摘要
神经系统可以对来自内、外环境的信息进行转化、编码、整合、储存和传递,这些过程主要是通过“全或无”的动作电位以规则或不规则放电的形式在神经纤维上的传导和突触之间的传递实现的。通常将动作电位大小不随刺激强度和传导距离而发生改变现象称作“全或无”,这一特性可以保证信息的精确性。目前,对动作电位串编码的方式虽然有多种学说,例如平均频率编码(meanrate code )、群体编码(population code )、时间编码(temporal code )、模式编码(pattern code )等等,但是关于动作电位串的模式与神经信息的确切关系问题一直存在着争论。最近的研究表明:大脑皮层神经元活动模式可能不是利用精确的活动模式来进行信息编码的。这对当前关于神经元节律编码信息的观点产生了极大的影响。是否存在节律编码以外的信息传递方式呢?对此,我们知之甚少。
伤害性信息通常被认为是在初级感觉神经元和终末的伤害性感受器激活后形成的动作电位串,可以将危害机体的刺激信息(模式、强度、时程)进行初级编码,然后以一定时间序列沿着躯体感觉神经纤维的中枢突向脊髓传递。与经典的动作电位不同,由于离子通道的多样性和多态性、特异性的离子通道阻断剂的缺乏以及离子通道活性调节的多因素性,伤害性感受器动作电位产生机制以及在病理性痛中的作用不甚清晰。更为重要的是,由于长期受到“全或无”动作电位编码信息理论的影响,目前对于病理过程中的单个动作电位时空参数(尤其是幅度和去极化时程)是否发生改变及其功能意义研究甚少。
本课题主要运用膜片钳技术,通过记录和分析痛觉信息传递的第一级神经元(DRG神经元)动作电位的空间参数(幅度)和时间参数(去极化时程和复极化时程),从离体细胞水平和在体水平两个层次上,分别研究神经元周围钙离子的稳态失衡和炎性病理性痛状态下,DRG神经元动作电位时空参数的变化特征。为了探讨动作电位产生与可塑性的离子通道机理,我们进一步研究了两种TTX-R钠通道(NaV1.8和NaV1.9)在动作电位时空可塑性中的作用及功能意义。通过对神经元单个动作电位时空参数的研究,我们得到以下结果:
1.初级感觉神经元动作电位不仅具有时间可塑性,还有空间可塑性
在没有外在刺激因素的状态下,自发放电的背根节(DRG)神经元所产生动作电位具有“全或无”的特征,其时间参数和空间参数相同。向DRG神经元内连续10次注入相同的电流(强度300 pA,间期:3 ms,间隔200 ms),所产生的动作电位具有“全或无”的特征,而向同一神经元内连续10次注入不同强度的电刺激(强度50-500 pA,50 pA递增),所产生的动作电位波形完全不同。为了进一步研究电刺激诱发动作电位的特性,我们采用了以下的电刺激参数:强度50-500 pA,每次递增50 pA,共10串电刺激,每串电刺激的波宽0.5-5 ms,每次递增0.5 ms,每个电刺激时间间隔200 ms,电刺激总计数100个。结果显示出以下特点:第一,动作电位的幅度在一定时间参数上不随电刺激波宽的增加而改变,但随电刺激强度的增加而增强。第二,动作电位的时程在同一阈上刺激强度时随电刺激波宽的增加而先缩短后恢复,但在同一波宽却随电刺激强度的增加而缩短。第三,动作电位的去极化时程在同一阈上刺激强度时不随电刺激波宽的增加而改变,但随电刺激强度的增加而缩短。第四,动作电位的复极化时程在同一阈上刺激强度时随电刺激波宽的增加而先缩短后恢复,但在同一波宽却随电刺激强度的增加而缩短。这些结果说明:初级感觉神经元动作电位具有时空可塑性。这种可塑性与电刺激参数有关,刺激电流的强度主要影响动作电位的幅度和去极化时程,而刺激电流波宽主要影响复极化时程。
2.细胞内外钙离子稳态的维持有利于初级感觉神经元动作电位保持‘全或无’的时空特征
1)细胞外钙离子浓度改变对DRG神经元动作电位的影响。形成全细胞模式后,依次将细胞外的钙离子调整为(mM):2.5 Ca,0 Ca,1 Ca, 5 Ca和10 Ca,分别记录电刺激(参数:强度500 pA,波宽3 ms)诱发的动作电位。其中0 Ca条件对动作电位的波形影响最大,可以使DRG神经元动作电位的幅度明显降低,复极化时程显著延长,而对去极化时程没有明显影响。由于细胞外0 Ca时, DRG神经元静息膜电位向去极化方向移动约10 mV,所以0 Ca条件对动作电位时空参数的影响可能是通过对膜电位的影响而实现的。在5 Ca和10 Ca条件下,除去极化时程显著延长外,其他参数没有显著变化。
2)细胞内钙离子浓度降低对DRG神经元动作电位的影响。用0 Ca外液和thapsigargin(Tg,1μM )共同孵育神经元15 min后,排空细胞内钙离子,记录电刺激(参数:强度500 pA,波宽3 ms)诱发的动作电位。结果显示:细胞内钙离子降低后,可以使DRG神经元产生的动作电位的幅度降低,总时程和复极化时程显著延长,而去极化时程没有明显改变。
3)细胞内钙离子浓度升高对DRG神经元动作电位时空参数的影响
首先,运用钙离子成像和膜片钳同步记录技术,确定辣椒素(capsaicin)诱发的钙离子升高持续时间、反应幅度以及神经元膜电位和内向电流变化时程等参数。Capsaicin(0.5μM, 10 s)诱发的钙离子浓度增加的峰值出现在给药后10 s,神经元内钙离子升高反应可以持续120 s以上,而内向电流和膜电位在30 s时,已经基本恢复到初始水平。IP3受体阻断剂2-APB(50μM)不能阻断capsaicin诱发的反应,而ryanodine受体阻断剂钌红(Ruthenium Red,RR)10μM可以完全阻断capsaicin诱发的钙离子升高反应,对内向电流也有显著的抑制作用。继而,我们分别记录capsaicin之前和停药30 s后,电刺(参数:强度500 pA,波宽3 ms)激诱发的动作电位,结果显示:细胞内钙离子升高可以使动作电位的幅度显著降低,而对去极化时程和复极化时程均没有影响。
3.外周炎症条件刺激可以改变初级感觉神经元动作电位的时空特性
前面的研究已经表明:细胞水平的钙离子稳态失衡可以引起动作电位的可塑性变化。那么在个体处于病理状态下,动作电位是否会发生变化?其意义如何呢?我们进一步运用炎性病理性痛模型,研究病理条件下动作电位的时空特性。
1)行为学试验表明,大鼠足底注射CFA(100μl,溶于生理盐水)1天或蜜蜂毒肽melittin (100μg/100μl,溶于生理盐水)2 h后,热刺激缩足反应潜伏期(PWTL)和机械刺激阈值(PWMT)均显著降低。在体电生理记录表明,CFA或melittin致炎处理后,伤害性热刺激(49℃水浴)以及毛刷(brush)、触压(pressure)和钳夹(pinch)等机械刺激诱发的脊髓背角广动力域(WDR)神经元放电反应均显著增强。这些结果说明,CFA和melittin足底注射可以诱发炎性痛反应。
2)CFA和melittin致炎处理对DRG神经元兴奋性的影响。通过足底注射荧光指示剂DiI ( 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate,17 mg/ml,溶于10% DMSO),我们可以鉴定出支配大鼠后肢足底的两类DRG神经元(即phasic神经元和tonic神经元)。结果显示:CFA和melittin致炎处理对两类DRG神经元兴奋性的影响不同,tonic神经元可以通过降低动作电位阈值和增加放电个数而提高神经元的兴奋性,而phasic神经元的兴奋性并未发生显著改变。结果提示:在炎性病理性痛状态下,伤害性信息可能同时以节律编码和非节律编码的方式进行传递。
3)CFA和melittin致炎处理对两类DRG神经元动作电位时空参数的影响。通过比较CFA或melittin致炎处理前后,相同的电流刺激(强度50-500 pA,波宽3 ms,时间间隔200 ms)诱发动作电位的波形,结果显示:tonic神经元的波形发生显著性改变,电刺激下动作电位的幅度和超射值显著增加,去极化时程和复极化时程均显著缩短。而phasic神经元动作电位波形的改变稍有不同:CFA致炎处理后phasic神经元动作电位的幅度和超射值显著增加,去极化时程和复极化时程均显著缩短,而melittin致炎处理后,phasic神经元动作电位的幅度和超射值显著增加,复极化时程显著缩短,但去极化时程没有明显变化。结果提示:除了上述神经元兴奋性改变以外,在炎性病理性痛状态下,DRG神经元动作电位的波形也可以发生显著变化,而这种改变可能是一种新的神经元可塑性的表现。
4)炎性病理性痛条件下,动作电位时空可塑性变化可能与TTX-R钠通道相关。在正常状态下,给予相同的电流刺激(强度50-500 pA,50 pA递增,波宽3 ms,时间间隔200 ms)诱发动作电位,TTX(1μM)可以使DRG神经元的动作电位去极化时程明显延长,幅度明显减小。而大鼠足底注射CFA (100μl) 1天或melittin (100μg/100μl)2小时后,TTX的对动作电位幅度和去极化时程的影响明显减弱了。结果提示:外周致炎处理后,形成动作电位的上升支和去极化时程的TTX-R钠电流成分显著增加。
4. TTX非敏感钠通道表达上调是导致初级感觉神经元动作电位空间可塑性的离子通道机制
1)免疫组织化学结果显示:正常情况下,大鼠NaV1.8主要表达在中小直径神经元,而NaV1.9仅在小直径神经元有表达。CFA或melittin致炎后,NaV1.8阳性神经元平均计数分别增加了93%和50%,NaV1.9阳性神经元平均计数分别增加了74%和47%。
2)Western blot结果显示:正常情况下,大鼠DRG神经元中NaV1.8和NaV1.9蛋白低表达。CFA处理组和melittin处理组中NaV1.8蛋白分别增加了84.25%和55.06%,而NaV1.9蛋白分别增加了51.97%和37.64%。
3)RT-PCR结果显示:正常情况下,大鼠DRG神经元中NaV1.8和NaV1.9的处于低水平表达。CFA或melittin处理后NaV1.8的基因水平分别增加了116.02%和82.01%,而NaV1.9分别增加了87.86%和43.36%。
4)膜片钳记录TTX-R钠电流的结果显示:大鼠足底注射CFA (100μl) 1天或melittin (100μg/100μl)2小时后,NaV1.8钠电流和NaV1.9钠电流均显著增加。
将上述结果与TTX的药理学作用相结合,可以提示:在炎性病理性痛状态下,TTX-R钠通道NaV1.8和NaV1.9基因和蛋白的表达上调和功能易化是引起DRG神经元动作电位空间可塑性的主要原因。
5.初级感觉神经元动作电位空间可塑性的病理意义
1)免疫组织化学和western blot的结果显示:鞘内给予羧基荧光素(Carboxyfluorescein,FAM)标记的NaV1.8或NaV1.9反义寡核苷酸(antisense oligodeoxynucleotide,AS OND)连续3天(45μg/次,2次/天),可以显著抑制CFA (100μl, 1d后)或melittin (100μg/100μl, 2 h后)引起的NaV1.8和NaV1.9阳性神经元的数量的增加和蛋白表达的上调。结果表明:反义寡核苷酸处理可以显著抑制炎性处理引起的蛋白表达增加反应。
2)行为学检测结果显示:鞘内给予NaV1.8 AS或NaV1.9 AS(连续3天,45μg/次,2次/天),对CFA (100μl, 1天后)或melittin (100μg/100μl,2 h后)诱发的炎性痛反应具有不同的作用。其中,NaV1.8 AS可以显著抑制CFA诱发的热痛敏和机械性痛敏,但对melittin诱发的热痛敏和机械性痛敏均没有明显影响。NaV1.9 AS对CFA诱发的热痛敏和机械性痛敏均没有明显影响,但可以显著抑制melittin诱发的热痛敏。这些结果提示:NaV1.8钠通道主要参与CFA诱发的慢性炎性热痛敏和机械性痛敏;而NaV1.9钠通道主要参与melittin诱发的急性炎性热痛敏。
3)膜片钳记录显示:鞘内给予NaV1.8 AS或NaV1.9 AS(连续3天,45μg/次,2次/天),对CFA (100μl, 1天后)或melittin (100μg/100μl, 2 h后)两种不同炎性痛状态下,DRG神经元的动作电位时空参数的影响不同。其中,NaV1.8 AS可以显著抑制CFA处理组动作电位的幅度,延长去极化时程,而NaV1.9 AS对CFA处理组动作电位的波形影响不大。相反,NaV1.8 AS对melittin处理组动作电位的波形影响不大,而NaV1.9 AS对melittin处理组动作电位的波形有一定程度的影响,主要表现在动作电位的幅度降低,去极化时程缩短。
上述结果说明:DRG神经元动作电位空间可塑性变化具有不同的分子基础,并且与不同的炎性病理性痛的状态密切相关。经过慢性致炎物质CFA处理后,NaV1.8钠通道通过影响动作电位的幅度而引起痛敏表现;而经过急性致炎物质melittin处理后,NaV1.9钠通道通过影响动作电位的幅度而引起痛敏表现。
结论
1、在某些条件下(如自发放电或接受恒定刺激),神经元动作电位具有“全或无”的时空特性,但是当刺激强度发生变化时,动作电位的空间参数(幅度)和时间参数(去极化和复极化时程)均可以发生显著变化,表明初级感觉神经元动作电位不仅具有时间可塑性,还有空间可塑性。
2、神经元周围钙离子稳态失衡可以通过直接或间接(膜电位)作用降低动作电位的幅度,一定程度地延长动作电位的时程,表明细胞内外钙离子稳态的维持有利于初级感觉神经元动作电位保持‘全或无’的时空特征。
3、在炎性病理性痛状态下,两类DRG神经元的动作电位的时空参数均发生可塑性变化。tonic神经元可能通过降低动作电位阈值和提高放电频率来提高兴奋性,参与伤害性信息传递,而phasic神经元的兴奋性并未发生显著变化,可能是通过幅度增大、时程缩短的动作电位来传递伤害性信息的。
4、在炎性病理性痛状态下,TTX-R钠通道NaV1.8和NaV1.9基因和蛋白表达上调和功能易化是初级感觉神经元动作电位时空可塑性的主要原因。
5、初级感觉神经元动作电位空间可塑性具有不同的分子基础,并且与不同的炎性病理性痛的状态密切相关。其中,NaV1.8钠通道表达异常引起的动作电位空间可塑性的变化与CFA诱发的慢性炎性热痛敏和机械性痛敏的发生密切相关,而NaV1.9钠通道表达异常引起的动作电位空间可塑性变化与melittin诱发的急性炎性热痛敏的发生密切相关。
Information from exogenous and endogenous environments can be transducted, encoded, integrated,stored and transmitted in the nervous system. The processes are completed through the generation and propagation of“all or none”action potentials with regular or irregular activities on cell bodies, axonal nerve fibers as well as trans-synaptic transmission. In general, the phenomenon of“all or none”means that amplitude of action potential (AP) should be constant under different stimulus intensities and distances. The property of“all or none”ensures the accuracy of information. At present, there are many theories on the information encoded by trains of APs, such as mean rate code, population code, temporal code, pattern code etc. However, the correlation between firing pattern and neuronal information remains controversial for many years. A recent study suggested that the patterns of neuronal activities in cortex might not through accurate firing patterns. The results seriously challenged the popular theory on neuronal rhythmic activity-encoded information. Is there any other means to transmit neuronal information beyond rhythmic patterns? It’s a poorly known field.
At present, noxious information seems to be mediated by different AP trains generated from nociceptors expressed in cell body and nerve terminal of primary sensory neurons, that can preliminarily encode the information of stimulus (such as pattern, intensity and duration). The latter could be transmitted to spinal cord along the central terminals of somatic sensory fiber in certain time course. Different from the classical AP, the generation and function of APs in nociceptor are poorly understood because of the variabilities and modalities of ion channels,lack of specific ion channel blockers and complex modulations of channel functions. Importantly, because of the theory of information encoded by AP with“all or none”property for a long time, there’s very little investigation on the characterization and significance of single AP waveform (especially amplitude and depolarization duration) during different pathologic-physiological conditions
In present study, we investigated the characterization of AP waveform generated in dorsal root ganglion (DRG) neurons, the first ones in the series of neurons that lead to pain sensation. By using the patch clamp recording, we recorded and analyzed the spatial parameter (amplitude) and temporal parameters (depolarization duration and repolarization duration) of DRG neuronal AP waveform under different in vitro and in vivo levels. The former were performed in the situation of disequilibrium of calcium homeostasis whereas the latter were performed in the situation of inflammatory pain. Furthermore, we investigated the functions and significance of two tetrodotoxin-resistant (TTX-R) sodium channels, NaV1.8 and NaV1.9, in the neuronal AP plasticity. Through researching on the single AP of DRG neurons, we got the following results.
1. Action potentials generated in primary sensory neuron perform spatiotemporal plasticity
Under normal situation and without any stimulation, APs produced in spontaneous discharging DRG neuron are“all or none”and having identical spatial parameter parameters and temporal parameters. APs in DRG neuron generated by 10 identical electrical stimulation (intensity, 300 pA; duration, 3 ms; interval, 200 ms) are“all or none”. APs in DRG neuron generated by 10 different electrical stimuli (intensity, 50-500 pA; duration, 3 ms; interval, 200 ms) are quite different in spatial and temporal parameters. To understand the characterizations of neuronal AP evoked by electrical stimulation, we apply a series of different electrical stimuli(intensity, 50-500 pA;△=50 pA, duration,0.5-5 ms; interval, 200 ms; number of trains, 10; total number of stimuli, 100 ) . We found the following characterizations: 1) Under certain temporal parameters,AP amplitude is not changed by duration increment but enhanced by intensity increment. 2) Under superthreshold stimulus intensitity, total duration of AP is initially shortented, then gradually recovered, whereas under the same stimulus duration, the values are shortened by stimulus intensity increment. 3) Under the same superthreshold stimulus intensitity, depolarization duration of AP is not changed, whereas the value is shortened by stimulus intensity increment. 4) Under the same superthreshold stimulus intensitity, repolarization duration of AP is initially shortented, then gradually recovered, whereas under the same stimulus duration, the values are shortened by stimulus intensity increment. These results indicated that AP generated in DRG neuron was plasticity. In addition, the plasticity of AP could be changed parameters of electrical stimuli. Intensity of electrical stimulation mainly affected AP amplitude and depolarization duration, while the duration of electrical stimulation mainly affected AP repolarization duration.
2. Calcium homeostasis is required for the maintainance of“all or none”property in primary sensory neuron
1) Effects of extracellular calcium change on AP of DRG neuron. After whole-cell performed, we recorded electrical stimuli-evoked APs (intensity, 500 pA; duration, 3 ms) under 2.5 Ca,0 Ca,1 Ca,5 Ca and 10 Ca in bath solution, respectively. Under 0 Ca condition, the AP amplitude was significantly decreased, total duration and repolarization duration were lengthened,whereas the depolarization duration was not changed. The suppressive effects might through membrane potential fluctuation because 0 Ca caused 10 mV resting membrane potential (RMP) change shifting to depolarization forward. In contrast, 5 Ca and 10 Ca had nearly no effects on AP spatial and temporal parameters,in addition to lengthen the depolarization duration.
2) Effects of intracellular calcium decrease on AP of DRG neuron. After pre-incubating neurons with thapsigargin (1μM )for 15 min to delete intracellular calcium,we recorded electrical stimuli-evoked APs (intensity, 500 pA; duration, 3 ms). Comparison with the 2.5 Ca condition, intracellular calcium decrease significantly inhibited AP amplitude generated with 500 pA stimulation, lengthened total duration repolarization duration, but had no effects on depolarization duration.
3) Effects of intracellular calcium increase on AP of DRG neuron. At first, we used combined voltage clamp and calcium imaging test to clarify parameters of capsaicin-induced responses, including duration and amplitude of intracellular calcium increase, duration of DRG neuronal membrane potential and inward current shifting. The peak reaction of intracellular calcium mobilization induced by capsaicin(0.5μM, 10 s duration)perfusion appeared at 10 s after drug adminidtration, while the duration of calcium response lasted more than 120 s. Duration of inward current and membrane potential shifting could return to baseline level within 30 s. IP3 receptor blocker, 2-APB(50μM)can not block capsaicin-induced responses. Ryanodine receptor blocker,ruthenium red (10μM)completely block capsaicin-induced calcium response and significantly suppressed capsaicin-induced inward current. Then, we recorded AP in DRG neurons induced by electrical stimuli (intensity, 500 pA; duration, 3 ms) under normal solution and after capsaicin perfusion. The data showed that AP amplitude was significantly decreased after capsaicin-induced intracellular calcium increase. In contrast, above treatments have no effects on depolarization or repolarization durations.
3. Spatiotemporal properties of action potential generated in primary sensory neuron can be modified under the state of inflammatory pain
Previous investigation had shown that disequilibrium of calcium homeostasis caused plasticity of AP waveform. Then we asked the question whether AP waveform changed under pathological situations? The potential functional significance of AP plasticity was valuable to be uncovered. Then we used inflammatory pain model to investigate the plasticity of AP waveform under pathological situations.
1) Behavioral tests demonstrated that paw withdrawal thermal latency (PWTL) and paw withdrawal mechanical threshold (PWTL) significantly decreased after subcutaneously injection of CFA (100μl, post 1 d) or melittin (100μg/100μl, post 2 h). In vivo electrophysiological recording tests demonstrated that noxious thermal and mechanical (brush, pressure and noxious pinch) stimuli cause increasing spike firing rates in spinal cord dorsal horn WDR neurons after CFA or melittin peripheral administration. The results showed that peripheral administration of CFA or melittin caused inflammatory pain.
2) Effects of CFA or melittin on the excitabilities of DRG neurons. By using DiI ( 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate17 mg/ml,10μl dissolved in 10% DMSO)s.c. injection, we identified two classes of DRG neurons ( phasic neuron and tonic neuron) innervating the glabrous skin of the hind paw of rat. The data showed that CFA and melittin treatment had different effects on the excitabilities of two classes of DRG neurons. Tonic neuron increased excitabilities through lowering rheobases of AP generation and increasing the number of spike firing, whereas the excitabilities of phasic neuron were not changed by inflammation treatment. The data indicated that noxious information could be transmitted by both nonrhythmic and rhythmic activities of neural coding under inflammatory pain.
3) Effects of CFA or melittin on spatial and temporal parameters of action potentials in two classes of DRG neurons. In tonic neuron, electrical stimuli-evoked(intensity, 50-500 pA; duration, 3 ms; interval, 200 ms) AP amplitude and overshoot were significantly increased, depolarization duration and depolarization duration were shortened after inflammation treatment by CFA or melittin. In phasic neuron, the effects of CFA and melittin were different. After CFA treatment, AP amplitude and overshoot were significantly increased, depolarization duration and repolarization durationare shortened. After melittin treatment , AP amplitude and overshoot were significantly increased, repolarization duration, but not depolarization duration were shortened. Above data indicated that,except for the changing of neuronal excitabilities, DRG neurons changed AP waveforms under inflammatory pain. The variability of AP waveform might be another novel activity of neuronal plasticity.
4) Potential roles of TTX on AP plasticity DRG neuron under inflammatory pain. In DRG neurons of naive rat,TTX (1μM)significantly lengthened depolarization duration and decreased amplitude of AP evoked by electrical stimuli-evoked(intensity, 50-500 pA; duration, 3 ms; interval, 200 ms). However, the effects of TTX on of AP waveform (depolarization duration and amplitude) were significantly decreased after inflammation treatment by CFA (100μl, post 1 d) or melittin (100μg/100μl, post 2 h). Above data indicated that TTX-R sodium current components contributing to AP amplitude and depolarization were increased after peripheral inflammation treatment.
4. Up-regulation of TIX-R sodium channels is one of important underlying mechanisms of spatial plasticity in primary sensory neurons
1) Immunohistochemistry data showed that NaV1.8-positive staining was mainly in medium- and small-sized DRG neurons, whereas NaV1.9-positive staining was only in small-sized DRG neurons in naive rat. After CFA (100μl, post 1 d) or melittin (100μg/100μl, post 2 h) s.c.injection, the mean numbers of NaV1.8-positive neurons increased 93% and 50%, respectively. The mean numbers of NaV1.9-positive neurons increased 74% and 47%, respectively.
2) Western blot data showed that both NaV1.8 and NaV1.9 proteins expressed in low levels in DRG naive rat. After CFA or melittin treatment, the relative intensities of NaV1.8 protein increased with 84.25% and 55.06%, respectively. NaV1.9 protein increased with 51.97% and 37.64%, respectively.
3) RT-PCR data showed that both NaV1.8 and NaV1.9 gene expressed in low levels in DRG naive rat. After CFA or melittin treatment, the relative intensities of NaV1.8 gene increased with 116.02% and 82.01%, respectively. NaV1.9 gene increased with 87.86% and 43.36%, respectively.
4) Patch clamp data showed that after CFA or melittin treatment, the densitity of NaV1.8 current was significantly increased with 55.12% and 64.48%, respectively. The densitity of NaV1.9 current was also significantly increased with 87.08% and 70.30%, respectively.
Above data indicated that up-regulation of NaV1.8 and NaV1.9 in gene and protein levels and facilitation of channel function were mainly responsible for the spatial plasticity of AP in DRG neuron.
5. Pathological significance of spatiotemporal plasticity of primary sensory neuronal action potentials
1) Immunohistochemistry and western blotdata showed that three days after twice daily i.t. administration (45μg) of fluorescence (Carboxyfluorescein,FAM)-labled antisense oligodeoxynucleotide (AS OND) to NaV1.8 and NaV1.9 could significantly decreased NaV1.8 or NaV1.9 up-regulation induced by CFA (100μl, post 1 d) or melittin (100μg/100μl, post 2 h) in both the number of positive neurons and protein levels. These data indicated that AS OND could significantly inhibited the NaV1.8 and NaV1.9 up-regulation induced by inflammation.
2) Behavioral tests showed three days after twice daily i.t. administration (45μg) of AS OND to NaV1.8 and NaV1.9 had different effects on inflammatory pain induced by CFA (100μl, post 1 d) or melittin (100μg/100μl, post 2 h). Comparison with mismatch (MM) treatment, i.t. administration of NaV1.8 AS significantly inhibited CFA, but not melittin, -induced heat and mechanical hypersensitivity. Conversely , NaV1.9 AS treatment significantly inhibited heat hypersensitivity induced by melittin,but not by CFA. Above results indicated that NaV1.8 mainly contributed to heat and mechanical hypersensitivity induced by CFA-induced chronic inflammatory pain, whereas NaV1.9 mainly contributed to heat hypersensitivity induced by melittin-induced acute inflammatory pain.
3) Patch clamp data showed that three days after twice daily i.t. administration (45μg) of AS OND to NaV1.8 and NaV1.9 had different effects on AP waveform generated in inflammatory pain induced by CFA (100μl, post 1 d) or melittin (100μg/100μl, post 2 h). NaV1.8 AS, but not NaV1.9 AS significantly inhibited AP amplitude and lengthened depolarization duration after CFA treatment. Conversely, NaV1.9 AS, but not NaV1.8 AS significantly inhibited AP amplitude and shortened depolarization duration after melittin treatment.
Above data indicated that plasticity of AP waveform generated in DRG neurons had different molecular basis and was close relevant to the different pathological states of inflammatory pain. During the process of CFA-induced chronic inflammatory pain, NaV1.8 contributed to pain hypersensitivity through changing neuronal AP waveform. During the process of melittin-induced acute inflammatory pain, NaV1.9 contributed to pain hypersensitivity through changing neuronal AP spatial plasticity.
Conclusions
1. Under certain condition (such as, spontaneous discharge or constant stimulation), action potential (AP) waveform is“all or none”. However, AP spatial parameter (amplitude) and temporal parameters (depolarization duration and repolarization duration) of DRG neuron are not identical given different stimulus intensities, demonstrating action potentials generated in primary sensory neuron perform spatiotemporal plasticity.
2. Disequilibrium of calcium homeostasis around DRG neuron decreases AP amplitude and lengthens duration in some degree, demonstrating that calcium homeostasis is required for the maintainance of“all or none”property in primary sensory neuron.
3. Under inflammatory pain, spatiotemporal plasticity of AP appears in two classes of DRG neurons. Tonic neuron increases excitabilities through lowering rheobases of AP generation and increasing the number of spike firing and contributes to the transmission of noxious information. However, phasic neuron showing no changes in excitabilities may transmit noxious information through APs with increased amplitude and shortened duration.
4. Up-regulation of NaV1.8 and NaV1.9 in both gene and protein levels and facilitation of channel function are mainly responsible for the spatiotemporal plasticity of AP generated in DRG neuron.
5. Spatial plasticity of AP generated in DRG neurons has different molecular basis and is closely relevant to the different pathological states of inflammatory pain. During the process of CFA-induced chronic inflammatory pain, NaV1.8 contributes to heat and mechanical hypersensitivity through changing neuronal AP spatial parameter. During the process of melittin-induced acute inflammatory pain, NaV1.9 contributes to heat hypersensitivity through changing neuronal AP AP spatial parameter.
引文
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