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Multielectrode Array Membrane Biophysics 9 November 2007 John Corthell and Kristal Tucker Two broad categories of multielectrode recordings • In vivo - KT – Recording and stimulation – Acute and Chronic – Heart, CNS, PNS and Retina • In vitro - JC – Organotypic and primary dissociated cultures • Heart, CNS, PNS, and retina Roadmap • • • • History Applications Techniques Representative articles Brain-Computer Interface Scott 2006. Chronic in vivo recordings Musallam et al 2007 Electrode fabrication Musallam et al 2007 Array insertion Musallam et al 2007 Data capture and analysis Musallam et al 2007 Variable depth arrays Sato et. al. 2007 Hochberg et al 2006 Hochberg et al 2006 Hochberg et al 2006 Hochberg et al 2006 Hochberg et al 2006 Hochberg et al 2006 Hochberg et al 2006 In vitro multielectrode array history • Gross, in 1979, first developed an array based on semiconductor technology • Regehr et al., 1989-first applied Aplysia, Hirudo (leech) and Helisoma (snail) cells to multielectrode array (MEA) chip for long-term recording • Masuda et al., in 1983, applied a linear electrode array to myoneural junctions • Linear electrode array recording Multielectrode array recording In vitro multielectrode applications • Olfactory processing-Christensen et al., 2000 • Long-term recording-Regehr et al., 1989 – Circadian rhythms-Abraham et al., 2005 • Neuromuscular junction activity-Masuda et al., 1983 • Network analysis – Long-term potentiation – Synaptic interaction Organotypic Slice Culture • A different type of cell culture that works with MEAs and preserves some circuitry (but not exactly native-synaptic rearrangement) • Ideal for long-term recording, as a culture can last from 3-4 weeks for recording to several months, depending on prep Duport et al., 1999 Organotypic Slice Cultures, cont. • Slice cultures preserve 3dimensional area for electrode preparation • Simple to prepare-remove brain (no more than 60s), place into cold solvent, cut into 425m thick slices, place onto MEA with media Spinal cord prep, from Bio-Rad website Fabrication • Commercially available, so you don’t have to make one yourself • TiN=titanium nitride Fabrication-MED • MED is newer than MEA-MED is a planar multielectrode array • MED is an attempt to lengthen recording time from previous MEAs MEA/MED Usage • Hooked up to amplifier, • Works like most A/D converter, and electrophysiology computer recordings-difference is • Typically software previous work to set up programs allow for array and post-experiment recording and stimulation work to analyze data near-simultaneously • Cells are usually grown in culture dish over the MEA, but can be organotypic Views of MEA chamber and amplifier plate -PP-probe pin -SC-stimulus connector -RA-recording area MEA + other techniques • MEA is often used in conjunction with other techniques, such as Ca imaging • MEA measures extracellular changes (as you cannot patch), so some things (like postsynaptic potentials and Ca flux) are missed • Optical recording techniques (identifying individual cells) are used with MEA to alleviate this Other shortcomings • MEA biochips are expensive to manufacture (may change with time), so researchers will clean the chip to attempt to salvage the product for future use ($250-$350) • Continued cleaning will result in degradation of chip until readings are no longer reliable Granados-Fuentes et al., “Olfactory bulb neurons express functional, entrainable circadian rhythms.” European J. Neuroscience, 19: 898-906, 2004. MEA and setup • Per1 transgenic rats (yes, rats) underwent bulbectomy from E15P37, cells were dispersed onto MEAs • MEAs had 60 electrodes, spaced 200m apart, with 10m tips (purchased from Germany) • SCN explants used as controls (P1-P7) • Cultures were covered with a membrane and transferred to a recording incubator • Recorded from 4 cultures for at least 5 days simultaneously • Used to establish spontaneous activity • Recording apparatus from inside the incubator neuro.gatech.edu/groups/ potter/realtimedac.html Other techniques used • Locomotor activity measured in normal vs. bulbectomized rats • Per1 activity measured by bioluminescence (Per1 gene is linked to luciferase gene [light from fireflies], add luciferin, and protein product will light up) from a photomultiplier tube • Temperature entrainment via incubator Results • Per1 expression in OB – Start showing rhythm at E19 • Top-firing of OB neuron • Bottom-firing of SCN control • OB neurons that fired rhythmically were found in the mitral cell layer but not the granule cell layer • Left axis is Firing Frequency • Different cells in the same culture can have different firing rhythms • TopMitral • BottomGranule • Removal of OB has no effect on running wheel behavior • Temperature changes work as zeitgebers (entraining signals) for OB culture cells Conclusions from paper • There is a rhythm of activity and Per1 expression in the olfactory bulb neurons of the mitral cell layer • This rhythm begins at E19 and matures over the first week postnatal • These oscillating neurons can have different rhythms from one another in the same culture Conclusions from in vitro MEA • Most modern MEA is the MED-the planar MEA biochip • Grow cells on biochip or use organotypic culture to study • Can be used to simultaneously record and stimulate extracellularly • Must be cared for-expensive • Should be used with other techniques to compensate for shortcomings