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  Laser-evoked synaptic transmission in cultured hippocampal neurons expressing Channelrhodopsin-2 delivered by adeno-associated virus

Wang, J., Hasan, M. T., & Seung, H. S. (2009). Laser-evoked synaptic transmission in cultured hippocampal neurons expressing Channelrhodopsin-2 delivered by adeno-associated virus. Journal of Neuroscience Methods, 183(2), 165-175. doi:10.1016/j.jneumeth.2009.06.024.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002C-4FD4-0 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002C-4FD5-E
Genre: Journal Article
Alternative Title : Laser-evoked synaptic transmission in cultured hippocampal neurons expressing Channelrhodopsin-2 delivered by adeno-associated virus

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 Creators:
Wang, Jennifer, Author
Hasan, Mazahir T.1, 2, Author              
Seung, H. Sebastian, Author
Affiliations:
1Department of Molecular Neurobiology, Max Planck Institute for Medical Research, Max Planck Society, ou_1497704              
2Department of Biomedical Optics, Max Planck Institute for Medical Research, Max Planck Society, ou_1497699              

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Free keywords: Channelrhodopsin-2 (ChR-2) Recombinant adeno-associated virus (rAAV) Synaptic physiology Primary hippocampal culture
 Abstract: We present a method for studying synaptic transmission in mass cultures of dissociated hippocampal neurons based on patch clamp recording combined with laser stimulation of neurons expressing channelrhodopsin-2 (ChR2). Our goal was to use the high spatial resolution of laser illumination to come as close as possible to the ideal of identifying monosynaptically coupled pairs of neurons, which is conventionally done using microisland rather than mass cultures. Using recombinant adeno-associated virus (rAAV) to deliver the ChR2 gene, we focused on the time period between 14 and 20 days in vitro, during which expression levels are high, and spontaneous bursting activity has not yet started. Stimulation by wide-field illumination is sufficient to make the majority of ChR2-expressing neurons spike. Stimulation with a laser spot at least 10 microm in diameter also produces action potentials, but in a reduced fraction of neurons. We studied synaptic transmission by voltage-clamping a neuron with low expression of ChR2 and scanning a 40 microm laser spot at surrounding locations. Responses were observed to stimulation at a subset of locations in the culture, indicating spatial localization of stimulation. Pharmacological means were used to identify responses that were synaptic. Many responses were of smaller amplitude than those typically found in microisland cultures. We were unable to find an entirely reliable criterion for distinguishing between monosynaptic and polysynaptic responses. However, we propose that postsynaptic currents with small amplitudes, simple shapes, and latencies not much greater than 8 ms are reasonable candidates for monosynaptic interactions.

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Language(s): eng - English
 Dates: 2009-06-172009-04-012009-06-182009-10-15
 Publication Status: Published in print
 Pages: 11
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: eDoc: 664715
DOI: 10.1016/j.jneumeth.2009.06.024
URI: http://www.ncbi.nlm.nih.gov/pubmed/19560489
Other: 7483
 Degree: -

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Title: Journal of Neuroscience Methods
  Other : J. Neurosci. Meth.
Source Genre: Journal
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Publ. Info: Amsterdam : Elsevier
Pages: - Volume / Issue: 183 (2) Sequence Number: - Start / End Page: 165 - 175 Identifier: ISSN: 0165-0270
CoNE: https://pure.mpg.de/cone/journals/resource/954925480594