Nagy Gergely G, Al-Ayyan Muna, Andrew David, Fukaya Masahiro, Watanabe Masahiko, Todd Andrew J
Spinal Cord Group, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom.
J Neurosci. 2004 Jun 23;24(25):5766-77. doi: 10.1523/JNEUROSCI.1237-04.2004.
Glutamate, the principal excitatory neurotransmitter in the spinal cord, acts primarily through AMPA receptors. Although all four AMPA subunits are expressed by spinal neurons, we know little about their distribution at glutamatergic synapses. We used an antigen-unmasking technique to reveal the synaptic distribution of glutamate receptor (GluR) 1-4 subunits with confocal microscopy. After pepsin treatment, punctate staining was seen with antibodies against each subunit: GluR2-immunoreactive puncta were distributed throughout the gray matter, whereas GluR1-immunoreactive puncta were restricted to the dorsal horn and were most numerous in laminas I-II. Punctate staining for GluR3 and GluR4 was found in all laminas but was weak in superficial dorsal horn. Colocalization studies showed that GluR2 was present at virtually all (98%) puncta that were GluR1, GluR3, or GluR4 immunoreactive and that most (>90%) immunoreactive puncta in laminas IV, V, and IX showed GluR2, GluR3, and GluR4 immunoreactivity. Evidence that these puncta represented synaptic receptors was obtained with electron microscopy and by examining the association of GluR2- and GluR1-immunoreactive puncta with glutamatergic boutons (identified with vesicular glutamate transporters or markers for unmyelinated afferents). The great majority (96%) of these boutons were associated with GluR2-immunoreactive puncta. Our findings suggest that GluR2 is almost universally present at AMPA-containing synapses, whereas GluR1 is preferentially associated with primary afferent terminals. We also found a substantial, rapid increase in staining for synaptic GluR1 subunits phosphorylated on the S845 residue in the ipsilateral dorsal horn after peripheral noxious stimulation. This finding demonstrates plastic changes, presumably contributing to central sensitization, at the synaptic level.
谷氨酸是脊髓中的主要兴奋性神经递质，主要通过AMPA受体发挥作用。尽管脊髓神经元表达所有四种AMPA亚基，但我们对它们在谷氨酸能突触处的分布了解甚少。我们使用抗原暴露技术，通过共聚焦显微镜揭示谷氨酸受体（GluR）1 - 4亚基的突触分布。胃蛋白酶处理后，用针对每个亚基的抗体可见点状染色：GluR2免疫反应性点状分布于整个灰质，而GluR1免疫反应性点状局限于背角，在I - II层最为密集。GluR3和GluR4的点状染色见于所有层，但在背角浅层较弱。共定位研究表明，GluR2几乎存在于所有（98%）GluR1、GluR3或GluR4免疫反应性点状处，并且IV、V和IX层中大多数（>90%）免疫反应性点状显示GluR2、GluR3和GluR4免疫反应性。通过电子显微镜以及检查GluR2和GluR1免疫反应性点状与谷氨酸能终扣（用囊泡谷氨酸转运体或无髓传入标记物鉴定）的关联，获得了这些点状代表突触受体的证据。这些终扣中的绝大多数（96%）与GluR2免疫反应性点状相关。我们的研究结果表明，GluR2几乎普遍存在于含AMPA的突触处，而GluR1优先与初级传入终末相关。我们还发现，外周伤害性刺激后，同侧背角中S845残基磷酸化的突触GluR1亚基染色显著快速增加。这一发现证明了突触水平的可塑性变化，可能有助于中枢敏化。
