＜0.001)and IA(F=7.760, P=0.002)are statistically significant,respectively.The changes of the three parameters in 5μmol/L group altered most obviously.But in 5μmol/L group the changes of RC,TE and IA at 3 min point were not statistically significant when they were compared with that at 5 min point,the value of P were 0.797、0.809、0.226, respectively.Steady drug response was observed 3～5 min after application.So the suitable concentration of A68930 in our experiments was 5μmol/L.
1.2 The effect of different concentration of SCH-23390 on RRDA Perfusing the slices with different concentration SCH-23390,a D1R antagonist, RC(F=70.561,P＜0.001)and TE(F=79.659,P=0.000)extended gradually, Concurrently,TI(F=21.800,P＜0.001)shortened gradually,IA(F=6.617,P=0.004) decreased gradually.Among the different concentration group of SCH-23390;RC (F=70.561,P＜0.001),TE(F=79.659,P=0.000),TI(F=21.800,P＜0.001)and IA (F=6.617,P=0.004)are statistically significant,respectively.The changes of the three parameters in 2μmol/L group are most obviously.But in 2μmol/L group the changes of RC,TI,TE and IA at 3 min point were not statistically significant when they were compared with that at 5 min point,the value of P were 0.874、0.854、0.865、0.238, respectively.Steady drug response was observed 3-5 min after application.So the suitable concentration of SCH-23390 in our experiments was 2μmol/L.
1.3 The effect of A68930 and A68930 + SCH-23390 on RRDA
After perfusing the slices with 5μmol/L A68930,compared to that of control group,RC and TE shortened markedly(P＜0.001),respectively.Concurrently,IA (F=6.617,P=0.004)increased noticeably.Moreover,the effect of A68930 on the respiratory rhythm was partially reversed by additional application of A68930+ SCH-23390.
2.The regulating effect of D1R on bisphsic expiratory(BE)neurons/inspiratory(I) neurons in brainstem-spinal cord preparation from newborn rat
2.1 D1R modulating the discharge activities of I neurons
After perfusing the slices with 5μmol/L A68930,when compared with control, RC and TE of I neurons shortened 20.90%(P=0.000)and 21.91%(P=0.000), respectively.Frequency(Fn)and IA increased 14.84%(P=0.001)and 14.27% (P=0.001),respectively.At the same time the change of TI was not statistically significant(P=0.825).These effects were reversed by subsequent application of D1R antagonist SCH-23390.
2.2 D1R affecting the discharge activities of BE neurons
Compard with that of control;RC and TE of BE neurons in A68930 group shortened 20.90%(P=0.000)and 21.91%(P=0.000),respectively;peak frequency(PFn) and IA increased 14.84%(P=0.001)and 14.27%(P=0.001),respectively;at the same time the change of TI was not statistically significant(P=0.844).These effects were reversed by subsequent application of D1R antagonist SCH-23390.
3.The role of cAMP in effect of D1R on BE neurons/I neurons in brainstem-spinal cord preparation from neonatal rats
3.1 cAMP increasing/decreasing agent acting the discharge activities of BE neurons/I neurons in mNRF
3.1.1 cAMP increasing/decreasing agent modulating the discharge activities of I neurons in mNRF
Firstly according to the results of previous of us and the literatures,we decided the suitable concentration of IBMX,clonidine,forskolin and Rp-cAMPS were 5,10, 5 and 10μmol/L,repectively.
3.1.1.1 IBMX and clonidine acting on the discharge activities of I neurons
After 5 min perfusing the slices with 5μmol/L IBMX,RC and TE of IBMX group compared with that of control;RC and TE of I neurons in IBMX group shortened 18.85%(P=0.003)and 20.18%(P=0.003),respectively;TI extended 8.82%(P=0.000);Fn and IA increased 17.16%(P=0.001)and 11.85%(P=0.001), respectively.Expectedly,these effects of IBMX were reversed by subsequent application of clonidine,a cAMP decreasing agent;the parameters above statement were returned to the level of control.
3.1.1.2 forskolin and Rp-cAMPS regulating the discharge activities of I neurons Compared with that of control,RC and TE of I neurons in forskolin group shortened 18.81%(P=0.004)and 20.18%(P=0.003),respectively;TI extended 8.34% (P=0.000);Fn and IA increased 18.96%(P=0.001)and 12.36%(P=0.001), respectively.Expectedly,these effects of forskolin on the discharge activities of I neurons were reversed by subsequent application of Rp-cAMPS,an inhibitor of PKA; the parameters above statement were recover to the level of control.
3.1.2.1 IBMX and clonidine acting on the discharge activities of BE neurons
After 5 min perfusing the slices with IBMX,RC and TE of IBMX group compared with that of control,RC and TE of I neurons in IBMX group all shortened markedly(P=0.000),TI extended significantly(P=0.003),PFn and IA all increased remarkably(P=0.000).Expectedly,these effects of IBMX were reversed by subsequent application of clonidine;the five parameters above mention were returned to the level of control.
3.1.2.2 forskolin and Rp-cAMPS modulating the discharge activities of BE neurons
Compared with that of control,RC and TE of BE neurons in forskolin group shortened 31.94%(P=0.000)and 23.53%(P=0.000),respectively;TI extended 10.17%(P=0.012);PFn and IA increased 19.55%(P=0.000)and 30.60%(P=0.000), respectively.Expectedly,these effects of forskolin on the discharge activities of BE neurons were reversed by subsequent application of Rp-cAMPS;the parameters above statement were recover to the level of control.
3.2 The role of clonidine and Rp-cAMPS in action of A68930 on BE neurons/I neurons in brainstem-spinal cord preparation from neonatal rats
3.2.1 The groups of inspiratory neurons
3.2.1.1 Clonidine and clonidine+A68930 regulating the discharge activities of I neurons
After 5 min perfusing the slices with clonidine,RC and TE of clonidine group compared with that of control;RC and TE of I neurons in clonidine group extended 21.68%(P=0.001)and 23.45%(P=0.000),respectively;TI shortened significantly (P=0.000);Fn and IA decreased 11.08%(P=0.000)and 12.35%(P=0.002), respectively.Notably,after pretreatment with clonidine,the excitatory effects of A68930 on I neurons were blocked;compared with the five parameters of clonidine group,the changes of RC,TI,TE,IA,Fn were not statistically significant and the values of P were 0.651,0.654,0.639,0.404 and 0.610,respectively.
3.2.1.2 Rp-cAMPS and Rp-cAMPS+A68930 modulating the discharge activities of I neurons
Compared with that of control;RC and TE of BE neurons in Rp-cAMPS group extended 22.04%(P=0.000)and 23.81%(P=0.000),respectively;TI shortened 14.29%(P=0.000);at the same time IA and Fn decreasing 13.86%(P=0.000)and 15.85%(P=0.000).Importantly,5 min after pretreatment with Rp-cAMPS,the excitatory effects of A68930 on I neurons were blocked.Compared with the five parameters of Rp-cAMPS group,the changes of RC,TI,TE,IA,Fn were not statistically significant and the values of P were 0.716,0.688,0.704,0.605 and 0.579, respectively.
3.2.2 The groups of BE neurons
3.2.2.1 Clonidine and clonidine+A68930 regulating the discharge activities of BE neurons
After 5 min perfusing the slices with clonidine,RC and TE of clonidine group compared with that of control;RC and TE of I neurons in clonidine group extended 25.93%(P=0.001)and 31.99%(P=0.000),respectively;TI shortened significantly (P=0.005);Fn and IA decreased 10.45%(P=0.001)and 11.37%(P=0.000), respectively.Notably,after pretreatment with clonidine,the excitatory effects of A68930 on I neurons were blocked;compared with the five parameters of clonidine group,the changes of RC,TI,TE,IA,PFn were not statistically significant and the values of P were 0.389,0.945,0.337,0.774 and 0.668,respectively.
3.2.2.2 Rp-cAMPS and Rp-cAMPS+A68930 modulating the discharge activities of BE neurons
Compared with that of control;RC and TE of BE neurons in Rp-cAMPS group extended 10.78%(P=0.001)and 13.41%(P=0.000),respectively;TI shortened 8.33% (P=0.035);meantime,IA and Fn decreasing 15.31%(P=0.000)and 17.41% (P=0.000),respectively.Importantly,5 min after pretreatment with Rp-cAMPS,the excitatory effects of A68930 on I neurons were blocked.Compared with the five parameters of Rp-cAMPS group,the changes of RC,TI,TE,IA,PFn were not statistically significant and the values of P were 0.942,0.759,0.881,0.688 and 0.751, respectively.
4.Determination of cAMP of neurons in mNRF“island”
Determination the concentration of cAMP of neurons in mNRF“island”by ELISA.After incubation the“island”with A68930,the concention of cAMP increased remakably when comparing with that of control.On the contrary,after incubation the“island”with SCH-23390,the concentration of cAMP decreased significantly when comparing with that of control.
5.The expression of mRNA of D1R gene in mNRF“island”
By real-time PCR we detected the expression of mRNA of D1R gene in mNRF“island”.The results suggested that mRNA of D1R gene expressed in mNRF“island”and the quantity of expression was 0.24 times of that of pre-frontal cortex,where the gene expressed plentifully according to the literatures reported.
Conclusion
1.D1R affected the respiratory rhythmic discharge activity(RRDA)of hypoglossal nerve roots.
2.D1R modulated the discharge activities of BE neurons/I neurons,the specific agonist of D1R excited the discharge activities of respiratory neurons and the specific antagonist of D1R had the opposite effect on the neurons.
3.Activating or inhibiting D1R influenced the intracellular level of cAMP of respiratory neurons.
4.mRNA of D1R gene expressed in mNRF“island”and the quantity of expression was about 0.24 times of that of pre-frontal cortex.
5.Forskolin and IBMX enhanced the discharge activities of BE neurons/I neurons,but clonidine and Rp-cAMPS had the opposite effect on the neurons.
6.Clonidine and Rp-cAMPS blocked the excitatory effects of A68930 on the respiratory neurons.
7.The intracellular level of cAMP acted to modulating the discharge activities of the neurons.
8.The current study indicated that D1R would modulate basic breathing rhythmgenesis via cAMP-dependent mechanisms.
为更好地研究呼吸节律,前人建立了新生大鼠离体延髓-脊髓标本模型、延髓脑片标本、pre-B(o|¨)tC“岛”,它们是研究呼吸节律的产生和调节机制良好的体外标本。正是借助这些标本,国内外的学者对呼吸中枢的确切发生部位又做了大量的研究工作。目前主要有以下几种观点:1.延髓面神经后核内侧区(the medialarea of nucleus retrofacialis,mNRF)1986年我室吴中海等提出mNRF是节律性呼吸产生的部位。mNRF包括面神经后核内侧、网状小细胞核腹外侧、网状巨细胞核背外侧和外侧网状核内侧部分。2.pre-B(o|¨)tzinger复合体(pre-B(o|¨)tzingercomplex,pre-B(o|¨)tC)1991年Smith等人的实验表明,在延髓吻端的一个部位对呼吸节律的发生极为重要,这个部位称为pre-B(o|¨)tC;在该Pre-B(o|¨)tzinger复合体内存在着一类电压依赖性的、能够产生节律性发放活动的神经元,称之为起步神经元(Pace-maker neurons);它对于产生节律性的呼吸活动可能具有重要的意义。3.延髓头腹外侧区(the rostral ventrolateral medulla,RVLM)Onimaru等学者认为可能RVLM是产生基本呼吸节律的关键部位。4.面神经核周围区(thevicinities of nucleus facialis,VFN)VFN包括面神经后核、斜方体后核、pre-B(o|¨)tC。这四种定位的区域全部位于延髓头腹外侧区,相互之间不完全相同,但有重叠。对于呼吸节律产生机制的认识上目前主要存在两种学说,即起步细胞学说(pacemaker neuron hypothesis)、神经网络学说(neurons network hypothesis)起步细胞学说认为在延髓存在具有“起步”性质的呼吸神经元,它们表现有内在的节律性活动,这种活动影响和决定了其它呼吸相关神经元的活动。我室在整体及新生鼠离体延髓脑薄片标本的mNRF记录到具有“起步”特征的呼气-吸气跨时相神经元(Expiratory-Inspiratory phase spanning neuron,E-I PS),这类神经元放电开始于吸气前(呼气相的中、晚期),以频率递增的形式发放至吸气开始,然后以恒频的形式持续,最后与吸气性放电同步结束。这类放电总是先于吸气放电的神经元可能就是“起步神经元”(Pacemaker neuron)。神经网络学说是以Richter等为代表,该学说认为由呼吸节律发生器(RRG)和吸气形式发生器(IPG)组成呼吸神经元网络,呼吸节律的产生依赖于延髓内呼吸神经元之间复杂的相互联系和相互作用。
诸多的神经递质、调质控制呼吸网络的活动。多巴胺(dopamine,DA)是一种小分子神经递质,属于儿茶酚胺类。在纹状体、延髓、大脑皮层等部位有大量的多巴胺受体分布。多巴胺参与呼吸活动、药物成瘾、痛觉以及缺血再灌注损伤等的调节。依据细胞内信号转导过程的差异和氨基酸序列的差异,DA受体可以分为多巴胺D1、D2、D3、D4、D5五种受体,它们都是G-蛋白耦联受体。D1、D5受体通过Gs蛋白与腺苷酸环化酶(adenylate cyclase,AC)正偶联,使AC的活力增加,使细胞内的cAMP水平升高,进而磷酸化效应蛋白,产生各种生理病理效应;多巴胺D2、D3、D4与Gi蛋白与AC负相关,从而抑制AC的活力,降低cAMP水平,以及提高磷脂酰肌醇代谢等,产生一系列的生理学效应。
多巴胺作为一种神经递质,参与呼吸的调节。在体研究表明,静脉途径给予多巴胺D1受体激动剂,可以增强膈神经和吸气神经元的放电活动;多巴胺D1受体阻断剂能够抑制膈神经放电、增强呼气神经元的兴奋性;多巴胺D1受体激动剂可以阻逆阿片类物质所引起的呼吸抑制,而不影响阿片类物质的镇痛效果,这些在体的研究结果提示多巴胺D1受体可能与呼吸的调节有关。
大量的研究表明激活或抑制多巴胺及其D1受体会影响cAMP的水平。ChengL等人在负鼠肾细胞的研究表明激活的多巴胺D1受体可激活细胞内腺苷酸环化酶使cAMP出现剂量依赖性的增加,并且在磷酸二酯酶抑制剂3-异丁基-1-甲基黄嘌呤存在的条件下,还会引起cAMP的显著增加;且这种作用可被D1受体阻断剂阻断。利用逆转录聚合酶链反应技术Smith DO等发现小鸡胚胎脊髓运动神经元表达多巴胺D1受体,将神经元用100 microM多巴胺抚育后,神经元内的cAMP水平将会增加33%。对家兔动脉血中cAMP含量进行测试的结果表明,多巴胺D1受体激动剂非诺多巴可以引起。肾、肺脏、肠系膜、股动脉cAMP出现剂量依赖性增加,多巴胺D1受体特异性拮抗剂SCH-23390可以显著地阻断这种效应。
cAMP作为一种第二信使,在细胞信号转导中起着重要的作用。研究表明cAMP在中枢呼吸节律产生的调节中同样起着重要的作用:在离体延髓脑片上clonidine通过α_2-肾上腺素能受体降低细胞内cAMP的水平,从而抑制C4和前吸气神经元的放电活动;甲基黄嘌呤、Db-cAMP与forskolin有相似的作用,都可以增加前吸气神经元的发放频率;cAMP可以通过调节前吸气神经元的内在发放特性来调控呼吸节律的产生。激活AC、增加细胞内cAMP的水平或者是激活多巴胺D1受体可以逆转阿片和PGE1所引起呼吸抑制。2003年Shao在离体延髓脑片研究了AMPA受体对吸气神经元放电活动的调节机制后指出,提高细胞内cAMP水平或者用蛋白激酶A抑制剂Rp-cAMPS阻断呼吸细胞内cAMP的作用可影响呼吸细胞节律性放电活动,cAMP-PKA信号转导途径可调控pre-B(o|¨)tC区吸气神经元的兴奋性,且内源性PKA的水平是影响静息呼吸频率的一个重要因素。
在离体脑片上多巴胺D1受体对呼吸基本节律性发生和调节有什么样的作用,通过什么样的途径起作用的,这些问题还不是很清楚,为了解决这些问题设计了本课题。旨在利用包含舌下神经根和mNRF的离体延髓脑薄片:①探讨多巴胺D1受体对舌下神经根呼吸节律性放电活动的影响;②观察多巴胺D1对舌下神经根及吸气神经元/双相呼气神经元放电活动的调制作用;③研究cAMP变化对呼吸神经元基本放电活动的影响。④观察pre-B(o|¨)C“岛”多巴胺D1受体的表达情况;⑤探讨多巴胺D1受体调控基本呼吸节律的可能机制。
1、多巴胺D1受体对舌下神经根呼吸节律性放电活动(the respiratoryrhythmic discharge activity,RRDA)的影响
1.1不同浓度A68930组对RRDA影响
用不同浓度A68930灌流脑片后,呼吸周期(respiratory cycle,RC)和呼气时间(expiratory time,TE)随浓度的增加逐渐缩短、放电积分幅度(integral amplitude,IA)逐渐增大;不同浓度组间RRDA的RC比较总体上有差别(F=48.663,P＜0.001),TE总体上有差别(F=53.655,P＜0.001),IA总体上有差别(F=7.760,P=0.002)。其中5μmol/L组的作用效果最明显;5μmol/L组3 min三相指标RC、TE、IA与5 min时的三项相应指标比较没有统计学差异,P值分别为0.797、0.809、0.226,即3-5分钟可以取得药物的稳定作用,选择5μmol/L作为实验的合适浓度。
1.2不同浓度SCH-23390组对RRDA影响
给予浓度SCH-23390灌流脑片后,RC(F=70.561,P＜0.001)和TE(F=79.659,P=0.000)随浓度的增加逐渐延长、吸气时间(inspiratory time,TI)(F=21.800,P＜0.001)逐渐缩短、IA(F=6.617,P=0.004)逐渐减小;不同浓度组间RRDA的RC总体上有差别(F=70.561,P＜0.001)、TE总体上有差别(F=79.659,P=0.000)、TI总体上有差别(F=21.800,P＜0.001),IA总体上有差别(F=6.617,P=0.004);其中2μmol/L组的作用效果最明显;2μmol/L组3 min时的RC、TI、TE、IA与5min相应指标比较没有统计学差异,P分别为0.874、0.854、0.865、0.238,即3-5分钟可以取得药物的稳定作用,选择2μmol/L作为实验的合适浓度。
1.3 A68930组与A68930+SCH-23390组
给予5μmol/LA68930灌流脑片,RC、TE显著缩短(P＜0.001)、IA显著增大(P＜0.001);而且A68930的作用可以被2μmol/L SCH-23390部分逆转。
2、多巴胺D1受体对mNRF区吸气神经元/双相呼气神经元放电活动的调控
2.1多巴胺D1受体对吸气神经元的影响
使用A68930灌流脑片5min时与正常对照比较,吸气神经元的RC缩短20.90%(P=0.000)、TE缩短21.91%(P=0.000)、Fn增加14.84%(P=0.001)、IA增加14.27%(P=0.001),但是TI的变化不显著(P=0.825);在A68930灌流条件下,用A68930+SCH-23390灌流脑片,A68930对吸气神经元的作用可以被SCH-23390逆转。
2.2多巴胺D1受体对双相呼气神经元的作用
A68930灌流脑片5min时与正常对照比较,双向呼气神经元的RC和TE均显著缩短(P=0.000)、IA和PFn均显著增加(P=0.000),而TI的变化不显著(P=0.844);用A68930灌流脑片条件下使用A68930+SCH-23390灌流脑片,A68930对双相呼气神经元的作用被SCH-23390所逆转。
3、cAMP在多巴胺D1受体对面神经后核内侧区吸气神经元/双相呼气神经元放电活动调控中的作用
3.1 IBMX、forskolin与clonidine、Rp-cAMPS对面神经后核内侧区吸气神经元/双相呼气神经元放电活动调控中的作用
3.1.1 IBMX与IBMX+clonidine、forskolin与forskolin+Rp-cAMPS对吸气神经元放电活动的作用
结合参考文献及所做的Rp-cAMPS、clonidine对舌下神经根节律放电活动量效曲线,实验确定IBMX、clonidine、forskolin、Rp-cAMPS四种试剂的最适浓度分别为5、10、5、10μmol/L。
3.1.1.1 IBMX与IBMX+clonidine对吸气神经元放电活动的作用
使用IBMX灌流脑片5min时与正常对照比较,吸气神经元的RC和TE分别缩短18.85%(P=0.003)和20.18%(P=0.003)、TI延长8.82%(P=0.000),IA和Fn分别增加17.16%(P=0.001)和11.85%(P=0.001)。在IBMX灌流条件下,用IBMX+可乐定灌流脑片,IBMX对吸气神经元的作用可以被可乐定逆转,各观察指标基本都恢复至对照水平。
3.1.1.2 forskolin与forskolin+Rp-cAMPS对吸气神经元放电活动的作用
使用forskolin灌流脑片5min时与正常对照比较,吸气神经元的RC缩短18.81%(P=0.004)、TE缩短20.18%(P=0.003)、TI延长8.34%(P=0.000),IA增加12.36%(P=0.001)、Fn增加18.96%(P=0.001);在forskolin灌流条件下,用forskolin+Rp-cAMPS灌流脑片,forskolin对吸气神经元的作用可以被Rp-cAMPS逆转,各观察指标基本都恢复至对照水平。
3.1.2 IBMX、clonidine、forskolin、Rp-cAMPS对双相呼气神经元放电活动的作用
3.1.2.1 IBMX与clonidine对双相呼气神经元放电活动的作用
IBMX灌流脑片5min时与正常对照相比较,双向呼气神经元的RC和TE均显著缩短(P=0.000),IA和PFn均增加(P=0.000),TI显著延长(P=0.003)。用IBMX灌流脑片条件下使用IBMX+clonidine灌流脑片,IBMX对双相呼气神经元的作用可以被可乐定逆转,各观察指标基本都恢复至对照水平。
3.1.2.2 forskolin、Rp-cAMPS对双相呼气神经元放电活动的作用
用forskolin灌流脑片5min时与正常对照相比较,双向呼气神经元的RC和TE均显著缩短(P=0.000),分别缩短31.94%、23.53%;IA和PFn显著增加(P=0.000),分别增加19.55%、30.60%;TI延长10.17%(P=0.012)。用forskolin灌流脑片条件下使用forskolin+Rp-cAMPS灌流脑片,与正常对照相比较,双相呼气神经元的RC、TI、TE、IA和PFn恢复至对照水平,P分别为0.746、0.683、0.679、0.815、0.827。
3.2 Clonidine与Rp-cAMPS在A68930调控吸气/双相呼气神经元放电活动中的作用
3.2.1吸气神经元组
3.2.1.1 clonidine与clonidine+A68930对mNRF区吸气神经元放电活动的作用
使用可乐定灌流脑片5min时与正常对照相比,RC和TE分别延长21.68%(P=0.001)和23.45%(P=0.000)、TI缩短12.86%(P=0.000),同时IA和Fn分别减小11.08%(P=0.000)和12.35%(P=0.002);用可乐定持续灌流脑片条件下,用可乐定+A68930灌流脑片,A68930对吸气神经元的兴奋作用可被可乐定阻断,与单独用可乐定灌流脑片相比较,观察指标RC、TE、TI、IA、Fn变化不显著,P值分别为0.651、0.654、0.639、0.404、0.610。
3.2.1.2 Rp-cAMPS与Rp-cAMPS+A68930对吸气神经元放电活动的作用
使用Rp-cAMPS灌流脑片5min时与正常对照比较,RC和TE分别延长22.04%(P=0.000)和23.81%(P=0.000)、TI缩短14.29%(P=0.000),同时IA和Fn分别减小13.86%(P=0.000)和15.85%(P=0.000);用Rp-cAMPS持续灌流脑片条件下,用Rp-cAMPS+A68930灌流脑片,A68930对吸气神经元的兴奋作用可被Rp-cAMPS阻断,与单独用Rp-cAMPS灌流脑片相比较,观察指标RC、TE、TI、IA、Fn变化不显著,P分别为0.716、0.688、0.704、0.605、0.579。
3.2.2双相呼气神经元组
3.2.2.1 clonidine与clonidine+A68930对双相呼气神经元放电活动的作用
使用可乐定灌流脑片5min后与正常对照相比,RC和TE分别延长25.93%和31.99%(P=0.000)、TI缩短14.57%(P=0.005),同时IA和Fn分别减小10.45%(P=0.001)和11.37%(P=0.000);用可乐定持续灌流脑片条件下,使用可乐定+A68930灌流脑片,A68930对吸气神经元的兴奋作用可以被可乐定阻断,与单独使用可乐定灌流脑片相比较,观察指标RC、TE、TI、IA、PFn变化不显著,P值分别为0.389、0.945、0.337、0.774、0.668。
3.2.2.2 Rp-cAMPS与Rp-cAMPS+A68930对双相呼气神经元放电活动的作用
使用Rp-cAMPS灌流脑片5min后与正常对照相比,RC和TE分别延长10.78%(P=0.001)和13.41%(P=0.000)、TI缩短8.33%(P=0.035),同时IA和PFn分别减小15.31%(P=0.000)和17.41%(P=0.000);用Rp-cAMPS持续灌流脑片条件下,使用Rp-cAMPS+A68930灌流脑片,A68930对双相呼气神经元的兴奋作用可以被Rp-cAMPS阻断,与单独使用Rp-cAMPS灌流脑片相比较,观察指标RC、TE、TI、IA、PFn变化不显著,P分别为0.942、0.759、0.881、0.688、0.751。
4、延髓面神经后核内侧区神经细胞cAMP浓度的测定
采用酶联免疫吸附实验测定延髓面神经后核内侧区神经细胞cAMP浓度的测定,将mNRF“岛”在灌流槽中分别用MKS、5μmol/L A68930、2μmol/LSCH-23390灌流20分钟后用酶联免疫试剂盒测定cAMP浓度。结果显示:激动剂A68930组cAMP的浓度升高,与对照组比较有统计学差异(P=0.000);拮抗剂SCH-23390组cAMP的浓度降低;与对照组比较有统计学差异(P=0.000)。
5、多巴胺D1受体在延髓面神经后核内侧区“岛”的表达
采用荧光定量PCR测定检测多巴胺D1受体在延髓面神经后核内侧区“岛”是否表达及表达的量。结果表明:mNRF“岛”区多巴胺D1受体基因有表达;与对照组(大脑前额皮质多巴胺D1受体基因的表达量)比较有统计出差异(t=-24.226,P=0.000),延髓面神经后核内侧区“岛”多巴胺D1受体基因的表达量低于大脑前额皮质的表达量,为大脑前额皮质表达量的2~(-2.09)倍。
结论:①多巴胺D1受体对舌下神经根呼吸节律性放电活动有调节作用。②多巴胺D1调制mNRF区吸气神经元/双相呼气神经元放电活动;特异性受体激动剂增强吸气神经元/双相呼气神经元放电活动,特异性受体抑制剂抑制吸气神经元/双相呼气神经元放电活动。③多巴胺D1受体影响mNRF区神经细胞内cAMP的水平。④pre-B(o|¨)tC“岛”多巴胺D1受体有表达,相对于大脑前额皮层的表达较少。⑤forskolin与IBMX等cAMP激动剂可以兴奋吸气神经元/双相呼气神经元放电活动;Rp-cAMPS与clonidine等cAMP抑制剂可以抑制呼吸节律性放电活动。⑥Rp-cAMPS与clonidine能够阻断多巴胺D1受体对呼吸神经元放电的兴奋作用。⑦呼吸细胞内cAMP的水平调控呼吸节律性放电活动。⑧多巴胺D1受体是通过cAMP-PKA信号转导途径来调控基本呼吸节律的。
Neuronal networks in the medulla oblongata generate basic respiratory rhythm in mammals and it is essential for life to keep normal rhythmic respiration.However, little is known about the precise site for respiratory rhythm generation and the mechanisms underlying respiratory rhythmogenesis.It is common in clinical work for opioid-or prostaglandin-induced respiratory depression and many diseases such as central respiratory failure and central respiratory rhythm disturbance,which induce the patient to die.So elucidating the mechanisms underlying respiratory rhythmogenesis clearly is singificant in not only pathophysiology but also preventing and curing respiratory central diseases.
In order to study mechanisms underlying respiratory rhythmogenesis conveniently,scholars made the model of medulla oblongata,spinal cord brainstem slices and pre-B(o|¨)tC“island”of neonatal rats.The researchers in the neural science field had paid much attention to the precise site for respiratory rhythm generation and up to now there are some points as below about it:Firstly,professor Zhong-Hai Wu has demonstrated that the medial area of nucleus retrofacialis(mNRF)was the site of respiratory rhythmogenesis in 1986,it was vital to respiratory rhythmogenesis. Sencendly,Smith conformed that the pre-B(o|¨)t complex(PBC)was the site of respiratory rhythmogenesis in 1991.Thirdly,the experimental results of Onimaru showed the rostral ventrolateral medulla(RVLM)was the critical site of respiratory rhythm generation.But in other worker ideas the vicinities of nucleus facialis(VFN) is the important site.The four site statemented as above are all located in the medullary lateral region of abdomen.The anatomical position of them has difference, but it overlapped.The site of basic respiratory rhythmogenesis has been ascertained in the medullary head lateral region of abdomen.There are two hypothesis for the mechanism of respiratory rhythmogenesis,pacemaker neuron hypothesis and neurons network hypothesis.Pacemaker neuron hypothesis is supported by majority of the present findings.We had found Expiratory-Inspiratory phase spanning(E-I PS) neuron characteristic of pacemaker on mNRF in vivo and in vitro in neonate rats.The type of E-I PS neurons begin to discharge before inspiration,burst to initiation inspiration by constant frequency,continue and terminate simultaneously with discharge of inspiration.The type of E-I PS neurons may be pacemaker neurons.
There are many neurotransmitters and neuromodulators modulating the respiratory network.As one of the important neuromodulators of catecholamines in the central nervous system(CNS),dopamine(DA)is involved in many physiological and pathophysiological activities such as learning and memory,respiration and blood pressure regulation as well as cocaine craving.The dopamine membrane receptors include five subtypes:D1R-D5R.D1R and D5R activate adenylate cyclase(AC)via Gs protein and increasing the intracellular cAMP level to generate biological effect but D2R-D4R have the opposite effect and decreasing the intracellular cAMP level.
As one of the neuromodulators,DA may be involved in the rhythmic discharges of respiration.D1R agonist increased the discharge activities of phrenic nerve and inspiratory neurons by intravenous administration in vivo,meanwhile D1R antagonist had the opposite influence on them.D1R agonists might be therapeutically useful for the treatment of opioid disturbances of breathing without impeding analgesia.Such results show the receptor may be modulate the respiratory rhythm.
Activation and suppress D1R would influence the intracellular cAMP level.The stimulation of cAMP production in opossum kidney cells by D1R agonist was dose dependent,and markedly higher levels were observed in the presence of dopamine plus a phosphodiesterase inhibitor,3-isobutyl-1-methylxanthine.The effect of D1R agonist on the opossum kidney cells blocked by a specific D1R antagonist, Sch-23390.Smith found that in cultured embryonic chick motoneurons intracellular cAMP levels increased by 33%following exposure to 100 microM dopamine. Another findings suggested the presence of D1R mediating the cAMP generation system in renal,pulmonary and mesenteric arteries,but SCH-23390 reversed the effect of D1R agonist.
As the second messager,cAMP play one important role in cell signal transduction as well as in modulating respiratory rhythmogenesis.Clonidine,which might decrease intracellular cAMP level viaα_2-receptors,induced long-lasting depression depression of the respiratory rhythm(C4 and Pre-I activity)in brainstem-spinal cord preparation from newborn rat.The effects of IBMX and Db-cAMP were similar to those of forskolin,they all enhanced the burst rate of isolated Pre-I neurons.cAMP is important in respiratory rhythm generation;this may be due to its regulation of the intrinsic burst generating properties of Pre-I neurons. Opioid-and PGE-evoked respiratory depression was reversed upon elevation of endogenous cAMP levels by stimulating adenylyl cyclase with forskolin,activating dopamine D1R or preventing cAMP breakdown with isobutylmethylxanthine. Increasing the intracellular cAMP level or supressing PKA by a PKA inhibitor affect the respiratory rhythm discharge activities,the cAMP-PKA pathway modulates excitability of inspiratory neurons in the preBotC and,therefore,regulates respiratory rhythm.Moreover,the basal level of endogenous PKA activity appears to be a determinant of resting respiratory frequency.
D1R plays an role in regulating respiratory activity in mammals,however,little is known about how this receptor acts to modulate the basic respiratory rhythmgenesis in brainstem-spinal cord preparation from neonatal rat in vitro.Here, by simultaneously recording the activities of biphasic expiratory(BE) neurons/inspiratory(Ⅰ)neurons and theⅫnerve rootlets from brainstem slices in order to solve the questions as below:To explore how D1R affect the respiratory rhythmic discharge activity(RRDA)ofⅫnerve rootlets;to research the modulating of D1R on BE neurons/I neurons;to study the effect of cAMP on respiratory neurons; to survey how D1R expresses in mNRF”island”,the last but important purpose is to explore the underlying mechanisms of D1R regulating the basic respiratory rhythmgenesis.
1.The influences of D1R on RRDA ofⅫnerve rootlets
1.1 The effect of different concentration of A68930 on RRDA
Perfusing the slices with different concentration A68930,a D1R agonist,the respiratory cycle(RC)and the expiratory time(TE)shortened gradually,Concurrently, integral amplitude(IA)increased gradually.Among the different concentration group of A68930;RC(F=48.663,P＜0.001),TE(F=53.655,P＜0.001)and IA(F=7.760, P=0.002)are statistically significant,respectively.The changes of the three parameters in 5μmol/L group altered most obviously.But in 5μmol/L group the changes of RC,TE and IA at 3 min point were not statistically significant when they were compared with that at 5 min point,the value of P were 0.797、0.809、0.226, respectively.Steady drug response was observed 3～5 min after application.So the suitable concentration of A68930 in our experiments was 5μmol/L.
1.2 The effect of different concentration of SCH-23390 on RRDA Perfusing the slices with different concentration SCH-23390,a D1R antagonist, RC(F=70.561,P＜0.001)and TE(F=79.659,P=0.000)extended gradually, Concurrently,TI(F=21.800,P＜0.001)shortened gradually,IA(F=6.617,P=0.004) decreased gradually.Among the different concentration group of SCH-23390;RC (F=70.561,P＜0.001),TE(F=79.659,P=0.000),TI(F=21.800,P＜0.001)and IA (F=6.617,P=0.004)are statistically significant,respectively.The changes of the three parameters in 2μmol/L group are most obviously.But in 2μmol/L group the changes of RC,TI,TE and IA at 3 min point were not statistically significant when they were compared with that at 5 min point,the value of P were 0.874、0.854、0.865、0.238, respectively.Steady drug response was observed 3-5 min after application.So the suitable concentration of SCH-23390 in our experiments was 2μmol/L.
1.3 The effect of A68930 and A68930 + SCH-23390 on RRDA
After perfusing the slices with 5μmol/L A68930,compared to that of control group,RC and TE shortened markedly(P＜0.001),respectively.Concurrently,IA (F=6.617,P=0.004)increased noticeably.Moreover,the effect of A68930 on the respiratory rhythm was partially reversed by additional application of A68930+ SCH-23390.
2.The regulating effect of D1R on bisphsic expiratory(BE)neurons/inspiratory(I) neurons in brainstem-spinal cord preparation from newborn rat
2.1 D1R modulating the discharge activities of I neurons
After perfusing the slices with 5μmol/L A68930,when compared with control, RC and TE of I neurons shortened 20.90%(P=0.000)and 21.91%(P=0.000), respectively.Frequency(Fn)and IA increased 14.84%(P=0.001)and 14.27% (P=0.001),respectively.At the same time the change of TI was not statistically significant(P=0.825).These effects were reversed by subsequent application of D1R antagonist SCH-23390.
2.2 D1R affecting the discharge activities of BE neurons
Compard with that of control;RC and TE of BE neurons in A68930 group shortened 20.90%(P=0.000)and 21.91%(P=0.000),respectively;peak frequency(PFn) and IA increased 14.84%(P=0.001)and 14.27%(P=0.001),respectively;at the same time the change of TI was not statistically significant(P=0.844).These effects were reversed by subsequent application of D1R antagonist SCH-23390.
3.The role of cAMP in effect of D1R on BE neurons/I neurons in brainstem-spinal cord preparation from neonatal rats
3.1 cAMP increasing/decreasing agent acting the discharge activities of BE neurons/I neurons in mNRF
3.1.1 cAMP increasing/decreasing agent modulating the discharge activities of I neurons in mNRF
Firstly according to the results of previous of us and the literatures,we decided the suitable concentration of IBMX,clonidine,forskolin and Rp-cAMPS were 5,10, 5 and 10μmol/L,repectively.
3.1.1.1 IBMX and clonidine acting on the discharge activities of I neurons
After 5 min perfusing the slices with 5μmol/L IBMX,RC and TE of IBMX group compared with that of control;RC and TE of I neurons in IBMX group shortened 18.85%(P=0.003)and 20.18%(P=0.003),respectively;TI extended 8.82%(P=0.000);Fn and IA increased 17.16%(P=0.001)and 11.85%(P=0.001), respectively.Expectedly,these effects of IBMX were reversed by subsequent application of clonidine,a cAMP decreasing agent;the parameters above statement were returned to the level of control.
3.1.1.2 forskolin and Rp-cAMPS regulating the discharge activities of I neurons Compared with that of control,RC and TE of I neurons in forskolin group shortened 18.81%(P=0.004)and 20.18%(P=0.003),respectively;TI extended 8.34% (P=0.000);Fn and IA increased 18.96%(P=0.001)and 12.36%(P=0.001), respectively.Expectedly,these effects of forskolin on the discharge activities of I neurons were reversed by subsequent application of Rp-cAMPS,an inhibitor of PKA; the parameters above statement were recover to the level of control.
3.1.2.1 IBMX and clonidine acting on the discharge activities of BE neurons
After 5 min perfusing the slices with IBMX,RC and TE of IBMX group compared with that of control,RC and TE of I neurons in IBMX group all shortened markedly(P=0.000),TI extended significantly(P=0.003),PFn and IA all increased remarkably(P=0.000).Expectedly,these effects of IBMX were reversed by subsequent application of clonidine;the five parameters above mention were returned to the level of control.
3.1.2.2 forskolin and Rp-cAMPS modulating the discharge activities of BE neurons
Compared with that of control,RC and TE of BE neurons in forskolin group shortened 31.94%(P=0.000)and 23.53%(P=0.000),respectively;TI extended 10.17%(P=0.012);PFn and IA increased 19.55%(P=0.000)and 30.60%(P=0.000), respectively.Expectedly,these effects of forskolin on the discharge activities of BE neurons were reversed by subsequent application of Rp-cAMPS;the parameters above statement were recover to the level of control.
3.2 The role of clonidine and Rp-cAMPS in action of A68930 on BE neurons/I neurons in brainstem-spinal cord preparation from neonatal rats
3.2.1 The groups of inspiratory neurons
3.2.1.1 Clonidine and clonidine+A68930 regulating the discharge activities of I neurons
After 5 min perfusing the slices with clonidine,RC and TE of clonidine group compared with that of control;RC and TE of I neurons in clonidine group extended 21.68%(P=0.001)and 23.45%(P=0.000),respectively;TI shortened significantly (P=0.000);Fn and IA decreased 11.08%(P=0.000)and 12.35%(P=0.002), respectively.Notably,after pretreatment with clonidine,the excitatory effects of A68930 on I neurons were blocked;compared with the five parameters of clonidine group,the changes of RC,TI,TE,IA,Fn were not statistically significant and the values of P were 0.651,0.654,0.639,0.404 and 0.610,respectively.
3.2.1.2 Rp-cAMPS and Rp-cAMPS+A68930 modulating the discharge activities of I neurons
Compared with that of control;RC and TE of BE neurons in Rp-cAMPS group extended 22.04%(P=0.000)and 23.81%(P=0.000),respectively;TI shortened 14.29%(P=0.000);at the same time IA and Fn decreasing 13.86%(P=0.000)and 15.85%(P=0.000).Importantly,5 min after pretreatment with Rp-cAMPS,the excitatory effects of A68930 on I neurons were blocked.Compared with the five parameters of Rp-cAMPS group,the changes of RC,TI,TE,IA,Fn were not statistically significant and the values of P were 0.716,0.688,0.704,0.605 and 0.579, respectively.
3.2.2 The groups of BE neurons
3.2.2.1 Clonidine and clonidine+A68930 regulating the discharge activities of BE neurons
After 5 min perfusing the slices with clonidine,RC and TE of clonidine group compared with that of control;RC and TE of I neurons in clonidine group extended 25.93%(P=0.001)and 31.99%(P=0.000),respectively;TI shortened significantly (P=0.005);Fn and IA decreased 10.45%(P=0.001)and 11.37%(P=0.000), respectively.Notably,after pretreatment with clonidine,the excitatory effects of A68930 on I neurons were blocked;compared with the five parameters of clonidine group,the changes of RC,TI,TE,IA,PFn were not statistically significant and the values of P were 0.389,0.945,0.337,0.774 and 0.668,respectively.
3.2.2.2 Rp-cAMPS and Rp-cAMPS+A68930 modulating the discharge activities of BE neurons
Compared with that of control;RC and TE of BE neurons in Rp-cAMPS group extended 10.78%(P=0.001)and 13.41%(P=0.000),respectively;TI shortened 8.33% (P=0.035);meantime,IA and Fn decreasing 15.31%(P=0.000)and 17.41% (P=0.000),respectively.Importantly,5 min after pretreatment with Rp-cAMPS,the excitatory effects of A68930 on I neurons were blocked.Compared with the five parameters of Rp-cAMPS group,the changes of RC,TI,TE,IA,PFn were not statistically significant and the values of P were 0.942,0.759,0.881,0.688 and 0.751, respectively.
4.Determination of cAMP of neurons in mNRF“island”
Determination the concentration of cAMP of neurons in mNRF“island”by ELISA.After incubation the“island”with A68930,the concention of cAMP increased remakably when comparing with that of control.On the contrary,after incubation the“island”with SCH-23390,the concentration of cAMP decreased significantly when comparing with that of control.
5.The expression of mRNA of D1R gene in mNRF“island”
By real-time PCR we detected the expression of mRNA of D1R gene in mNRF“island”.The results suggested that mRNA of D1R gene expressed in mNRF“island”and the quantity of expression was 0.24 times of that of pre-frontal cortex,where the gene expressed plentifully according to the literatures reported.
Conclusion
1.D1R affected the respiratory rhythmic discharge activity(RRDA)of hypoglossal nerve roots.
2.D1R modulated the discharge activities of BE neurons/I neurons,the specific agonist of D1R excited the discharge activities of respiratory neurons and the specific antagonist of D1R had the opposite effect on the neurons.
3.Activating or inhibiting D1R influenced the intracellular level of cAMP of respiratory neurons.
4.mRNA of D1R gene expressed in mNRF“island”and the quantity of expression was about 0.24 times of that of pre-frontal cortex.
5.Forskolin and IBMX enhanced the discharge activities of BE neurons/I neurons,but clonidine and Rp-cAMPS had the opposite effect on the neurons.
6.Clonidine and Rp-cAMPS blocked the excitatory effects of A68930 on the respiratory neurons.
7.The intracellular level of cAMP acted to modulating the discharge activities of the neurons.
8.The current study indicated that D1R would modulate basic breathing rhythmgenesis via cAMP-dependent mechanisms.
引文
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