Figure 1.
Summary flowchart for the whole-cell access methods described in the text.
The orange diamonds highlight an explicit decision point by the experimenter (e.g., the first orange diamond depends on the choice to employ the WS method or a Touch and Zap variant), whereas the gray diamonds depend on the observed recording properties.
Table 1.
Total number of recorded neurons and glial cells for the rat and the cat, as a function of whole-cell access method.
Table 2.
Relative proportion of neuron physiological types and glial cells in the rat and cat for the WS and pooled Touch and Zap methods (TZ, TZS and TS).
Figure 2.
Regular-spiking neuron in cat visual cortex in vivo recorded with the TZ method.
(A) Voltage and current recordings under current clamp mode during the approach to the neuron, detection of contact (“touch”; at ∼6 seconds), release of pressure with immediate seal formation, electroporation of the membrane and establishment of whole-cell access (“zap”), and initial measures of the voltage response to current steps. (B–C), expanded traces from (A) highlighting the access to the whole-cell configuration. (B) corresponds to the region marked by the orange square in (A), and (C) corresponds to the region marked by the grey square in (B). The “touch” is characterized by a small increase in the electrode resistance (C). The positive pressure in the pipette is released within a second after the touch, followed by an immediate increase in the electrode resistance as the seal spontaneously forms. A first “zap” occurs within about one second (B), followed by a second “zap” which results in the whole-cell configuration, evidenced by the much smaller voltage deflections, and the hyperpolarization of the voltage envelope as the cell's resting potential establishes a DC bias. At this point the current pulses are typically set to 100 pA, switching polarity as necessary to estimate and compensate for Ra. (D) Voltage responses to current steps (−100, 0, 150 and 250 pA), with the response to 250 pA showing a regular-spiking firing pattern. (E) Left – Examples of preferred (180°) and non-preferred (90°) responses (10 trials overlaid) to a moving sinusoidal grating (4 Hz, 100% contrast, 0.5 second duration). Right - Total spike count over the 10 trials as a function of stimulus direction, showing that this neuron is strongly tuned to stimulus orientation and modestly tuned to stimulus direction. The on-line corrected Ra for this recording was 24 MΩ, which was subsequently corrected off-line to 21 MΩ. The input resistance was 102 MΩ, giving a relative Ra of 0.21.
Figure 3.
Bursting neuron in cat visual cortex in vivo recorded with the TZ method.
(A–B). As in Figure 2, voltage and current recordings during the approach to the neuron, the “touch” (at ∼16 seconds), release of pressure with immediate seal formation, the first “zap” about two seconds later, followed by a second “zap” and establishment of whole-cell access, and initial measures of the voltage response to current steps. (C) Voltage responses to current steps (−50, 150 and 250 pA), showing a bursting firing pattern in response to the 250 pA step. (D) Left – Examples of preferred (0°) and non-preferred (90°) responses (10 trials overlaid) to a moving sinusoidal grating (4 Hz, 100% contrast, 1 second duration). Right – Total spike count over the 10 trials as a function of stimulus direction, showing the strong orientation tuning of this neuron. The on-line corrected Ra was 44 MΩ, which was subsequently corrected off-line to 61 MΩ. The input resistance was 70 MΩ, giving a relative Ra of 0.87.
Figure 4.
Fast-spiking neuron in cat visual cortex in vivo recorded with the TS method.
(A–B). As in Figure 2, voltage and current recordings during the approach to the neuron, with the “touch” at ∼36 seconds. As can be appreciated in (B), and in distinction to the TZ endpoints, here there is only a slow increase in electrode resistance after the release of the pipette pressure. At approximately 51 seconds, the “zap” attempt is abandoned, and the standard methods of suction and hyperpolarization for obtaining a gigaseal were used, with the whole-cell access achieved by mouth suction at ∼70 seconds. (C) Voltage responses to current steps (0 and 800 pA), showing a fast-spiking firing pattern to the suprathreshold stimulus. (D) Left – Examples of preferred (230°) and non-preferred (50°) responses (10 trials overlaid) to a moving sinusoidal grating (4 Hz, 100% contrast, 1 second duration). Right – Total spike count over the 10 trials as a function of stimulus direction, showing that this neuron is mainly tuned to stimulus direction. The on-line corrected Ra was 37 MΩ, which was validated off-line. The input resistance was 26 MΩ, giving a relative Ra of 1.4.
Figure 5.
Glial cell in cat visual cortex in vivo recorded with the TZ method.
(A–B). As in Figure 2, voltage and current recordings during the approach to the cell, the “touch” (at ∼33 seconds), release of pressure with immediate seal formation, the first “zap” about two seconds later. The current was reduced to 100 pA about one second later, and final whole-cell access was achieved by a small suction. As for neurons (e.g. Figures 2 and 3), the “zap” is distinguished by the sudden reduction in resistance while the strong hyperpolarizing current pulses are applied. (C) Voltage responses to current steps (100 nA increments from −100 to 700 nA), showing the lack of spikes and a fast membrane time constant that are characteristic of glial cells. (D) Left - Preferred (45°) and non-preferred (135°) current clamp responses (average of 10 trials) to a moving sinusoidal grating (4 Hz, 100% contrast, 2 second duration). Right – Average standard deviation of the visual responses (over the 10 trials) as a function of stimulus direction, showing that the membrane voltage fluctuations of this cell are tuned to stimulus direction. The Ra estimated on-line was 45 MΩ (validated off-line), and the cell input resistance was 105 MΩ, thus a relative Ra of 0.43.
Figure 6.
Glial cell in cat visual cortex in vivo recorded with the TZS method.
(A–B) As in Figure 2, voltage and current recordings during the approach to the cell and subsequent whole-cell access. Here, an initial spontaneous seal with a subsequent “zap” was accomplished, but in distinction to the TZ endpoints illustrated in Figures 2, 3 and 5, the membrane resealed (between 24 and 27 seconds), and whole-cell access was re-established by suction at ∼46 seconds (not visible in (B)). (C) Voltage responses to current steps (100 nA increments from −200 to 700 nA). (D) Left – Preferred (270°) and non-preferred (0°) voltage responses (average of 10 trials) to a moving sinusoidal grating (4 Hz, 100% contrast, 5 second duration). Right – Average standard deviation of the visual responses (over the 10 trials) as a function of stimulus direction, showing that this cell is tuned to stimulus direction, with a small bias for stimulus orientation. The on-line corrected Ra was 40 MΩ, which was validated off-line. The input resistance was 99 MΩ, giving a relative Ra of 0.40.
Figure 7.
Gial cell in cat visual cortex in vivo recorded with the WS method.
(A) Voltage responses to current steps (100 nA increments from −100 to 700 nA). (B) Left – Preferred (135°) and non-preferred (45°) voltage responses (average of 10 trials) to a moving sinusoidal grating (4 Hz, 100% contrast, 5 second duration). Right – Average standard deviation of the visual responses (over the 10 trials) as a function of stimulus direction, showing that this cell is tuned to stimulus orientation, with a small bias for stimulus direction. The on-line corrected Ra was 48 MΩ, which was validated off-line. The input resistance was 100 MΩ, giving a relative Ra of 0.48.
Figure 8.
Distribution of recording durations for all neuronal and glial recordings in this study.
Table 3.
Protocol parameters for whole-cell patch recordings of neurons and glial cells in-vivo.
Table 4.
Cell and recording parameters for cortical neurons and glial cells in vivo according to the WS and the pooled TZ and TZS whole-cell access methods.
Figure 9.
Neuron parameter dependencies on estimated access resistance Ra.
(A) Rat neurons closed circles, cat neurons open circles; Top left, cell input resistance versus Ra (r2 = 0.08). Top right, cell membrane time constant versus Ra (r2 = 0.06). Bottom left, peak spike voltage for regular-spiking neurons versus Ra (r2 = 0.22). Bottom right, peak spike depolarization for regular-spiking neurons versus Ra (r2 = 0.14). (B) Dependence of absolute and relative Ra on recording depth. Left, absolute value of Ra. Right, Ra normalized to the cell input resistance Rin. Pearson correlation coefficients (r2) are calculated over the entire dataset in each plot.
Table 5.
Reported access resistance values from in vivo whole-cell patch recordings of neurons in mouse, rat and cat cortex, compared with values from the present study.