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. 2019 Nov 19;8:e48178. doi: 10.7554/eLife.48178

Figure 3. Pipette cleaning protocol.

(A) Design of custom-made recording chamber with one centre well for the brain slice and recording solution and another circular outer well for the cleaning solution, see construction design in Appendix 1—figure 1 and 2. (B–H) Experimental steps for pipette cleaning. Tree diagrams on the top left depict the configuration of the pressure system with the following pressure levels: LOW, ATMOSPHERE, HIGH, PATCH, CLEAN. (B) Pipettes are moved into the recording solution with LOW pressure. (C) Cells are approached with HIGH pressure. (D) Formation of gigaseal and membrane rupture with pressures applied through the PATCH channel with either mouth piece or syringe. (E) Cells are kept at ATMOSPHERE pressure after successful patch. (F) Pipettes are moved into the cleaning solution and switched to CLEAN pressure. (G) Cleaning pressure sequence is applied in the outer well. (H) Pipettes are moved back into the recording solution above the slice and switched to LOW pressure. (I) Current clamp traces at resting membrane potential exhibiting spontaneous EPSPs recorded using the same pipette before (blue) and after cleaning (red and yellow). (J) Box plots of cellular, synaptic and recording properties of cells recorded with fresh (blue, n = 25, except n = 23 for synaptic parameters) and cleaned pipettes (red, n = 21 and yellow, n = 21). (K) Action potential firing patterns elicited through step current injection and enlarged action potential traces recorded on the same pipette before (blue) and after cleaning (red and yellow). (L) Box plots of action potential properties of cells recorded with fresh (blue, n = 25) and cleaned pipettes (red and yellow, n = 21). Further statistical information and analysis are shown in Figure 3—source data 1.

Figure 3—source data 1. Statistical summary intrinsic parameters.
Figure 3—source data 2. Statistical summary synaptic parameters.

Figure 3.

Figure 3—figure supplement 1. Fresh pipette vs. cleaned pipette.

Figure 3—figure supplement 1.

Boxplots depict the intrinsic electrophysiological properties of different human neurons patched either with fresh pipettes (blue, n = 24) or cleaned pipettes (red, n = 9). Further statistical data in Figure 3—source data 1.
Figure 3—figure supplement 2. Fresh pipette vs. repatch of same cell with cleaned pipette.

Figure 3—figure supplement 2.

Left panel with line plots shows the intrinsic electrophysiological properties of the same human neuron (each line) patched with a fresh pipette (‘P1’, blue) and repatched with the same pipette after cleaning (‘RE’, red). Right panel shows the relative change for each neuron. Out of 15 neurons, 9 were included (black lines and circles) and 6 were excluded due to depolarised resting membrane potential after repatch (gray lines and circles). Further statistical data in Figure 3—source data 1.
Figure 3—figure supplement 3. Fresh pipette vs. repatch with fresh pipette.

Figure 3—figure supplement 3.

Left panel with line plots shows the intrinsic electrophysiological properties of the same human neuron (n = 5, each line) patched with a fresh pipette (‘P1’, blue) and repatched with another fresh pipette (‘RE’, blue). Right panel shows the relative change for each neuron. Further statistical data in Figure 3—source data 1.
Figure 3—figure supplement 4. ACSF before vs. after rinsing during cleaning.

Figure 3—figure supplement 4.

Left panel with line plots shows the intrinsic electrophysiological properties of the same human neuron (each line) patched with a fresh pipette in fresh ACSF (‘Before’, blue) and consecutively held in ACSF which has been subject to rinsing during the cleaning of two other pipettes (‘After’, red). Right panel shows the relative change for each neuron. Out of 22 neurons, 19 were included (black lines and circles) and three were excluded due to depolarised resting membrane potential in the initial recording (gray lines and circles). Further statistical data in Figure 3—source data 1.
Figure 3—figure supplement 5. Synaptic amplitudes recorded in ACSF before vs. after rinsing during cleaning.

Figure 3—figure supplement 5.

Boxplots show distribution of individual excitatory postsynaptic potential amplitudes acquired from connected human neuronal pairs (n = 20 individual sweeps). Seven synaptic pairs were recorded in fresh ACSF (blue) and again recorded in ACSF which has been subject to rinsing during the cleaning of other pipettes (red). Synaptic pair two showed a significant increase, while synaptic pair three showed a significant decrease in amplitude (Mann-Whitney U Test). Overall, neither a significant trend toward increasing or decreasing amplitudes were found. Further statistical data in Figure 3—source data 2.