Figure 3.

Validity of firing rate measurement by use of loose-seal cell-attached recordings. (A–D) The procedure used to verify the lack of the pipette interference with the measurement. (A) Time-course of a recording illustrating the procedure. During the first 30 s, gentle suction was applied to the pipette to establish loose-seal cell-attached recording configuration. The pressure was then released and after 30 s, left to allow relaxation of the patched cell membrane, a 3-min-long segment (denoted a, from 1 to 4 min) was acquired for the measurement. Afterward, additional suction was slowly applied until the spike amplitude approximately doubled (lower panel) and the recording was prolonged for an additional 1.5 min (denoted b). Since the firing rate remained essentially the same after the test (second suction), the recording was considered reliable, i.e., free of pipette interference. (B) Superimposed average spikes of the same experiment. Spike duration, measured as the interval from the upstroke peak (U) to the second downstroke peak (D₂), was unchanged by the additional suction. (C) Box plot of the same experiments shows no changes in distribution of interspike intervals (ISI). Boxes represent median and the interquartile range (IQR). Whiskers denote 1.5 IQR. (D) Trace shows a segment (4:10–4:30 min) of the original recording during which additional (test) suction was applied. Scale bars: 50 pA, 1 s. (E) Post hoc analysis performed to verify that firing rate of recorded neurons was not influenced by the time passed between slicing and recording. Symbols represent individual neurons. Pearson correlation revealed no correlation between the time after slicing and the firing rate (r = −0.037, 95% CI −0.096 to 0.023, r² = 0.0014, P = 0.23). Lines represent linear regression (Slope −0.017 Hz h⁻¹).