Addiction Biology Unit, Molecular Targets and Medications Discovery Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, 21224, USA.
Synaptic Plasticity Section, Cellular Neurobiology Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, 21224, USA.
Acta Pharmacol Sin. 2019 Mar;40(3):398-409. doi: 10.1038/s41401-018-0037-3. Epub 2018 Jul 2.
Cannabinoid CB receptors are highly expressed in the brain and functionally modulate presynaptic neurotransmitter release, while cannabinoid CB receptors (CBRs) were initially identified in the spleen and regarded as peripheral cannabinoid receptors. Recently, growing evidence indicates the presence of functional CBRs in the brain. However, this finding is disputed because of the specificity of CBR antibody signals. We used two strains of currently available partial CB-knockout (CB-KO) mice as controls, four anti-rat or anti-mouse CBR antibodies, and mRNA quantification to further address this issue. Western blot assays using the four antibodies detected a CBR-like band at ~40 kD in both the brain and spleen. Notably, more bands were detected in the brain than in the spleen, and specific immune peptides blocked band detection. Immunohistochemical assays also detected CB-like immunostaining in mouse midbrain dopamine neurons. CBR deletion in CB-KO mice may reduce or leave CBR-like immunoreactivity unaltered depending on antibody epitope. Antibodies with epitopes at the receptor-deleted region detected a significant reduction in CBR band density and immunostaining in N-terminal-deleted Deltagen and C-terminal-deleted Zimmer strain CB-KO mice. Other antibodies with epitopes at the predicted receptor-undeleted regions detected similar band densities and immunostaining in wild-type and CB-KO mice. Quantitative RT-PCR assays detected CB mRNA expression using probes that targeted upstream or downstream gene sequences but not the probe that targeted the gene-deleted sequence in Deltagen or Zimmer CB-KO mice. These findings suggest that none of the tested four polyclonal antibodies are highly mouse CBR-specific. Non-specific binding may be related to the expression of mutant or truncated CBR-like proteins in partial CB-KO mice and the use of anti-rat CB antibodies because the epitopes are different between rat and mouse CBRs.
大麻素 CB 受体在大脑中高度表达，并对突触前神经递质释放具有功能性调节作用，而大麻素 CB 受体 (CBR) 最初在脾脏中被鉴定，并被认为是外周大麻素受体。最近，越来越多的证据表明大脑中存在功能性 CBR。然而，由于 CBR 抗体信号的特异性，这一发现存在争议。我们使用两种现有的部分 CB 敲除 (CB-KO) 小鼠作为对照，四种抗大鼠或抗小鼠 CBR 抗体，并通过 mRNA 定量进一步解决这个问题。使用这四种抗体的 Western blot 检测在大脑和脾脏中均检测到约 40kD 的 CBR 样带。值得注意的是，大脑中检测到的带比脾脏中多，并且特异性免疫肽阻断了带的检测。免疫组织化学检测也在小鼠中脑多巴胺神经元中检测到类似 CB 的免疫染色。CB-KO 小鼠中的 CBR 缺失可能会根据抗体表位减少或使 CBR 样免疫反应保持不变。在 N 端缺失的 Deltagen 和 C 端缺失的 Zimmer 株 CB-KO 小鼠中，针对受体缺失区域的抗体检测到 CBR 带密度和免疫染色的显著减少。针对预测的受体未缺失区域的其他抗体在野生型和 CB-KO 小鼠中检测到相似的带密度和免疫染色。使用针对基因缺失序列而不是针对 Deltagen 或 Zimmer CB-KO 小鼠中的基因缺失序列的探针进行的定量 RT-PCR 检测显示 CB mRNA 表达。这些发现表明，四种测试的多克隆抗体都不是高度特异性的小鼠 CBR。非特异性结合可能与部分 CB-KO 小鼠中突变体或截短的 CBR 样蛋白的表达以及使用抗大鼠 CB 抗体有关，因为大鼠和小鼠 CBR 之间的表位不同。
