Matsumura Shinji, Taniguchi Wataru, Nishida Kazuhiko, Nakatsuka Terumasa, Ito Seiji
Department of Medical Chemistry, Kansai Medical University, 2-5-1 Shin-machi, Hirakata, 573-1010, Japan.
Eur J Neurosci. 2015 Apr;41(7):989-97. doi: 10.1111/ejn.12837. Epub 2015 Jan 29.
Two-photon microscopy imaging has recently been applied to the brain to clarify functional and structural synaptic plasticity in adult neural circuits. Whereas the pain system in the spinal cord is phylogenetically primitive and easily exhibits behavioral changes such as hyperalgesia in response to inflammation, the structural dynamics of dendrites has not been analysed in the spinal cord mainly due to tissue movements associated with breathing and heart beats. Here we present experimental procedures to prepare the spinal cord sufficiently to follow morphological changes of neuronal processes in vivo by using two-photon microscopy and transgenic mice expressing fluorescent protein specific to the nervous system. Structural changes such as the formation of spine-like structures and swelling of dendrites were observed in the spinal dorsal horn within 30 min after the multiple-site injections of complete Freund's adjuvant (a chemical irritant) to a leg, and these changes continued for 5 h. Both AMPA and N-methyl-D-aspartate receptor antagonists, and gabapentin, a presynaptic Ca(2+) channel blocker, completely suppressed the inflammation-induced structural changes in the dendrites in the spinal dorsal horn. The present study first demonstrated by in vivo two-photon microscopy imaging that structural synaptic plasticity occurred in the spinal dorsal horn immediately after the injection of complete Freund's adjuvant and may be involved in inflammatory pain. Furthermore, acute inflammation-associated structural changes in the spinal dorsal horn were shown to be mediated by glutamate receptor activation.
双光子显微镜成像技术最近已应用于大脑，以阐明成年神经回路中的功能和结构突触可塑性。脊髓中的疼痛系统在系统发育上较为原始，容易表现出行为变化，如对炎症产生痛觉过敏，然而，由于与呼吸和心跳相关的组织运动，脊髓中树突的结构动力学尚未得到分析。在此，我们介绍了一些实验步骤，通过使用双光子显微镜和表达神经系统特异性荧光蛋白的转基因小鼠，充分准备脊髓，以追踪体内神经元突起的形态变化。在将完全弗氏佐剂（一种化学刺激物）多点注射到腿部后30分钟内，在脊髓背角观察到了诸如棘状结构形成和树突肿胀等结构变化，这些变化持续了5小时。AMPA和N-甲基-D-天冬氨酸受体拮抗剂以及突触前Ca(2+)通道阻滞剂加巴喷丁，均完全抑制了炎症诱导的脊髓背角树突结构变化。本研究首次通过体内双光子显微镜成像证明，在注射完全弗氏佐剂后，脊髓背角立即发生了结构突触可塑性，并且可能与炎性疼痛有关。此外，急性炎症相关的脊髓背角结构变化被证明是由谷氨酸受体激活介导的。
