摘要
正常的节律性呼吸是维持生命的基本条件，确定基本节律性呼吸的发生部位、揭示其发生和调控机制是神经科学领域的一项重要理论课题。婴儿猝死综合征(sudden infant death syndrome，SIDS)、先天性中枢性肺换气不足综合征(congenital central hypoventilation syndrome，CCHS)、中枢性呼吸衰竭(centralrespiratory failure，CRF)、中枢性呼吸节律紊乱(central respiratory rhythmdisturbance，CRD)等不但是临床上常见的疾病或病理生理学过程，也是常见的致死原因。因此，研究基本节律性呼吸发生及其调控机制不仅是呼吸生理学的一项重大理论突破，同时对临床防治中枢性呼吸疾病也具有重要理论指导意义。
近来的研究已证明基本节律性呼吸发生部位位于延髓头腹外侧区。但对于基本节律性呼吸发生部位的精确定位还存在不同看法，目前主要有以下三种观点：1．延髓面神经后核内侧区(the medial area of nucleus retrofacialis，mNRF)1986年吴中海等提出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．前包钦格复合体(pre-B(?)tzinger complex，pre-B(?)tC)1991年Smith提出前包钦格复合体是呼吸节律发生的部位。pre-B(?)tC位于腹侧呼吸组的吻端，包括疑核的腹侧、面神经核和闩之间、面神经后核尾部。3．延髓头腹外侧区(the rostrolventrolateral medulla,RVLM)Onimaru等认为RVLM是呼吸节律发生的部位，它位于从面神经后核吻端到外侧网状核吻端，腹面从腹侧表面到疑核或面神经后核的腹侧面。mNRF、pre-B(?)tC和RVLM定位的区域全部位于延髓的头腹外侧区，相互之间不完全相同，但有部分重叠。
对于基本节律性呼吸产生机制目前主要存在两种学说，即起步神经元学说(pacemaker neuron hypothesis)和神经网络学说(neurons network hypothesis)。1．起步神经元学说该学说认为在延髓中存在具有“起步”性质的神经元，它们表现内在的节律性自动去极化特性，这种活动影响和决定了其它呼吸相关神经元的活动。2．神经元网络学说即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。
尼可刹米化学名称为N，N-二乙基-3-吡啶甲酰胺即二乙基尼克酰胺，化学结构式为(?)。在临床治疗上被应用在以下几方面：1．最常用做非特异性呼吸中枢兴奋剂，尼可刹米可选择性兴奋延髓呼吸中枢，也可通过作用于颈动脉体和主动脉体化学感受器反射性地兴奋呼吸中枢，并提高呼吸中枢对二氧化碳的敏感性，使呼吸加深加快，增加中枢吸气驱动；2．降低肝脏转氨酶和黄疸，常用来治疗新生儿黄疸；3．作为溶解性较差药物的促溶剂，尼可刹米的分子结构含极性部分和非极性部分，可以促进水溶性差药物的溶解和在体内的释放。尼可刹米虽然属于常用药，但其作用机制鲜见报道，尤其尼可刹米作用于呼吸中枢的细胞机制尚未见报道。
呼吸神经元膜上存在诸多膜受体和通道，这些受体和通道对维持神经元的存活、完成相应的生理活动至关重要。5-HT_(2A)受体是G蛋白偶联受体，受体被激活后通过激活细胞膜上磷脂酶C(phospholipase C，PLC)引起细胞内[Ca~(2+)]升高、蛋白激酶C(protein kinase C，PKC)活性增加来产生效应。GABA_A受体是Cl~-通道受体，GABA通过激活GABA_A受体，使Cl~-内流产生快速抑制性突触后电位(fast IPSP)发挥抑制作用。瞬时性钠电流参与动作电位的发生和传导，持续性钠电流因其持续时间长、激活电位低的特点在调节细胞动作电位发生的节律性和重复性方面起着重要作用。
为研究尼可刹米兴奋延髓呼吸中枢的作用机制和基本节律性呼吸的发生、调节机制，本实验利用包含舌下神经根和mNRF的离体延髓脑薄片①观察尼可刹米对舌下神经根节律性呼吸放电和吸气神经元放电的作用；②观察5-HT_(2A)受体对吸气神经元放电的调制作用；⑨观察5-HT_(2A)受体对基本节律性呼吸的调制作用及其在尼可刹米兴奋呼吸过程中的作用；④观察GABA_A受体对基本节律性呼吸的调制作用及其在尼可刹米兴奋呼吸过程中的作用；⑤观察尼可刹米对呼吸起步神经元电活动及呼吸起步神经元、吸气神经元钠电流和钠通道的作用。从而探讨尼可刹米兴奋延髓呼吸中枢的作用机制及基本节律性呼吸的发生和调节机制，为中枢性呼吸疾病的治疗和尼可刹米的应用提供实验依据。
一、尼可刹米对舌下神经根节律性呼吸放电活动(respiratory-related rhythmicdischarge activity,RRDA)和吸气神经元放电的作用
尼可刹米在浓度0．5-7μg／mL范围内对RRDA有兴奋作用：延长吸气时程(inspiratory time，TI)(F=7．263，P=0．000)、增加放电积分幅度(integral amplitude，IA)(F=31．80，P=0．000)、缩短呼吸周期(respiratory cycle，RC)(F=6．430，P=0．001)(n=6，重复测量方差分析)。5μg／mL的尼可刹米对RRDA的TI、IA、RC作用效果最显著，当尼可刹米浓度达到10μg／mL，RRDA放电与对照组相比TI延长、IA增加，RC显著延长，RRDA放电形式变为长周期长吸呼吸即深慢呼吸，提示该浓度的尼可刹米已对呼吸产生抑制效应。5μg／mL的尼可刹米是对新生SD大鼠延髓脑片标本RRDA作用的最适浓度，并作为以后实验中使用的浓度。
mNRF区吸气神经元放电对5μg／mL的尼可刹米的反应有两种情况：1．11个吸气神经元中有6个神经元放电幅度增加(t=12．346，P=0．000)，放电频率变化无显著性意义(t=0．502，P=0．637)(n=6，配对t检验)。2．11个吸气神经元中有5个放电频率增加有显著性意义(t=6．576，P=0．003)，放电幅度变化无显著性意义(t=1．832，P=0．126)(n-5，配对t检验)。
实验结果提示：尼可刹米对呼吸的兴奋作用是通过兴奋基本呼吸节律产生部位的吸气神经元和呼吸节律性放电实现的。为探讨尼可刹米对呼吸兴奋效应与呼吸神经元细胞膜上的受体和钠通道是否有关，我们进行了以下实验。
二、5-HT_(2A)受体对mNRF区吸气神经元放电的调制作用
使用5-HT_(2A)受体激动剂2,5-二甲氧基-4-碘苯基丙烷-2胺盐酸盐[1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane，DOI]灌流脑片标本延长了吸气神经元的TI(P=0．004 vs control group)，使用5-HT_(2A)受体阻断剂酮舍林(ketansrine)缩短了吸气神经元的TI(P=0．002 vs control group)(F=50．593，P=-0．000)；DOI增强了IA(P=0．001 vs control group)，酮舍林降低了IA(P=0．004vs control group)(F=50．694，P=0．000)；DOI缩短了RC(P=008 vs controlgroup)，酮舍林延长了RC(P=0．003 vs control group)(F=68．778，P=0．000)；DOI增加了神经元放电的峰频率(peak frequency,PF)(P=0．008 vs controlgroup)、酮舍林降低了PF(P=0．003 vs control group)(F=33．208，P=0．000)(n=7，重复测量方差分析)。
实验结果证实5-HT_(2A)受体调节了mNRF区吸气神经元的活动，激活5-HT_(2A)受体对吸气神经元电活动有兴奋效应。
三、5-HT_(2A)受体在尼可刹米引起呼吸兴奋中的作用
DOI延长了RRDA的TI(t=10．225，P=0．000)、增强了IA(t=14．143，P=0．000)、缩短了RC (t=7．817，P=0．001)。5-HT_(2A)受体阻断剂酮舍林缩短了RRDA的TI(t=11．62，P=0．000)、降低了IA(t=12．147，P=0．000)、延长了RC(t=7．21，P=0．001)。联合使用酮舍林和DOI对RRDA无作用(TI t=0．342P=0．746；IA t=1．179 P=0．291；RC t=0．168 P=0．873)(n=6，配对t检验)。
联合应用尼可刹米和酮舍林，与单独使用尼可刹米相比，酮舍林可完全阻断尼可刹米对RC的作用(P=0．002 vs nikethamide group，F=44．132 P=0．000)，部分阻断尼可刹米对IA的作用(P=0．001 vs nikethamide group，F=81．025P=0．000)，对尼可刹米作用的TI(P=0．83 vs nikathamide group，F=47．762P=0．000)无影响(n=6，重复测量方差分析)。
实验结果显示5-HT_(2A)受体阻断剂酮舍林可部分阻断尼可刹米对呼吸的兴奋作用，提示5-HT_(2A)受体是尼可刹米兴奋呼吸中枢的途径之一。
四、GABA_A受体在尼可刹米引起呼吸兴奋中的作用
0、10、20、40、60μmol／L等浓度GABA对RRDA的作用显示：GABA对RRDA有抑制作用，且抑制作用呈浓度依赖性。GABA缩短TI(F=43．07，P=0．000)、降低IA(F=93．97，P=0．000)、降低呼吸频率(respiratory frequency,RF)(F=28．669，P=0．000)、延长RC(F=23．54，P=0．000)(n=6，重复测量方差分析)。10μmol／LGABA即对TI和IA产生抑制作用，60μmol／LGABA使6只脑片中的2只RRDA消失。40μmol／L是GABA对RRDA的最适浓度，并作为以下实验的浓度。
GABA_A受体阻断剂bicueulline对RRDA产生兴奋作用：延长TI(t=8．257，P=0．001)、增强IA(t=10．786，P=0．000)、缩短RC(t=8．968，P=0．001)。联合使用GABA和bicuculline对RRDA无作用：TI(t=0．384，P=0．721)、IA(t=1．596，P=0．186)、RC(t=1．295，P=0．265)(n=6，配对t检验)。联合使用尼可刹米和bicuculline，与单独使用尼可刹米相比TI(P=0．006 vs nikethamide group，F=63．837 P=0．000)、IA(P=-0．003 vs nikethamide group，F=126．395 P=0．000)增加，RC变化无显著性意义(P=0．642 vs nikethamide group，F=44．23 P=0．000)(n=6，重复测量方差分析)。
Bicueulline对RRDA有兴奋作用表明在mNRF区有内源性的GABA和GABA_A受体参与基本节律性呼吸的形成与调节。联合使用尼可刹米和bicuculline比单独使用bicuculline TI、IA增加的实验结果提示GABA_A受体是尼可刹米兴奋呼吸中枢的作用途径之一。
五、尼可刹米对呼吸神经元钠电流、钠通道的作用
(一)、尼可刹米对Cd~(2+)非敏感呼吸起步神经元burst发放的作用
记录4个Cd~(2+)非敏感呼吸起步神经元，尼可刹米增加Cd~(2+)非敏感呼吸起步神经元burst幅度、延长burst持续时间、增加spike频率。Burst幅度从54．68±2．20mV增加到59．22±3．87mV(t=4．885 P=0．016)；burst持续时间从2．38±0．20s增加到2．67±0．23s(t=11．676 P=0．001)；spike频率从2．62±0．34s增加到3．37±0．52s(t=4．886 P=0．016)。(配对t检验，n=4)
(二)、尼可刹米对Cd~(2+)非敏感呼吸起步神经元和吸气神经元瞬时性和持续性钠电流的作用
记录4个Cd~(2+)非敏感呼吸起步神经元和6个吸气神经元。在观察尼可刹米对Cd~(2+)非敏感性呼吸起步神经元和吸气神经元的瞬时性和持续性钠电流的影响时，相应采用了Voltage steps(-80～+20 mV)记录瞬时性钠电流和Voltageramp(-80～+20 mV，90 mV／s)记录持续性钠电流，比较钠电流幅度在使用尼可刹米前后的变化。
起步神经元瞬时性钠电流峰值从加药前的1552．50±135．74 pA增加到1962．50±130．22 pA(配对t检验，t=15．135 P=0．001，n=4)；吸气神经元瞬时性钠电流从加药前的1628．33±276．94 pA增加到1961．67±252．86 pA(配对t检验，t=6．798 P=0．001，n=6)。起步神经元持续性钠电流从加药前的339±37 pA增加到370±35 pA(配对t检验，t=9．886 P=0．002，n=4)；吸气神经元持续性钠电流从加药前的295±58 pA增加到320±71 pA(配对t检验，t=4．077 P=0．010，n=6)。这两类电流可以被1μmol／L TTX完全抑制，确定所记录的为钠电流。起步神经元和吸气神经元的瞬时性和持续性钠电流加药前后的变化值和变化百分比差异无显著性意义。(瞬时性钠电流t=1．177 P=0．273，持续性钠电流t=1．291 P=0．233独立样本t检验)实验结果显示尼可刹米可以增加起步神经元和吸气神经元的瞬时性和持续性钠电流；起步神经元持续性钠电流幅度在使用尼可刹米前后均大于吸气神经元持续性钠电流幅度，且起步神经元持续性钠电流增加百分比也大于吸气神经元续性钠电流增加百分比；但这两类神经元钠电流在尼可刹米作用前后的变化程度无显著性意义。实验结果提示：由瞬时性和持续性钠电流增加导致的呼吸神经元兴奋性增加可能是尼可刹米兴奋呼吸中枢的作用机制之一；持续性钠电流参与形成呼吸起步神经元的“起步”机制，但不起决定性作用，还有其它因素参与形成呼吸起步神经元的“起步”机制。
(三)尼可刹米对Cd~(2+)非敏感呼吸起步神经元稳态激活曲线、稳态失活曲线的作用
Cd~(2+)非敏感呼吸起步神经元激活曲线V_(1/2)和κ从尼可刹米作用前的-34．49±0．73mV、5．67±0．47，变为-40．45±2．00mV、4．54±0．32，曲线向超极化方向移动。钠通道激活门开放阈值变负，通道激活门易开放。
Cd~(2+)非敏感呼吸起步神经元失活曲线V_(1/2)和κ从尼可刹米作用前的-46．85±1．59mV、5．34±0．33变为-35．02±1．55mV、6．67±0．30，曲线向去极化方向移动，钠通道失活门关闭电位升高，失活门不易关闭。
结论：
1、尼可刹米对延髓脑片基本节律性呼吸放电和吸气神经元放电有兴奋作用。
2、在mNRF区，生理状态下内源性5-HT和5-HT_(2A)受体参与基本呼吸节律的发生和调节，激活5-HT_(2A)受体对延髓脑片基本节律性呼吸放电和吸气神经元放电有兴奋作用。
3、尼可刹米对延髓脑片基本节律性呼吸放电的兴奋作用与5-HT_(2A)受体存在交互作用，5-HT_(2A)受体是尼可刹米兴奋呼吸作用的途径之一。
4、在mNRF，生理状态下内源性GABA和GABA_A受体参与基本呼吸节律的发生和调节，激活GABA_A受体对延髓脑片基本节律性呼吸放电有抑制作用。
5、尼可刹米对延髓脑片基本节律性呼吸放电的兴奋作用与GABA_A受体存在交互作用，GABA_A受体是尼可刹米兴奋呼吸作用的途径之一。
6、尼可刹米增加了呼吸起步神经元burst发放的频率、幅度、持续时间，spike频率和数量，对呼吸起步神经元电活动有兴奋作用。
7、尼可刹米增加吸气神经元和呼吸起步神经元瞬时性和持续性钠电流，尼可刹米对呼吸的兴奋作用与增加神经元钠电流有关。尼可刹米改变了呼吸起步神经元钠通道的动力学特征，促进钠通道激活门开放，抑制失活门关闭。尼可刹米提高了神经元的兴奋性。
8、持续性钠电流参与形成呼吸起步神经元的“起步”机制，但不起主要作用，还有其它因素参与形成“起步”机制。
Keeping normal rhythmic respiration is essential for life. It is important to make it clear that which site is the precise site for respiratory rhythm generation and the mechanisms underlying respiratory rhythmogenesis. There are many diseases such as sudden infant death syndrome (SIDS), congenital central hypoventialation syndrome (CCHS), central respiratory failture, central respiratory rhythm disturbance, which are due to abnormality of respiratory center. So making the mechanisms underlying respiratory rhythmogenesis clear is not only meanful in physiology but also for prevention and cure respiratory central diseases.
Our researches have demonstrated that the medial area of nucleus retrofacialis (mNRF) is the site of respiratory rhythmogenesis. Smith stated that the pre-Botzinger complex (PBC) was the site of respiratory rhythmogenesis in 1991.mNRF and PBC are located in rostrol ventrolateral medulla. The anatomical position of them is of difference, but they are overlapped partly. The site of basic respiratory rhythmogenesis has been ascertained in the rostrol ventrolateral medulla. There are two hypothesis for the mechanism of respiratory rhythmogenesis: pacemaker neuron hypothesis and neurons network hypothesis. Pacemaker neuron hypothesis has been supported by a majority of the present findings. We had found Expiratory-Inspiratory phase spanning (E-I PS) neuron maybe is pacemaker on mNRF in vivo and in vitro in neonate rats. The 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. Foreign scholars haveidentified pacemaker neuron as cadmium-insensitive and cadmium-sensitive. Cadmium-insensitive pacemakers are characterized by bursting that persist in the presence of Cadmium, dependent on persistent and transient sodium current (I_(NaP) and I_(NaT)) and blocked by TTX. Cadmium-sensitive pacemakers are characterized by bursting that is blocked in the presence of Cadmium, dependent on Ca~(2+)-activated nonspecific cationic current (I_(CAN)) and blocked by flufenamic acid (FFA).
Nikethamide (N, N-diethylnicotinamide) has been usedwidely in clinic. It is generally used in three directions as follow. First, it is used as a respiratory central stimulator, nikethamide can excite respiratory center selectively. Second nikethamide can decrease the aminopherase and jaundice levels of infant Thirdly, niketnamide comprise polarity part and nonpolarity part, it can be used as chaotropic agent to dissolve drugs which is difficult to dissolve in water. Although nikethamide has been used widerly but its mechanisms is still unknown.
5-HT_(2A) receptors have been found in many positions of the CNS, including the cerebral cortex, basal ganglia, hippocampus, thalamus, cerebellum, and hypothalamus. 5-HT_(2A) receptror agonists have excitory effects on respiratory activity. Activation of 5-HT_(2A) receptors with l-(2,5-dimethoxy-4-iodophenyl)-2-am inopropane (DOI) increases the frequency of respiratory activity. Blockade of endogenously activated 5-HT_(2A) receptors decreases the frequency, amplitude, and regularity of respiratory population activity. An ERK activation pathway has been confirmed in rat renal mesangial cells as follows: 5-HT _(2A) receptor-> G(q) protein-> phospholipase C-> diacylglycerol-> protein kinase C (PKC)->NAD(P)H oxidase-> reactive oxygen species (ROS, i.e., H2O2 and superoxide) -> mitogen-activated extracellular signal-regulated kinase-> extracellular signal-regulated kinase (ERK).
To explore the effects of nikethamide on respiratory-related rhythmic discharge activity and respiratory neurons in the mNRF in vitro, and explore whether exist interaction between nikethamide and 5-HT_(2A) receptors pathway, we designed this study as following: 1. To explore the effects of nikethamide on activity (RRDA) and discharge activity of respiratory neurons, simultaneous recording of the hypoglossal nerve (Xlln) respiratory-related rhythmic discharge (RRDA) with suction electrode and the respiratory neuronal discharge with microelectrodes in the mNRF on the brainstem slices in vitro. 2. Simultaneous recording of RRDA and the respiratory neuronal discharge. To observe the roles of 5-HT_(2A) receptors in respiratory neurons. 3. To explore whether the interaction exist between nikethamide and 5-HT_(2A) receptors pathway on respiratory center, RRDA was recorded with suction electrode on brainstem slices. 4. To explore weather exist the interaction between nikethamide and GABAa receptors pathway on respiratory center, RRDA was recorded with suction on brainstem slices. 5. Simultaneous recording of RRDA and the respiratory neuronal discharge was performed with whole cell patch in the mNRF on the brainstem slice in vitro. To observe transient and the persistent sodium current changes of respiratory pacemaker neurons and respiratory neurons respectively before and after nikethamide perfusion.
1. Effects of nikethamide on respiratory-related rhythmic discharge activity recorded from hypoglossal nerve rootlets and discharge activity of respiratory neurons in mNRF.
The possible role of nikethamide in RRDA was investigated by perfused different concentration of nikethamide, 0, 0.5,1, 3, 5, 7, 10μg/mL in modified Kreb's solution (ACSF). Nikethamide increased RRDA at the range of concentration from 0.5 to 7μg/mL, 5μg/mL being the most effective concentration of nikethamide on RRDA of transverse medullary slices, in inspiratory time (TI)( F=7.263, P=0.000), integral amplitude (IA)( F=31.80, P=0.000), and respiratory cycle (RC)( F=6.430, P=0.001)(Repeat measures, n=6). While the concentration of nikethamide reachedlOμg/mL RRDA changed into the deep, very slow inspiratory mode. It suggested nikethamide has a depressive effect on RRDA in this concentration.
According to the response to nikethamide, the respiratory neurons in mNRF were classified into two groups. One group was the changed in discharge amplitude (t=12.346, P=0.000) it was significant, but change in discharge frequency was nonsignificant (t=0.502, P=0.637) (paired-samples t test, n=6); and another group was changed with significance in discharge frequency (t=6.576, P=0.003) but not in discharge amplitude (t=1.832, P=0.126) (paired-samples t test, n=5).
On the basis of those experimental results, nikethamide may stimulate respiration by exciteing respiratory neuron and neurons in respiratory related nucleus.
2. Effects of DOI and ketanserine on inspiratory neurons
DOI prolonged TI( P=0.004 vs control group), ketanserine shortened TI(P=0.002 vs control group) (F=50.593, P=0.000); DOI increased IA (P=0.001 vs control group), ketanserine decreased IA (P=0.004 vs control group) (F=50.694, P=0.000); DOI shortend RC (P=0.008 vs control group), ketanserine prolonged RC (P=0.003 vs control group) (F=68.778, P=0.000); DOI increased peak discharge frequency (PF) (P=0.008 vs control group), ketanserine decreased PF (P=0.003 vs control group) (Repeat measures, n=7).
5-TH_(2A) receptors are involved in the modulation of discharge activity of inspiratory neurons. Actived 5-TH_(2A) receptors play a role of stimulation on inspiratory neurons and RRDA, block 5-TH_(2A) receptors have an opposite effect.
3. Effects of 5-HT_(2A) receptor in the increased of RRDA by nikethamide in neonatal rats transverse medullary slice.
DOI increased RRDA in TI (t=10.225, P=0.000), IA (t=14.143, P=0.000), and RC (t=7.817, P=0.001). Ketanserine decreased RRDA in TI (t=11.62, P=0.000), IA (t=12.147, P=0.000) and RC (t=7.21, P=0.001). Ketanserine plus DOI had no significant effects on RRDA (TI t=0.342 P=0.746; IA t=1. 179 P=0.291; RC t=0.168 P=0.873). (Those groups were texted by paired-samples t test, n=6.) The effects of nikethamide on RC (P=0.002 vs nikethamide group, F=44.132 P=0.000) and IA (P=0.001 vs nikethamide group, F=81.025 P=0.000) were totally and partially reversed respectively by additional application of ketanserine, but the effect of nikethamide on TI (P=0.83 vs nikethamide group, F=47.762 P=0.000) was not influenced by ketanserine (Repeat measures, n=6).
Experimental results show ketanserine can partially block the stimulating effects of nikethamide on RRDA, it suggests nikethamide can increase the RRDA partly via 5-TH_(2A) receptor.
4. Roles of GABAa receptor in respiratory enhancement induced by nikethamide in neonatal rats.
Within the concentration range of 10 to 40μmol/L, GABA decreased RRDA in TI (F=43.07, P=0.000), IA (F=93.97, P=0.000), RC (F=8.968, P=0.001) (Repeat measures, n=5). 40μmol/L GABA was the most effective concentration decreasing RRDA in TI, IA, RC. 10μmol/L bicuculline could increase RRDA in TI (t=8.257, P=0.001), IA (t=10.786, P=0.000) and RC (t=8.968, P=0.001). 10μmol/L bicuculline plus 40μmol/L GABA had no significant effects on RRDA [TI (t=0.384, P=0.721), IA (t=1.596, P=0.186), RC (t=1.295, P=0.265)] (Those two groups were texted by paired-samples t test, n=5). The effects of nikethamide plus bicuculline were significantly increased in TI (P=0.006 vs nikethamide group, F=63.837 P=0.000) and IA (P=0.003 vs nikethamide group, F=126.395 P=0.000) compared with nikethamide alone, but RC (P=0.642 vs nikethamide group, F=44.23 P=0.000) had no significant influence by nikethamide plus bicuculline compared with nikethamide alone (Repeated measures, n=6).
Experimental results show GABAa receptors were involved in the modulation of RRDA, 40μmol/L GABA was the most effective concentration to depress RRDA. Blocking GABAa receptors can stimulate RRDA based on the role of nikethamide.It suggests nikethamide can increase the RRDA partly via GABAa receptor.
5. Effects of nikethamide on sodium current and sodium channel of respiratory pacemaker neurons and inspiratory neurons.
5.1 Effects of nikethamide on burst of respiratory pacemaker neurons
Nikethamide significantly increased the bursts duration (t=4.885 P=0.016), burst amplitude (t=11.676 P=0.001) and spike frequency (t=4.886 P=0.016) of respiratory pacemaker neurons and inspiratory neuron (paired-samples t test, n=4).
5.2 Effects of nikethamike on transient sodium current and persistent sodium current of respiratory pacemaker neurons and inspiratory neurons.
Experiment was performed in voltage-clamp configuration. Voltage steps from -80 to +20 mV and slow voltage ramps from -80 to +20 mV (90 mV/ sec) were applied to elicit transient and persistent sodium currents, respectively. The persistent sodium current elicited by Voltage ramp were increased by nikethamide too. Both the persistent and transient inward currents recorded in the Cd-insensitive pacemaker neurons were sensitive to TTX. The transient sodium currents were increased by nikethamide in pacemaker neurons (t=15.135 P=0.001) (paired-samples t test, n=4) and inspiratory neuron (t=6.798 P=0.001) (paired-samples t test, n=6). The transient sodium currents were increased by nikethamide in pacemaker neurons (t=9.886 P=0.002) (paired-samples t test, n=4) and inspiratory neuron (t=4.077 P=0.01) (paired-samples t test, n=4) too. There were no differences between pacemaker neurons and inspiratory neurons in transient (t=1.177 P=0.273) and persistent (t=1.291 P=0.233) sodium currents (independent-samples t test). The experimental results suggest that nikethamide stimulates respiratory neurons due to increasing sodium conductance.
The ampulite of persistent sodium currents and the change ratio of persistent sodium currents before and after nikethamide of pacemaker neurons are larger than those of inspiratory neuron. So we think that persistent sodium current are involved with "pacemaker" properties, but its role is not decisive, there are other factors contribute to "pacemaker" properties of pacemaker neuron.
5.3 Effects of nikethamide on steady activation curves and steady inactivation curves of sodium channel in respiratory pacemaker neurons.
Nikethamide makes sodium channel steady activation curves shift left vs control. This means the threshold of sodium channel is lower than control, channel begin open at a lower potential. Nikethamide makes sodium channel steady inactivation curves shift right vs control. This means the inactivation threshold of sodium channel is higher than control, channel begin to close at a higher potential
From those experimental results we thought nikethamide increase sodium current due to it change the kinetics of sodium channel, insreases its open probability, decreases its close probability.
Conclusion:①Nikethamide can increase discharge activity of respiratory-relate rhythm discharge activity and inspiratory neurons.②Endogenous 5-HT and 5-HT_(2A) receptors are involved in the modulation of discharge activity of inspiratory neurons and respiratory rhythmogenesis.③Activation of 5-HT _(2a) receptors have excitory effects on rhythmic respiration, nikethamide increases the RRDA partly via 5-TH_(2A) receptors.④Activation of GABA_a receptors in mNRF have depressive effects on rhythmic respiration, nikethamide increases the RRDA partly via GABA receptors.⑤Nikethamide increases transient and persistent sodium currents, perhaps it is the reason why nikethamide can increase RRDA level.Nikethamide changes kinetics to increase the excitability of neurons.⑥Persistent sodium currents is are involved with "pacemaker" property, but its role is not decisive, there are other factor contribute to "pacemaker" property of pacemaker neuron.
引文
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