小脳平行線維（PF）―プルキンエ細胞（PC）シナプスに局在するδ2型グルタミン酸受容体（GluD2）について、①同シナプスの長期可塑性（LTD）に必須であること、②特に幼若期において同シナプスの活動に応じてバーグマングリアから放出されるDセリンがリガンドとして結合することでLTDを誘導すること、近年の構造解析から、③Dセリン結合部位付近にCa結合部位があり、Dセリンに対する親和性に影響すること、が知られている。すなわち、PF―PCシナプスの間隙の神経活動依存的なCa動態がDセリン依存性LTDの新規制御機構である可能性がある。本研究は、この可能性について検討を行った。
PF―PCシナプスには、間隙のCa濃度に影響を及ぼす様々な分子が発現している。例えば、電位依存性Caチャネルは主にシナプス前終末（PF）に発現し、活性化すると伝達物質放出をトリガーするが、その際シナプス間隙からCaを細胞内に流入させる。そこで本年度は、PF―PCシナプスにおけるLTDに対して電位依存性Caチャネルが果たす役割を検討した。
N型Caチャネルを薬理学的に阻害したところ、定常状態におけるシナプス伝達が有意に減少したが、LTDはコントロール条件と同程度に誘導することができた。
次に、R型Caチャネルを薬理学的に阻害したところ、定常状態におけるシナプス伝達は同様に減少したが、驚くべき事にLTDも有意に抑制された。つまり、R型Caチャネルは単に伝達物質放出に関わるだけでなく、LTD誘導にも重要な役割を担っていることが示唆された。更に、GluD2欠損マウスではR型Caチャネルの局在が変化していることも明らかになった。つまりシナプス間隙の、GluD2近傍のCa濃度を、R型Caチャネルが活動依存的に制御している可能性が考えられる。今後、R型CaチャネルがどのようにしてPF―PCシナプスのLTDを制御しているのか、さらなる研究が必要である。
Delta type 2 glutamate receptor (GluD2), one of ionotropic glutamate receptor, is specifically localized to parallel fiber (PF) – Purkinje cell (PC) synapse in cerebellum. Accumulated evidence has shown that (1) GluD2 is essential for induction of long term depression (LTD) at this synapse, (2) its ligand is D-serine, which is released from Bergmann glia (BG) in activity dependent manner, and binding to the D-serine can induce LTD at this synapse especially when mice are juvenile, and that (3) GluD2 has Ca2+ binding site near the D-serine binding site at its ligand binding domain, which can affect its affinity to D-serine. These findings suggest that the activity dependent Ca2+ dynamics at the synaptic cleft of PF-PC synapse can be a novel regulation mechanism for LTD induction at this synapse through the Ca binding site of GluD2.
At PF-PC synapse, many molecules are expressed to regulate Ca concentration at its cleft ([Ca]cleft). Among them, voltage gated Ca channels (VGCCs), which are mainly expressed at presynaptic (= PF) terminal, can potentially work as a "Ca sink" for [Ca]cleft when the PF-PC synapses are activated; when action potentials are generated, the VGCCs are activated and flow Ca2+ into the PF terminal from the synaptic cleft, resulting in the reduction in [Ca]cleft. Therefore, there is a possibility that presynaptic VGCCs can affect the LTD induction at the PF-PC synapse via Ca binding site of GluD2. Thus, in this year, this possibility was tested by electrophysiological experiments.
Acute cerebellar slice preparations were made from wild type mice and whole-cell clamp recordings were performed from PCs to get recordings of PF-PC synaptic transmission. First, the contribution of N type VGCC was tested. When N type VGCC was blocked pharmacologically, the PF-PC synaptic transmission was slightly but significantly reduced. On the other hand, LTD, which was induced by repetitive PF stimulation combined with post PC depolarization, was normal.
Next, R type VGCC was pharmacologically blocked and this time again the synaptic transmission was reduced. However, when the LTD induction stimulus was applied, LTD was significantly inhibited. Moreover, when the same experiment was performed using GluD2 KO mice in stead of WT mice, R type VGCC contribution to the synaptic transmission was impaired. These results suggest that the localization of R type VGCC could be regulated by GluD2 and could play an important role for LTD induction at PF-PC synapse. One of possible mechanisms how R type VGCC regulates LTD induction is that R type VGCC is localized near GluD2 and work as a Ca sink to regulate D-serine dependent LTD. Further investigation is necessary to elucidate how R type VGCC regulates the LTD induction at PF-PC synapse.
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