In vivo two-photon uncaging of glutamate revealing the structure-function relationships of dendritic spines in the neocortex of adult mice
In vivo two-photon uncaging of glutamate revealing the structure-function relationships of dendritic spines in the neocortex of adult mice
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DOI：
10.1113/jphysiol.2011.207100
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发表时间：
2011-05-15
影响因子：
5.5
通讯作者：
Kasai, Haruo
中科院分区：
文献类型：
--
作者：
Noguchi, Jun;Nagaoka, Akira;Kasai, Haruo
双光子(2P)去化笼养神经递质可以有效地刺激单个突触，被广泛用于表征脑片的突触功能。在此，我们将2P去势扩展到成年小鼠的新皮质锥体神经元，在活体中，笼养的谷氨酸从软膜表面施加。为了验证方法学，我们用同样的方法应用了一个小的荧光探针，并确认其浓度在大脑皮层表面以下200微米处大致均匀，新皮质的细胞外空间高达22%。事实上，活体全细胞记录显示，2P谷氨酸去化可以诱发与兴奋性突触后电流(EPSCs)非常相似的瞬时电流(2pEPSCs)。谷氨酸去化的空间分辨率为0.6-0.8微米，最深可达200微米，体内2P去化可刺激单个已识别的脊椎。对这种覆盖在树突分支表面的2pEPSCs进行自动三维(3-D)定位显示，功能性AMPA受体的表达稳定，并与脊髓体积成正比。此外，活体2P钙成像和去势显示，谷氨酸诱导的钙瞬变的幅度与脊柱体积成反比。因此，关键的结构-功能关系在体内成人新皮质的树突棘中存在，就像在年轻的海马片制备中一样。在体内，2P去化将是研究新皮质突触特性的有力工具。
Two-photon (2P) uncaging of caged neurotransmitters can efficiently stimulate individual synapses and is widely used to characterize synaptic functions in brain slice preparations. Here we extended 2P uncaging to neocortical pyramidal neurons in adult mice in vivo where caged glutamate was applied from the pial surface. To validate the methodology, we applied a small fluorescent probe using the same method, and confirmed that its concentrations were approximately homogenous up to 200 mu m below the cortical surface, and that the extracellular space of the neocortex was as large as 22%. In fact, in vivo whole-cell recording revealed that 2P glutamate uncaging could elicit transient currents (2pEPSCs) very similar to excitatory postsynaptic currents (EPSCs). A spatial resolution of glutamate uncaging was 0.6-0.8 mu m up to the depth of 200 mu m, and in vivo 2P uncaging was able to stimulate single identified spines. Automated three-dimensional (3-D) mapping of such 2pEPSCs which covered the surfaces of dendritic branches revealed that functional AMPA receptor expression was stable and proportional to spine volume. Moreover, in vivo 2P Ca2+ imaging and uncaging suggested that the amplitudes of glutamate-induced Ca2+ transients were inversely proportional to spine volume. Thus, the key structure-function relationships hold in dendritic spines in adult neocortex in vivo, as in young hippocampal slice preparations. In vivo 2P uncaging will be a powerful tool to investigate properties of synapses in the neocortex.