Cortical functional subnetworks in the fosGFP+ mouse

这个建议的中心问题是：L2锥体神经元形成功能连接的子网络的兴奋性神经元具有不同的感觉处理特性的fosGFP小鼠？fosGFP小鼠中的神经元在活性依赖性即刻早期基因c-fos的调节下表达GFP。约10%的L2和L 6锥体神经元更强烈地被GFP标记。在之前的资助期间，我们已经开发了体内双光子和三光子靶向记录，以常规记录麻醉的fosGFP转基因小鼠中附近（<100 μm）的L2锥体神经元。在我们的报告中，我们表明，与fosGFP-神经元相比，体内表达fosGFP+的L2锥体神经元在upstates期间具有更大幅度的突触输入，自发放电率增加，并显示出不同的感觉反应。在GFP表达神经元中对触须刺激的感觉反应比GFP-锥体神经元中的反应更早并且具有更大的幅度。来自相同小鼠系的脑切片连接性数据表明，fosGFP+神经元比fosGFP-神经元彼此连接得更多。总之，这提出了一种可能性，即fosGFP+神经元是更多连接的锥体神经元的功能不同的子网络。（i）使用体内双光子靶向全细胞记录将突触连接与麻醉小鼠L2中连接/未连接的fosGFP+/ fosGFP-神经元中的感觉反应和自发活动相关联;（ii）在麻醉和清醒的小鼠中进行L2和L 6 fosGFP+神经元的体内细胞内记录和染色，与Dirk Feldmeyer教授和Jochen Staiger教授的研究小组合作，研究fosGFP+神经元在不同皮质层中是否具有独特的解剖特征和相似的功能特性。总之，解剖学和生理学数据将帮助我们检查fosGFP+神经元是否在体内形成功能性子网络。

The central question of this proposal is: do L2 pyramidal neurons form functional connected subnetworks of excitatory neurons with distinct sensory processing properties in the fosGFP mouse? Neurons in the fosGFP mouse express GFP under the regulation of the activity dependent, immediate early gene c-fos. About 10% of L2 and L6 pyramidal neurons are more strongly labelled with GFP. Over the previous funding period we have developed in vivo double and triple two-photon targeted recordings to record routinely from nearby (<100 μm) L2 pyramidal neurons in the anaesthetised fosGFP transgenic mouse. In our report, we show that in vivo fosGFP+ expressing L2 pyramidal neurons have increased spontaneous firing rates with larger amplitude synaptic input during upstates and show distinct sensory responses as compared to fosGFP- neurons. The sensory response to whisker stimulation in GFP expressing neurons is earlier and has larger amplitude than the response in GFP- pyramidal neurons. Brain slice connectivity data from the same mouse line suggests that fosGFP+ neurons are more connected to each other than to fosGFP- neurons. Together this raises the possibility that the fosGFP+ neurons are a functionally distinct subnetwork of more connected pyramidal neurons. Here we propose to use this mouse line to perform two central experiments: (i) use in vivo 2-photon targeted whole-cell recordings to correlate synaptic connectivity with sensory responses and spontaneous activity in connected / unconnected fosGFP+/ fosGFP- neurons in L2 of anaesthetised mice; (ii) perform in vivo juxtacellular recordings and stainings of L2 and L6 fosGFP+ neurons in anaesthetised and awake mice, in collaboration with the group of Prof. Dirk Feldmeyer and Prof Jochen Staiger, to investigate whether fosGFP+ neurons have distinct anatomical features and similar functional properties in different cortical layers. Taken together, the anatomical and physiological data will help us examine whether the fosGFP+ neurons form functional subnetworks in vivo.