Roles of specific glutamate receptor subunits in the cerebellar long-term depression

小脑长期抑郁(LTD)是运动学习的一种候选机制。它是颗粒神经元-浦肯野神经元(G-P)突触传递效率的长期下降，由下橄榄核神经元和颗粒神经元的反复联合激活引起。我们一直在分析LTD在分离细胞培养准备中的细胞和分子诱导机制，并已证实mGluR1亚型代谢性谷氨酸受体、δ2亚基和IP3参与LTD的诱导已有报道。我们检测了MAP激酶是否参与，发现MAP激酶级联的抑制剂PD98059抑制LTD的诱导，而条件性刺激诱导LTD激活MAP激酶。PD98059抑制了应用mGluR1激动剂对Purkinje细胞产生的内向电流，这表明MAP激酶活性支持mGluR1级联反应。我们还成功地在LTD诱导后一周内通过测量LTD诱导前和LTD诱导后培养的小型EPSCs的幅度来监测突触效率。通过这种方法，我们证明了LTD诱导后两天内突触效应恢复，LTD由早期和晚期组成，其诱导机制是不同的。后期的诱导需要长时间的条件性刺激，依赖于mRNA和蛋白质的合成。为了阐明δ2亚单位在LTD诱导中的作用，利用从δ2基因敲除小鼠制备的培养浦肯野神经元和腺病毒载体表达突变的δ2蛋白的研究正在进行中。

The cerebellar long-term depression (LTD) has been a candidate mechanism for motor learning. It is the long-lasting reduction of transmission efficacy at the granule neuron-Purkinje neuron (G-P) synapses induced by repeated conjunctive activation of an inferior olivary neuron and granule neurons. We have been analyzing cellular and molecular induction mechanism of the LTD in dissociated cell culture preparation, and have demonstrated the involvement of mGluR1 subtype of metabotropic glutamate receptors, δ2 subunit of ionotropic glutamate receptors and IP3 in the LTD induction have been reported. We examined whether MAP kinase is involved or not, and have found that PD98059, an inhibitor of MAP kinase cascade, inhibits the LTD induction, and that the conditioning stimulation inducing LTD activates MAP kinase. PD98059 suppressed the inward current induced by application of a mGluR1 agonist to a Purkinje cell, which suggests that MAP kinase activity supports the mGluR1 cascade. We also succeeded to monitor synaptic efficacy for a week after the LTD induction by measuring the miniature EPSCs amplitude before and after the LTD induction in the culture. By this method, we demonstrated that the synaptic efficacy recovers in two days after the LTD induction and that the LTD is consist of early and late phases whose induction mechanisms are distinct. The late phase required the prolonged conditioning stimulation for the induction and was dependent on both mRNA and protein syntheses. The study to clarify roles of δ2 subunit in the LTD induction is on going, using cultured Purkinje neurons prepared form δ2 knockout mice and the adeno-virus vector to express mutant δ2 proteins.