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

小脑长期抑郁（LTD）被认为是运动学习的潜在机制。它是由下橄榄神经元和颗粒神经元的反复连接激活引起的颗粒神经元-浦肯野神经元（G-P）突触传递效率的长期降低。我们在解离细胞培养制备中分析了LTD的细胞和分子诱导机制，并证实了代谢性谷氨酸受体的mGluR1亚型、嗜离子性谷氨酸受体的δ2亚基和IP3参与了LTD的诱导。我们研究了MAP激酶是否参与其中，发现MAP激酶级联抑制剂PD98059抑制LTD的诱导，而诱导LTD的条件刺激激活MAP激酶。PD98059抑制了mGluR1激动剂对浦肯野细胞诱导的内向电流，这表明MAP激酶活性支持mGluR1级联反应。我们还通过在培养中测量LTD诱导前后的微型EPSCs振幅，成功地监测LTD诱导后一周的突触效能。通过这种方法，我们证明了突触效能在LTD诱导后2天恢复，LTD分为早期和晚期，其诱导机制不同。后期需要长时间的条件刺激来诱导，并且依赖于mRNA和蛋白质的合成。利用δ2敲除小鼠制备的浦肯野神经元和腺病毒载体表达突变体δ2蛋白，进一步阐明δ2亚基在LTD诱导中的作用。

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.