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Patch clamp recordings of fast‐acting ion channels in rat dorsal root ganglion cells (654.3)
Author(s) -
Svensson Dalén Jennie,
Sinclair Jon,
Karlsson Anna,
Pihl Johan,
Karila Paul
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.654.3
Subject(s) - dorsal root ganglion , ion channel , patch clamp , pipette , electrophysiology , biophysics , chemistry , biomedical engineering , neuroscience , materials science , sensory system , nanotechnology , biology , biochemistry , medicine , receptor
Manual patch clamp recordings offer direct insight into ion channel properties through the characterization of ion channel activity. Due to the high quality data it is considered the gold standard for ion channel research. Still, the limited throughput means that maximizing data from each cell, especially sensitive primary cells, is important. We have developed a patch clamp‐based assay for characterization fast‐acting ion channels, in this case exemplified by P2X, in primary dorsal root ganglion (DRG) neurons. This assay is based on the use of primary DRG neurons in culture as a cell model for chronic pain. These neurons retain their sensory functionality and remain responsive to thermal, mechanical and functional stimuli, and when supplemented with nerve growth factor (NGF) they can be used to mimic peripheral sensitization. The assay utilizes a microfluidic perfusion system, Dynaflow Resolve, to facilitate a stable recording situation and fast and programmable solution exchange. At the start of the experiment, compounds and buffer are loaded in the 16 wells of the Dynaflow Resolve chip. Micro‐channels connect each well to a recording chamber where the cells are added. To be able to provide fast solution exchange the DRG neuron is lifted using the patch pipette and positioned in front of the micro‐channel outlets. Then, the cell is scanned through the discrete flow zones formed outside the channels. A comparison between conventional bath perfusion and the automated Dynaflow Resolve system showed that both systems have similar capacity when it comes to obtaining successful high resistance seals. However, for collecting full 5‐8 point dose‐responses from single cells, the success rate for the Dynaflow Resolve superseded the conventional system. This enables a dramatically increased productivity when performing more complex pharmacological characterizations.