Presynaptically localized cyclic GMP-dependent protein kinase 1 is a key determinant of spinal synaptic potentiation and pain hypersensitivity.
Presynaptically localized cyclic GMP-dependent protein kinase 1 is a key determinant of spinal synaptic potentiation and pain hypersensitivity.
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DOI：
10.1371/journal.pbio.1001283
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发表时间：
2012
期刊：
影响因子：
9.8
通讯作者：
Kuner R
中科院分区：
文献类型：
--
作者：
Luo C;Gangadharan V;Bali KK;Xie RG;Agarwal N;Kurejova M;Tappe-Theodor A;Tegeder I;Feil S;Lewin G;Polgar E;Todd AJ;Schlossmann J;Hofmann F;Liu DL;Hu SJ;Feil R;Kuner T;Kuner R
小鼠的电生理和行为实验表明，cGMP依赖性激酶放大了外周疼痛传感器的神经递质释放，增强了脊髓突触，并导致夸张的疼痛。突触长时程增强（Synaptic long-term potentiation，LTP）是脊髓神经元将疼痛特异性信息从外周直接传递到大脑的一种机制。以前的研究在功能上涉及NMDA受体-NO通路和下游第二信使cGMP，在这些过程中。由于cGMP可以广泛地影响多种离子通道、激酶和磷酸二酯酶，在突触前和突触后，cGMP介导脊髓LTP的靶点的精确身份、它们的作用机制以及它们在脊髓回路中的位置仍然不清楚。在这里，我们发现，蛋白激酶G1（PKG-I）定位于突触前的伤害性感受器末梢在脊髓LTP的表达中起着至关重要的作用。使用Cre-lox P系统，我们产生了伤害感受器特异性敲除小鼠，其在脊髓伤害感受器的突触前末端特异性缺乏PKG-I，但在突触后神经元或其他地方没有（SNS-PKG-I−/−小鼠）。膜片钳记录显示，在SNS-PKG-I−/−小鼠中，在伤害感受器和投射到导水管周围灰质（PAG）的脊髓神经元之间的已识别突触处，活动诱导的LTP被完全消除，尽管基础突触传递不受影响。突触失败率和成对脉冲比的分析表明，突触前PKG-I在调节神经递质释放的可能性中起作用。肌醇1，4，5-三磷酸受体1和肌球蛋白轻链激酶被招募为伤害性神经元中突触前PKG-I的关键磷酸化靶点。最后，体内行为分析显示，在几种活动诱导的伤害性超敏反应模型中，SNS-PKG-I−/−小鼠存在明显缺陷，药理学研究确定了PKG-I在伤害性感受器脊髓末端表达的明确贡献。因此，我们的研究结果表明，突触前机制，涉及增加释放概率从伤害性感受器是可操作的突触LTP的表达脊髓PAG投射神经元和PKG-I定位在突触前伤害性感受器终端在这一过程中起着至关重要的作用，以调节疼痛的敏感性。疼痛是一种重要的生理功能，可以保护我们的身体免受伤害。疼痛感受神经元，称为伤害感受器，将有害刺激转化为电信号，并通过脊髓将此信息传递到大脑。当伤害感受器被持续激活时，例如在受伤后，它们与脊髓中神经元的连接在称为突触长时程增强（LTP）的过程中发生改变。在这项研究中，我们研究了LTP的分子和细胞机制的伤害性感受器到脊髓神经元的突触。我们在小鼠中使用了多种实验方法，从遗传到行为，以表明这种形式的LTP涉及突触前事件，当伤害感受器被重复激活时，这些事件会在伤害感受器中展开。特别地，由第二信使cGMP激活的酶（称为蛋白激酶G-I）使突触前蛋白磷酸化并增加神经递质从脊髓中的伤害感受器末端的释放。当我们在基因上沉默蛋白激酶G-I或阻断其在伤害感受器中的激活时，炎症性疼痛在行为水平上显著降低。这些结果阐明了病理性疼痛的基本机制，并为新的治疗方法铺平了道路。
Electrophysiological and behavioral experiments in mice reveal that a cGMP-dependent kinase amplifies neurotransmitter release from peripheral pain sensors, potentiates spinal synapses, and leads to exaggerated pain. Synaptic long-term potentiation (LTP) at spinal neurons directly communicating pain-specific inputs from the periphery to the brain has been proposed to serve as a trigger for pain hypersensitivity in pathological states. Previous studies have functionally implicated the NMDA receptor-NO pathway and the downstream second messenger, cGMP, in these processes. Because cGMP can broadly influence diverse ion-channels, kinases, and phosphodiesterases, pre- as well as post-synaptically, the precise identity of cGMP targets mediating spinal LTP, their mechanisms of action, and their locus in the spinal circuitry are still unclear. Here, we found that Protein Kinase G1 (PKG-I) localized presynaptically in nociceptor terminals plays an essential role in the expression of spinal LTP. Using the Cre-lox P system, we generated nociceptor-specific knockout mice lacking PKG-I specifically in presynaptic terminals of nociceptors in the spinal cord, but not in post-synaptic neurons or elsewhere (SNS-PKG-I−/− mice). Patch clamp recordings showed that activity-induced LTP at identified synapses between nociceptors and spinal neurons projecting to the periaqueductal grey (PAG) was completely abolished in SNS-PKG-I−/− mice, although basal synaptic transmission was not affected. Analyses of synaptic failure rates and paired-pulse ratios indicated a role for presynaptic PKG-I in regulating the probability of neurotransmitter release. Inositol 1,4,5-triphosphate receptor 1 and myosin light chain kinase were recruited as key phosphorylation targets of presynaptic PKG-I in nociceptive neurons. Finally, behavioural analyses in vivo showed marked defects in SNS-PKG-I−/− mice in several models of activity-induced nociceptive hypersensitivity, and pharmacological studies identified a clear contribution of PKG-I expressed in spinal terminals of nociceptors. Our results thus indicate that presynaptic mechanisms involving an increase in release probability from nociceptors are operational in the expression of synaptic LTP on spinal-PAG projection neurons and that PKG-I localized in presynaptic nociceptor terminals plays an essential role in this process to regulate pain sensitivity. Pain is an important physiological function that protects our body from harm. Pain-sensing neurons, called nociceptors, transduce harmful stimuli into electrical signals and transmit this information to the brain via the spinal cord. When nociceptors are persistently activated, such as after injury, the connections they make with neurons in the spinal cord are altered in a process called synaptic long-term potentiation (LTP). In this study, we examine the molecular and cellular mechanisms of LTP at synapses from nociceptors onto spinal neurons. We use multiple experimental approaches in mice, from genetic to behavioural, to show that this form of LTP involves presynaptic events that unfold in nociceptors when they are repetitively activated. In particular, an enzyme activated by the second messenger cGMP, referred to as Protein Kinase G-I, phosphorylates presynaptic proteins and increases the release of neurotransmitters from nociceptor endings in the spinal cord. When we genetically silence Protein Kinase G-I or block its activation in nociceptors, inflammatory pain is markedly reduced at the behavioural level. These results clarify basic mechanisms of pathological pain and pave the way for new therapeutic approaches.
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