Optimal Pipette Resistance, Seal Resistance, and Zero-Current Membrane Potential for Loose Patch or Breakthrough Whole-Cell Recording in vivo

Abstract

In vivo loose patch and breakthrough whole-cell recordings are useful tools for investigating the intrinsic and synaptic properties of neurons. However, the correlation among pipette resistance, seal condition, and recording time is not thoroughly clear. Presently, we investigated the recording time of different pipette resistances and seal conditions in loose patch and breakthrough whole-cell recordings. The recording time did not change with pipette resistance for loose patch recording (R_p-loose) and first increased and then decreased as seal resistance for loose patch recording (R_s-loose) increased. For a high probability of a recording time ≥30 min, the low and high cutoff values of R_s-loose were 21.5 and 36 MΩ, respectively. For neurons with R_s-loose values of 21.5-36 MΩ, the action potential (AP) amplitudes changed slightly 30 min after the seal. The recording time increased as seal resistance for whole-cell recording (R_s-tight) increased and the zero-current membrane potential for breakthrough whole-cell recording (MP_zero-current) decreased. For a high probability of a recording time ≥30 min, the cutoff values of R_s-tight and MP_zero-current were 2.35 GΩ and -53.5 mV, respectively. The area under the curve (AUC) of the MP_zero-current receiver operating characteristic (ROC) curve was larger than that of the R_s-tight ROC curve. For neurons with MP_zero-current values ≤ -53.5 mV, the inhibitory or excitatory postsynaptic current amplitudes did not show significant changes 30 min after the seal. In neurons with R_s-tight values ≥2.35 GΩ, the recording time gradually increased and then decreased as the pipette resistance for whole-cell recording (R_p-tight) increased. For the high probability of a recording time ≥30 min, the low and high cutoff values of R_p-tight were 6.15 and 6.45 MΩ, respectively. Together, we concluded that the optimal R_s-loose range is 21.5-36 MΩ, the optimal R_p-tight range is 6.15-6.45 MΩ, and the optimal R_s-tight and MP_zero-current values are ≥2.35 GΩ and ≤ -53.5 mV, respectively. Compared with R_s-tight, the MP_zero-current value can more accurately discriminate recording times ≥30 min and <30 min.

Keywords: in vivo recordings; membrane potential; pipette resistance; recording time; seal resistance.