Forsythe I D, Coates R T
Laboratory of Developmental Neurobiology, NICHD, Bethesda, MD 20892.
J Neurosci Methods. 1988 Aug;25(1):19-27. doi: 10.1016/0165-0270(88)90116-1.
With the increasing use of cell culture in electrophysiology there has arisen a need for more rigorous control of the extracellular fluid composition and temperature. We describe here an environmental chamber designed to permit perfusion and temperature control of the extracellular medium during electrophysiological studies of cultured or acutely dissociated neurones in 35 mm Petri dishes. The chamber consists of a Peltier driven heat exchange, which allows temperature control above and below ambient. The Petri dish temperature is controlled in 3 ways: (1) through direct conduction from the controlled surface; (2) by flow of gas around the dish; and (3) by flow of extracellular medium to the Petri dish. We demonstrate the temperature control achieved with this equipment and the ability to exchange the extracellular medium in the whole dish. Using the whole-cell-patch recording technique to record from hippocampal neurones, Q10 parameters were estimated for the membrane time constant, input resistance and action potential halfwidth, over a range of 15-36 degrees C. The mean Q10 values (+/- S.E.M.) were 0.71 +/- 0.05, 0.58 +/- 0.03 and 0.35 +/- 0.1, respectively. Because of rapid diffusion and mixing of drugs when using puffer pipettes, quantitative pharmacological studies or manipulations of the extracellular recording medium are not possible. By perfusing the whole recording chamber the final concentration can be accurately known. Using this method we obtained an estimate of the relative potency of the non-specific excitatory amino acid antagonist, kynurenate, for synaptically activated N-methyl-d-aspartate (NMDA) receptors of 1:0.62.
