Takahashi Hirokazu, Sakurai Takeshi, Sakai Hideo, Bakkum Douglas J, Suzurikawa Jun, Kanzaki Ryohei
Research Center for Advanced Science and Technology, The University of Tokyo, Komaba 4-6-1, Meguro-ku, Tokyo 153-8904, Japan.
Biosystems. 2012 Feb;107(2):106-12. doi: 10.1016/j.biosystems.2011.10.002. Epub 2011 Oct 14.
Individual neurons are heterogeneous and have profound impact on population activity in a complex cortical network. Precise experimental control of the firing of multiple neurons would be therefore beneficial to advance our understanding of cell-network interactions. Except for direct intracellular stimulation, however, it is difficult to gain precise control of targeted neurons without inducing antidromic activation of untargeted neurons. To overcome this problem, we attempt to create a sparse group of photosensitized neurons via transfection of Channelrhodopsin-2 (ChR2) in primary dissociated cultures and then deliver light-addressed stimulation exclusively to these target neurons. We first show that liposome transfection was able to express ChR2 in 0.3-1.9% of cells plated depending on cell density. This spatially sparse but robust expression in our neuronal cultures offered the capability of single cell activation by illuminating a spot of light. We then demonstrated that delivering a pulsed train to photo-activate a single neuron had a substantial effect on the activity level of an entire neuronal culture. Furthermore, the activity level was controllable by altering the frequency of light illumination when 4 neurons were recruited as stimulation targets. These results suggest that organized activation of a very small population of neurons can provide better control over global activity of neuronal circuits than can single-neuron activities by themselves.
单个神经元具有异质性，并且对复杂皮质网络中的群体活动有深远影响。因此，精确实验控制多个神经元的放电将有助于推进我们对细胞 - 网络相互作用的理解。然而，除了直接细胞内刺激外，在不诱导非靶向神经元的逆向激活的情况下，很难对靶向神经元进行精确控制。为了克服这个问题，我们试图通过在原代解离培养物中转染通道视紫红质 -2（ChR2）来创建一组稀疏的光敏神经元，然后仅对这些靶神经元进行光寻址刺激。我们首先表明，脂质体转染能够根据细胞密度在0.3 - 1.9%的接种细胞中表达ChR2。在我们的神经元培养物中这种空间上稀疏但稳定的表达提供了通过照射一个光点来激活单个细胞的能力。然后我们证明，向光激活单个神经元传递脉冲序列对整个神经元培养物的活动水平有显著影响。此外，当将4个神经元作为刺激靶点时，通过改变光照频率可以控制活动水平。这些结果表明，与单个神经元活动本身相比，对非常小的神经元群体进行有组织的激活能够更好地控制神经元回路的全局活动。
