Laser-evoked synaptic transmission in cultured hippocampal neurons channelrhodopsin-2 delivered by adeno-associated virus
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...
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| Format: | Article |
| Language: | en_US |
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Elsevier
2012
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| Online Access: | http://hdl.handle.net/1721.1/74024 |
| _version_ | 1826204578160312320 |
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| author | Wang, Jennifer Hasan, Mazahir T. Seung, H. Sebastian |
| author2 | Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences |
| author_facet | Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences Wang, Jennifer Hasan, Mazahir T. Seung, H. Sebastian |
| author_sort | Wang, Jennifer |
| collection | MIT |
| description | 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 μm 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 μm 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. |
| first_indexed | 2024-09-23T12:57:41Z |
| format | Article |
| id | mit-1721.1/74024 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T12:57:41Z |
| publishDate | 2012 |
| publisher | Elsevier |
| record_format | dspace |
| spelling | mit-1721.1/740242022-10-01T12:10:35Z Laser-evoked synaptic transmission in cultured hippocampal neurons channelrhodopsin-2 delivered by adeno-associated virus Wang, Jennifer Hasan, Mazahir T. Seung, H. Sebastian Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences Massachusetts Institute of Technology. Department of Physics Wang, Jennifer Seung, H. Sebastian 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 μm 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 μm 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. Howard Hughes Medical Institute 2012-10-16T15:28:54Z 2012-10-16T15:28:54Z 2009-10 2009-06 Article http://purl.org/eprint/type/JournalArticle 0165-0270 http://hdl.handle.net/1721.1/74024 Wang, Jennifer, Mazahir T. Hasan, and H. Sebastian Seung. “Laser-evoked Synaptic Transmission in Cultured Hippocampal Neurons Expressing Channelrhodopsin-2 Delivered by Adeno-associated Virus.” Journal of Neuroscience Methods 183.2 (2009): 165–175. en_US http://dx.doi.org/10.1016/j.jneumeth.2009.06.024 Journal of Neuroscience Methods Creative Commons Attribution-Noncommercial-Share Alike 3.0 http://creativecommons.org/licenses/by-nc-sa/3.0/ application/pdf Elsevier PubMed Central |
| spellingShingle | Wang, Jennifer Hasan, Mazahir T. Seung, H. Sebastian Laser-evoked synaptic transmission in cultured hippocampal neurons channelrhodopsin-2 delivered by adeno-associated virus |
| title | Laser-evoked synaptic transmission in cultured hippocampal neurons channelrhodopsin-2 delivered by adeno-associated virus |
| title_full | Laser-evoked synaptic transmission in cultured hippocampal neurons channelrhodopsin-2 delivered by adeno-associated virus |
| title_fullStr | Laser-evoked synaptic transmission in cultured hippocampal neurons channelrhodopsin-2 delivered by adeno-associated virus |
| title_full_unstemmed | Laser-evoked synaptic transmission in cultured hippocampal neurons channelrhodopsin-2 delivered by adeno-associated virus |
| title_short | Laser-evoked synaptic transmission in cultured hippocampal neurons channelrhodopsin-2 delivered by adeno-associated virus |
| title_sort | laser evoked synaptic transmission in cultured hippocampal neurons channelrhodopsin 2 delivered by adeno associated virus |
| url | http://hdl.handle.net/1721.1/74024 |
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