title: Laser-evoked synaptic transmission in cultured hippocampal neurons expressing channelrhodopsin-2 delivered by adeno-associated virus.
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.
authors: Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, 77 Massachusetts Avenue 46-5065, Cambridge, MA 02139, USA. jenwang@mit.edu
authors: Jennifer
authors: J
authors: Wang
authors: Mazahir T
authors: MT
authors: Hasan
authors: H Sebastian
authors: HS
authors: Seung
mesh_terms: D000200:Action Potentials / Q000502:physiology / Q000528:radiation effects; D000818:Animals; D000831:Animals, Newborn; D001426:Bacterial Proteins / Q000235:genetics; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics*; D006624:Hippocampus / Q000166:cytology; D007834:Lasers; D008164:Luminescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal / Q000379:methods; D009474:Neurons / Q000378:metabolism* / Q000528:radiation effects; D018408:Patch-Clamp Techniques; D051381:Rats; D011930:Reaction Time / Q000528:radiation effects; D012243:Rhodopsin / Q000235:genetics / Q000378:metabolism*; D009435:Synaptic Transmission / Q000528:radiation effects*; D013997:Time Factors; D014162:Transfection / Q000379:methods
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D012243:Rhodopsin