Vallano Mary Lou, Beaman-Hall Carol M, Bui Cuong J, Middleton Frank A
Department of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY 13210, USA.
Neuropharmacology. 2006 May;50(6):651-60. doi: 10.1016/j.neuropharm.2005.11.012. Epub 2006 Jan 18.
Presynaptic terminals of cerebellar granule neurons are primary targets of cannabinoids, which act through type 1 G alpha(i/o)-coupled cannabinoid receptors (CB1) to modulate glutamate release. To study CB1 signaling investigators use primary cultures of granule neurons, typically grown in medium supplemented with elevated KCl to improve long-term survival. Herein, we demonstrate that CB1 expression and signaling are perturbed under these conditions. Specifically, immunochemical and RT-PCR assays indicate that depolarizing growth conditions decrease CB1 protein, mRNA and CB1-mediated inhibition of adenylyl cyclase compared to cultures grown in physiologic medium containing 5mM KCl. Depolarization-dependent downregulation of CB1 mRNA, like survival, is attenuated by L-type VDCC antagonists but not the Na(+)-channel antagonist, tetrodotoxin. Comparison of oligonucleotide microarrays from cultures grown in 5mM versus 25 mM KCl confirms that depolarization reduces CB1 mRNA, but not mRNAs encoding several G-protein subunits or adenylyl cyclases. However, significant alterations in synaptic signaling proteins that likely lie downstream of CB1 are observed, including K(+) channels, alpha-neurexins, cAMP-GEFII, Munc13-3, secretogranin and synaptotagmin. These findings make a compelling argument to adopt cultures grown in 5mM KCl for future study of CB1 signaling in granule neurons. Further, they suggest that a depolarization and Ca(2+)-dependent signaling pathway represses CB1 gene transcription.
小脑颗粒神经元的突触前终末是大麻素的主要作用靶点，大麻素通过1型与Gα(i/o)偶联的大麻素受体（CB1）发挥作用，调节谷氨酸释放。为了研究CB1信号传导，研究人员使用颗粒神经元的原代培养物，这些培养物通常在添加了高浓度氯化钾的培养基中生长，以提高长期存活率。在此，我们证明在这些条件下CB1的表达和信号传导会受到干扰。具体而言，免疫化学和RT-PCR分析表明，与在含有5mM氯化钾的生理培养基中生长的培养物相比，去极化生长条件会降低CB1蛋白、mRNA以及CB1介导的腺苷酸环化酶抑制作用。CB1 mRNA的去极化依赖性下调与存活率一样，会被L型电压依赖性钙通道拮抗剂减弱，但不会被钠通道拮抗剂河豚毒素减弱。对在5mM和25mM氯化钾中生长的培养物的寡核苷酸微阵列进行比较，证实去极化会降低CB1 mRNA，但不会降低编码几种G蛋白亚基或腺苷酸环化酶的mRNA。然而，观察到可能位于CB1下游的突触信号蛋白有显著变化，包括钾通道、α-神经连接素、cAMP-GEFII、Munc13-3、分泌粒蛋白和突触结合蛋白。这些发现有力地支持在未来对颗粒神经元中CB1信号传导的研究中采用在5mM氯化钾中生长的培养物。此外，它们表明去极化和钙依赖性信号通路会抑制CB1基因转录。
