Whole-cell recording of anoxic effects on hippocampal neurons in slices

Abstract

1. In 400-microns-thick slices from young adult Sprague-Dawley rats, CA1 pyramidal layer neurons were studied by the whole-cell recording technique. The patch pipettes were filled most often with (in mM) 140 potassium gluconate, 2 MgCl2, and 0.2 guanosine triphosphate (GTP): in many cases, 2 mM ATP and/or 1.1 mM EGTA and 0.1 mM Ca were added. The slices were kept at 30–32 degrees C. 2. Cells recorded with ATP-containing electrodes had a much higher input resistance (RN, 101 +/- 5.6 M omega, mean +/- SE) and somewhat less negative resting potentials (Vm; -59.8 +/- 1.1 mV) than cells recorded with ATP-free electrodes (71 +/- 2.7 M omega and -63.1 +/- 0.8 mV). The presence or absence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or the substitution of KCl for potassium gluconate did not significantly affect Vm or RN. 3. Overall changes in Vm and RN elicited by anoxia (95% N2-5% CO2 for 3–6 min) were much less pronounced than those seen previously with intracellular electrodes: instead of a hyperpolarization and a approximately 50% fall in RN, there was only a minor depolarization (by 2.4 +/- 0.7 mV) and a small reduction in RN (by 12 +/- 2.4%). During voltage clamp, at holding potentials approximately -35 mV, anoxia evoked only very small outward currents, especially when we recorded with ATP-containing electrodes. 4. The remaining anoxic changes in RN (but not Vm) were very significantly smaller (P < 0.001) when recorded with ATP-containing electrodes (-6 +/- 1.4%) than with ATP-free electrodes (-19 +/- 2.7%). The presence of internal EGTA (1.1-11 mM) was associated with significantly smaller (P < 0.05) anoxic changes in RN: -9.7 +/- 2.0% versus -17 +/- 3.1% in its absence. EGTA also reduced slow afterhyperpolarizations by 80%, though even 11 mM EGTA did not abolish them. However, EGTA had no significant effect on anoxic changes in Vm and did not suppress voltage sags observed during applications of hyperpolarizing current pulses. 5. Judging by these observations, it appears that 1) the much greater anoxic changes in Vm and RN recorded with intracellular electrodes are probably mediated by a diffusible cytosolic agent and 2) during whole-cell recording, both resting RN and the anoxic fall in RN are more strongly determined by cytosolic [ATP] than [Ca]. How ATP affects RN and anoxic changes in RN remains to be established.