Voltage-sensitive dye recording of action potentials and synaptic potentials from sympathetic microcultures.
Given the appropriate multicell electrophysiological techniques, small networks of cultured neurons (microcultures) are well suited to long-term studies of synaptic plasticity. To this end, we have developed an apparatus for optical recording from cultured vertebrate neurons using voltage-sensitive fluorescent dyes (Chien, C.-B., and J. Pine. 1991. J. Neurosci. Methods. 38:93-105). We evaluate here the usefulness of this technique for recording action potentials and synaptic potentials in microcultures of neurons from the rat superior cervical ganglion (SCG). After extensive dye screening and optimization of conditions, we chose the styryl dye RH423, which gave fast linear fluorescence changes of approximately 1%/100 mV for typical recordings. The root mean square noise of the apparatus (limited by shot noise) was typically 0.03%, equivalent to 3 mV of membrane potential. Illumination for at least 100 flashes of 100 ms each caused no noticeable photodynamic damage. Our results show that voltage-sensitive dyes can be used to record from microcultures of vertebrate neurons with high sensitivity. Dye signals were detected from both cell bodies and neurites. Signals from presumptive dendrites showed hyperpolarizations and action potentials simultaneous with those in the cell body, while those from presumptive axons showed delayed propagating action potentials. Subthreshold synaptic potentials in the cell body were occasionally detectable optically; however, they were usually masked by signals from axons passing through the same pixel. This is due to the complex anatomy of SCG microcultures, which have many crisscrossing neurites that often pass over cell bodies. Given a simpler microculture system with fewer neurites, it should be possible to use dye recording to routinely measure subthreshold synaptic strengths.
Department of Physics, California Institute of Technology, Pasadena 91125.
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Chien
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Pine
D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D003712:Dendrites / Q000502:physiology; D005456:Fluorescent Dyes; D005728:Ganglia, Sympathetic / Q000502:physiology*; D018738:Hexamethonium; D006584:Hexamethonium Compounds / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence / Q000379:methods; D013569:Synapses / Q000187:drug effects / Q000502:physiology*
D005456:Fluorescent Dyes; D006584:Hexamethonium Compounds; D018738:Hexamethonium