Light-addressed single-neuron stimulation in dissociated neuronal cultures with sparse expression of ChR2.
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.
Research Center for Advanced Science and Technology, The University of Tokyo, Komaba 4-6-1, Meguro-ku, Tokyo 153-8904, Japan. takahashi@i.u-tokyo.ac.jp
Hirokazu
H
Takahashi
Takeshi
T
Sakurai
Hideo
H
Sakai
Douglas J
DJ
Bakkum
Jun
J
Suzurikawa
Ryohei
R
Kanzaki
D000200:Action Potentials; D000818:Animals; D002118:Calcium / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D018920:Coculture Techniques / Q000662:veterinary; D004558:Electric Stimulation / Q000295:instrumentation* / Q000379:methods*; D008081:Liposomes / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009457:Neuroglia / Q000166:cytology / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D010775:Photic Stimulation; D051381:Rats; D017208:Rats, Wistar; D012243:Rhodopsin / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000662:veterinary
D008081:Liposomes; D009419:Nerve Tissue Proteins; D012243:Rhodopsin; D002118:Calcium