Site-Specific and Time-Dependent Activation of the Endocannabinoid System after Transection of Long-Range Projections

BackgroundAfter focal neuronal injury the endocannabinioid system becomes activated and protects or harms neurons depending on cannabinoid derivates and receptor subtypes. Endocannabinoids (eCBs) play a central role in controlling local responses and influencing neural plasticity and survival. However, little is known about the functional relevance of eCBs in long-range projection damage as observed in stroke or spinal cord injury (SCI). MethodsIn rat organotypic entorhino-hippocampal slice cultures (OHSC) as a relevant and suitable model for investigating projection fibers in the CNS we performed perforant pathway transection (PPT) and subsequently analyzed the spatial and temporal dynamics of eCB levels. This approach allows proper distinction of responses in originating neurons (entorhinal cortex), areas of deafferentiation/anterograde axonal degeneration (dentate gyrus) and putative changes in more distant but synaptically connected subfields (cornu ammonis (CA) 1 region). ResultsUsing LC-MS/MS, we measured a strong increase in arachidonoylethanolamide (AEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) levels in the denervation zone (dentate gyrus) 24 hours post lesion (hpl), whereas entorhinal cortex and CA1 region exhibited little if any changes. NAPE-PLD, responsible for biosynthesis of eCBs, was increased early, whereas FAAH, a catabolizing enzyme, was up-regulated 48hpl. ConclusionNeuronal damage as assessed by transection of long-range projections apparently provides a strong time-dependent and area-confined signal for de novo synthesis of eCB, presumably to restrict neuronal damage. The present data underlines the importance of activation of the eCB system in CNS pathologies and identifies a novel site-specific intrinsic regulation of eCBs after long-range projection damage.背景：局灶性神经元损伤后，内源性大麻素系统（endocannabinoid system）会被激活，其对神经元的保护或损伤效应取决于大麻素衍生物及受体亚型。内源性大麻素（endocannabinoids, eCBs）在调控局部应答、影响神经可塑性与神经元存活过程中发挥核心作用。然而，目前对于内源性大麻素在卒中或脊髓损伤（spinal cord injury, SCI）所致的长距离投射损伤中的功能相关性仍知之甚少。方法：以大鼠器官型内嗅皮层-海马脑片培养物（organotypic entorhino-hippocampal slice cultures, OHSC）——这是研究中枢神经系统（central nervous system, CNS）投射纤维的经典且适用的实验模型——为研究对象，我们实施了穿通通路横断术（perforant pathway transection, PPT），随后分析了内源性大麻素水平的时空动态变化。该实验方案可清晰区分始发神经元（内嗅皮层）、去传入/顺行性轴突退变区域（齿状回）以及更远端但存在突触连接的亚区（海马角（cornu ammonis, CA）1区）的应答变化。结果：采用液相色谱-串联质谱（LC-MS/MS）检测，我们发现损伤后24小时（hours post lesion, hpl），去神经支配区域（齿状回）内的花生四烯酸乙醇胺（arachidonoylethanolamide, AEA）、油酰乙醇胺（oleoylethanolamide, OEA）以及棕榈酰乙醇胺（palmitoylethanolamide, PEA）水平显著升高；而内嗅皮层与CA1区的相关水平几乎无变化。负责内源性大麻素生物合成的NAPE-PLD表达早期上调，而代谢分解酶FAAH则在损伤后48小时（48hpl）出现上调。结论：通过长距离投射横断所模拟的神经元损伤，可明显触发依赖于时间且局限于特定区域的内源性大麻素从头合成信号，其潜在作用为限制神经元损伤程度。本研究数据凸显了内源性大麻素系统在中枢神经系统病理过程中的激活意义，并揭示了长距离投射损伤后内源性大麻素的一种全新的位点特异性内在调控机制。