Howorth Patrick W, Teschemacher Anja G, Pickering Anthony E
Department of Physiology, University of Bristol, Bristol, BS8 1TD, UK.
J Comp Neurol. 2009 Jan 10;512(2):141-57. doi: 10.1002/cne.21879.
The spinal dorsal horn receives a dense innervation of noradrenaline-containing fibers that originate from pontine neurons in the A5, locus coeruleus (LC), and A7 cell groups. These pontospinal neurons are believed to constitute a component of the endogenous analgesic system. We used an adenoviral vector with a catecholaminergic-selective promoter (AVV-PRS) to retrogradely label the noradrenergic neurons projecting to the lumbar (L4-L5) dorsal horn with enhanced green fluorescent protein (EGFP) or monomeric red fluorescent protein (mRFP). Retrogradely labeled neurons (145 +/- 12, n = 14) were found in A5-12%, LC-80% and A7-8% after injection of AVV-PRS-EGFP to the dorsal horn of L4-L5. These neurons were immunopositive for dopamine beta-hydroxylase, indicating that they were catecholaminergic. Retrograde labeling was optimal 7 days after injection, persisted for over 4 weeks, and was dependent on viral vector titer. The spinal topography of the noradrenergic projection was examined using EGFP- and mRFP-expressing adenoviral vectors. Pontospinal neurons provide bilateral innervation of the cord and there was little overlap in the distribution of neurons projecting to the cervical and lumbar regions. The axonal arbor of the pontospinal neurons was visualized with GFP immunocytochemistry to show projections to the inferior olive, cerebellum, thalamus, and cortex but not to the hippocampus or caudate putamen. Formalin testing evoked c-fos expression in these pontospinal neurons, suggesting that they were nociresponsive (A5-21%, LC-16%, and A7-26%, n = 8). Thus, we have developed a viral vector-based strategy to selectively, retrogradely target the pontospinal noradrenergic neurons that are likely to be involved in the descending control of nociception.
脊髓背角接受来自A5、蓝斑（LC）和A7细胞群中脑桥神经元的去甲肾上腺素能纤维的密集支配。这些脑桥脊髓神经元被认为是内源性镇痛系统的一个组成部分。我们使用带有儿茶酚胺能选择性启动子的腺病毒载体（AVV-PRS），用增强型绿色荧光蛋白（EGFP）或单体红色荧光蛋白（mRFP）逆行标记投射到腰段（L4-L5）背角的去甲肾上腺素能神经元。将AVV-PRS-EGFP注射到L4-L5背角后，在A5区域发现12%的逆行标记神经元，LC区域发现80%，A7区域发现8%（n = 14）。这些神经元对多巴胺β-羟化酶呈免疫阳性，表明它们是儿茶酚胺能的。注射后7天逆行标记效果最佳，持续超过4周，且依赖于病毒载体滴度。使用表达EGFP和mRFP的腺病毒载体检查去甲肾上腺素能投射的脊髓拓扑结构。脑桥脊髓神经元对脊髓提供双侧支配，投射到颈段和腰段区域的神经元分布几乎没有重叠。通过GFP免疫细胞化学观察脑桥脊髓神经元的轴突分支，显示其投射到下橄榄核、小脑、丘脑和皮质，但不投射到海马体或尾状壳核。福尔马林测试诱发这些脑桥脊髓神经元中c-fos表达，表明它们对伤害性刺激有反应（A5区域为21%，LC区域为16%，A7区域为26%，n = 8）。因此，我们开发了一种基于病毒载体的策略，以选择性地、逆行靶向可能参与伤害性感受下行控制的脑桥脊髓去甲肾上腺素能神经元。
