Polgár Erika, Watanabe Masahiko, Hartmann Bettina, Grant Seth Gn, Todd Andrew J
Spinal Cord Group, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
Mol Pain. 2008 Jan 23;4:5. doi: 10.1186/1744-8069-4-5.
Glutamate receptors of the AMPA type (AMPArs) mediate fast excitatory transmission in the dorsal horn and are thought to underlie perception of both acute and chronic pain. They are tetrameric structures made up from 4 subunits (GluR1-4), and subunit composition determines properties of the receptor. Antigen retrieval with pepsin can be used to reveal the receptors with immunocytochemistry, and in this study we have investigated the subunit composition at synapses within laminae I-III of the dorsal horn. In addition, we have compared staining of AMPArs with that for PSD-95, a major constituent of glutamatergic synapses. We also examined tissue from knock-out mice to confirm the validity of the immunostaining.
As we have shown previously, virtually all AMPAr-immunoreactive puncta were immunostained for GluR2. In laminae I-II, approximately 65% were GluR1-positive and approximately 60% were GluR3-positive, while in lamina III the corresponding values were 34% (GluR1) and 80% (GluR3). Puncta stained with antibody against the C-terminus of GluR4 (which only detects the long form of this subunit) made up 23% of the AMPAr-containing puncta in lamina I, approximately 8% of those in lamina II and 46% of those in lamina III. Some overlap between GluR1 and GluR3 was seen in each region, but in lamina I GluR1 and GluR4 were present in largely non-overlapping populations. The GluR4 puncta often appeared to outline dendrites of individual neurons in the superficial laminae. Virtually all of the AMPAr-positive puncta were immunostained for PSD-95, and 98% of PSD-95 puncta contained AMPAr-immunoreactivity. Staining for GluR1, GluR2 and GluR3 was absent in sections from mice in which these subunits had been knocked out, while the punctate staining for PSD-95 was absent in mice with a mutation that prevents accumulation of PSD-95 at synapses.
Our results suggest that virtually all glutamatergic synapses in laminae I-III of adult rat spinal cord contain AMPArs. They show that synapses in laminae I-II contain GluR2 together with GluR1 and/or GluR3, while the long form of GluR4 is restricted to specific neuronal populations, which may include some lamina I projection cells. They also provide further evidence that immunostaining for AMPAr subunits following antigen retrieval is a reliable method for detecting these receptors at glutamatergic synapses.
AMPA型谷氨酸受体（AMPArs）介导脊髓背角的快速兴奋性传递，被认为是急性和慢性疼痛感知的基础。它们是由4个亚基（GluR1 - 4）组成的四聚体结构，亚基组成决定受体的特性。用胃蛋白酶进行抗原修复可用于免疫细胞化学显示这些受体，在本研究中，我们调查了脊髓背角I - III层内突触处的亚基组成。此外，我们比较了AMPArs与PSD - 95（谷氨酸能突触的主要成分）的染色情况。我们还检查了基因敲除小鼠的组织以确认免疫染色的有效性。
如我们之前所示，几乎所有AMPAr免疫反应性斑点都对GluR2进行了免疫染色。在I - II层中，约65%为GluR1阳性，约60%为GluR3阳性，而在III层中，相应的值分别为34%（GluR1）和80%（GluR3）。用针对GluR4 C末端的抗体染色的斑点（该抗体仅检测该亚基的长形式）在I层中占含AMPAr斑点的23%，在II层中约占8%，在III层中占46%。在每个区域都观察到GluR1和GluR3之间有一些重叠，但在I层中，GluR1和GluR4存在于大部分不重叠的群体中。GluR4斑点通常似乎勾勒出浅层层中单个神经元的树突。几乎所有AMPAr阳性斑点都对PSD - 95进行了免疫染色，并且98%的PSD - 95斑点含有AMPAr免疫反应性。在敲除这些亚基的小鼠的切片中，未观察到GluR1、GluR2和GluR3的染色，而在具有阻止PSD - 95在突触处积累的突变的小鼠中，未观察到PSD - 95的点状染色。
我们的结果表明，成年大鼠脊髓I - III层中几乎所有谷氨酸能突触都含有AMPArs。结果显示，I - II层中的突触含有GluR2以及GluR1和/或GluR3，而GluR4的长形式仅限于特定的神经元群体，这可能包括一些I层投射细胞。它们还进一步证明，抗原修复后对AMPAr亚基进行免疫染色是在谷氨酸能突触处检测这些受体的可靠方法。
