Recordings of cultured neurons and synaptic activity using patch-clamp chips

Planar patch-clamp chip technology has been developed to enhance the assessment of novel compounds for therapeutic efficacy and safety. However, this technology has been limited to recording ion channels expressed in isolated suspended cells, making the study of ion channel function in synaptic transmission impractical. Recently, we developed single- and dual-recording site planar patch-clamp chips and demonstrated their capacity to record ion channel activity from neurons established in culture. Such capacity provides the opportunity to record from synaptically connected neurons cultured on-chip. In this study we reconstructed, on-chip, a simple synaptic circuit between cultured pre-synaptic visceral dorsal 4 neurons and post-synaptic left pedal dorsal 1 neurons isolated from the mollusk Lymnaea stagnalis. Here we report the first planar patch-clamp chip recordings of synaptic phenomena from these paired neurons and pharmacologically demonstrate the cholinergic nature of this synapse. We also report simultaneous dual-site recordings from paired neurons, and demonstrate dedicated cytoplasmic perfusion of individual neurons via on-chip subterranean microfluidics. This is the first application of planar patch-clamp technology to examine synaptic communication.

[1]  K. Lukowiak,et al.  In Vitro Synaptogenesis between the Somata of Identified Lymnaea Neurons Requires Protein Synthesis But Not Extrinsic Growth Factors or Substrate Adhesion Molecules , 1997, The Journal of Neuroscience.

[2]  Christophe Py,et al.  A novel silicon patch‐clamp chip permits high‐fidelity recording of ion channel activity from functionally defined neurons , 2010, Biotechnology and bioengineering.

[3]  A. Bulloch,et al.  In vitro reconstruction of the respiratory central pattern generator of the mollusk Lymnaea. , 1990, Science.

[4]  Jürgen Rühe,et al.  Planar microelectrode-cavity array for high-resolution and parallel electrical recording of membrane ionic currents. , 2008, Lab on a chip.

[5]  N. Syed,et al.  Trophic Factor-Induced Intracellular Calcium Oscillations Are Required for the Expression of Postsynaptic Acetylcholine Receptors during Synapse Formation between Lymnaea Neurons , 2009, The Journal of Neuroscience.

[6]  F. Sigworth,et al.  Microchip technology in ion-channel research , 2005, IEEE Transactions on NanoBioscience.

[7]  K. Lukowiak,et al.  Electrophysiological differences in the CpG aerial respiratory behavior between juvenile and adult Lymnaea. , 2003, Journal of neurophysiology.

[8]  C. Shatz.  Impulse activity and the patterning of connections during cns development , 1990, Neuron.

[9]  M. Woodin,et al.  Trophic factor-induced plasticity of synaptic connections between identified Lymnaea neurons. , 1999, Learning & memory.

[10]  A. Craig,et al.  How to build a central synapse: clues from cell culture , 2006, Trends in Neurosciences.

[11]  C. Shatz,et al.  Synaptic Activity and the Construction of Cortical Circuits , 1996, Science.

[12]  M Segal,et al.  Neurotrophins Induce Formation of Functional Excitatory and Inhibitory Synapses between Cultured Hippocampal Neurons , 1998, The Journal of Neuroscience.

[13]  A. Brown,et al.  Patch and whole cell calcium currents recorded simultaneously in snail neurons , 1984, The Journal of general physiology.

[14]  F. Qin,et al.  Gated, ion-selective channels observed with patch pipettes in the absence of membranes: novel properties of a gigaseal. , 1993, Biophysical journal.

[15]  Robert H Blick,et al.  Whole cell patch clamp recording performed on a planar glass chip. , 2002, Biophysical journal.

[16]  J. W. Goh,et al.  Pharmacological and physiological properties of the after‐hyperpolarization current of bullfrog ganglion neurones. , 1987, The Journal of physiology.

[17]  Bert Sakmann,et al.  The extracellular patch clamp: A method for resolving currents through individual open channels in biological membranes , 1978, Pflügers Archiv.

[18]  Christophe Py,et al.  High-fidelity patch-clamp recordings from neurons cultured on a polymer microchip , 2010, Biomedical microdevices.

[19]  Celeste A. Morris,et al.  Stretch-activated ion channels in growth cones of snail neurons , 1989, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[20]  Michel De Waard,et al.  Hourglass SiO2 coating increases the performance of planar patch-clamp. , 2006, Journal of biotechnology.

[21]  N. Syed,et al.  Activity‐induced large amplitude postsynaptic mPSPs at soma–soma synapses between Lymnaea neurons , 2008, Synapse.

[22]  Chang-Yu Chen,et al.  Patch clamping on plane glass-fabrication of hourglass aperture and high-yield ion channel recording. , 2009, Lab on a chip.

[23]  Y. Dan,et al.  Spike Timing-Dependent Plasticity of Neural Circuits , 2004, Neuron.

[24]  R. Gillette,et al.  Fast and slow activation kinetics of voltage-gated sodium channels in molluscan neurons. , 1997, Journal of neurophysiology.

[25]  Frederick Sachs,et al.  Biophysics and structure of the patch and the gigaseal. , 2009, Biophysical journal.

[26]  A missing factor in chip-based patch clamp assay: gigaseal , 2006 .

[27]  Z. Gil,et al.  Ionic requirements for membrane-glass adhesion and giga seal formation in patch-clamp recording. , 2007, Biophysical journal.

[28]  N. Syed,et al.  Synaptogenesis in the CNS: An Odyssey from Wiring Together to Firing Together , 2003, The Journal of physiology.

[29]  P. Scheiffele.  Cell-cell signaling during synapse formation in the CNS. , 2003, Annual review of neuroscience.