Cell dialysis by sharp electrodes can cause nonphysiological changes in neuron properties

Abstract

Cell dialysis by sharp electrodes can cause nonphysiological changes in neuron properties. J Neurophysiol 114: 1255-1271, 2015. First published June 10, 2015 doi:10.1152/jn.01010.2014.-We recorded from lobster and leech neurons with two sharp electrodes filled with solutions often used with these preparations (lobster: 0.6 M K 2 SO 4 or 2.5 M KAc; leech: 4 M KAc), with solutions approximately matching neuron cytoplasm ion concentrations, and with 6.5 M KAc (lobster, leech) and 0.6 M KAc (lobster). We measured membrane potential, input resistance, and transient and sustained depolarization-activated outward current amplitudes in leech and these neuron properties and hyperpolarization-activated current time constant in lobster, every 10 min for 60 min after electrode penetration. Neuron properties varied with electrode fill. For fills with molarities Ն2.5 M, neuron properties also varied strongly with time after electrode penetration. Depending on the property being examined, these variations could be large. In leech, cell size also increased with noncytoplasmic fills. The changes in neuron properties could be due to the ions being injected from the electrodes during current injection. We tested this possibility in lobster with the 2.5 M KAc electrode fill by making measurements only 10 and 60 min after penetration. Neuron properties still changed, although the changes were less extreme. Making measurements every 2 min showed that the time-dependent variations in neuron properties occurred in concert with each other. Neuron property changes with high molarity electrode-fill solutions were great enough to decrease neuron firing strongly. An experiment with 14 C-glucose electrode fill confirmed earlier work showing substantial leak from sharp electrodes. Sharp electrode work should thus be performed with cytoplasm-matched electrode fills. current injection; leech; neuron properties; stomatogastric IN WHOLE-CELL PATCH CLAMP, electrode fill solution ion composition is matched as closely as possible to neuron cytoplasmic composition because of the high leak and consequent cell dialysis that occur with these large-tip diameter electrodes (Hille 2003; Pusch and Neher 1988). Despite their much smaller-tip diameters, substantial leak of electrode contents (Fromm and Schultz 1981), sufficient to cause cell swelling (Stoner et al. 1984) and chloride loading (Eisen and Marder 1982), also occurs with sharp microelectrodes. Nonetheless, sharp electrodes filled with 3 M KCl (much greater than the ionic strength of neuron cytoplasm) solutions were used in the second paper, demonstrating that sharp electrode physiology was possible (Nastuk and Hodgkin 1950). Nastuk and Hodgkin (1950) made this choice to reduce the resistance of their electrodes, because it obviated the need to correct for liquid junctional potentials (see MATERIALS AND METHODS). They were aware of the danger of electrode leak using such a high ionic strength electrode fill solution but reasoned that it would have very little effect with the volumes of the muscle ...

Choline and NMDG directly reduce outward currents: reduced outward current when these substances replace Na+is alone not evidence of Na+-activated K+currents

“…As has been observed previously (Hooper et al 2015), in the PD neurons the current necessary to achieve 0 mV showed a large decrease during the 10 depolarizing steps ( Fig. 2A).…”

“…We therefore filled the microelectrodes with solutions whose ion concentration matched the cytoplasmic ion concentration of the neuron type being recorded from: for lobster (in mM), 20 NaCl, 15 Na 2 SO 4 , 400 K-gluconate, 10 MgCl 2 , and 10 HEPES, pH 7.2; for leech (in mM), 7.6 NaCl, 1.4 Na 2 SO 4 , 112 K-gluconate, 0.2 MgCl 2 , and 10 HEPES, pH 7.2. When recorded from with electrodes filled with these solutions, outward current amplitudes are stable for the entire recording times used in the experiments reported here (Hooper et al 2015).…”

“…Intracellular neuronal recordings were made with glass microelectrodes. High-ionic strength electrode fill solutions such as 2.5 M KCl internally dialyze pyloric and leech neurons and cause their depolarization-activated outward currents to decrease over time, increase the time constants of some conductances in lobster, and even make leech neurons swell (Hooper et al 2015). We therefore filled the microelectrodes with solutions whose ion concentration matched the cytoplasmic ion concentration of the neuron type being recorded from: for lobster (in mM), 20 NaCl, 15 Na 2 SO 4 , 400 K-gluconate, 10 MgCl 2 , and 10 HEPES, pH 7.2; for leech (in mM), 7.6 NaCl, 1.4 Na 2 SO 4 , 112 K-gluconate, 0.2 MgCl 2 , and 10 HEPES, pH 7.2.…”

Rapid adaptation to elevated extracellular potassium in the pyloric circuit of the crab, Cancer borealis

“…Intracellular recordings from somata were performed in the desheathed STG with 10-30 MΩ sharp glass microelectrodes filled with internal solution (10 mM MgCl2, 400 mM potassium gluconate, 10 mM HEPES buffer, 15 mM NaSO4, 20 mM NaCl as in (Hooper et al, 2015). Intracellular signals were amplified with an Axoclamp 900A amplifier (Molecular Devices).…”

Neuronal morphologies built for reliable physiology in a rhythmic motor circuit

“…The STG was desheathed for access to somata for intracellular recordings. These recordings were executed with glass micropipettes (20-30 MΩ) filled with internal solution: 10 mM MgCl2, 400 mM potassium gluconate, 10 mM HEPES buffer, 15 mM NaSO4, 20 mM NaCl (Hooper et al, 2015). Intracellular recordings signals were amplified with an Axoclamp 900A amplifier (Molecular Devices, as described in Otopalik et al 2017b).…”