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. Author manuscript; available in PMC: 2023 Feb 9.
Published in final edited form as: Methods Mol Biol. 2023;2565:239–260. doi: 10.1007/978-1-0716-2671-9_17

Patch Amperometry and Intracellular Patch Electrochemistry

Eugene V Mosharov 1, Manfred Lindau 2
PMCID: PMC9910029  NIHMSID: NIHMS1849708  PMID: 36205899

Abstract

Both, patch amperometry (PA) and intracellular patch electrochemistry (IPE) take advantage of a recording configuration where an electrochemical detector – carbon fiber electrode (CFE) – is housed inside a patch pipette. PA, which is employed in cell-attached or excised inside-out patch clamp configuration, offers high resolution patch capacitance measurements with simultaneous amperometric detection of catecholamines released during exocytosis. The method provides precise information on single vesicle size and quantal content, fusion pore conductance and permeability of the pore for catecholamines. IPE, on the other hand, measures cytosolic catecholamines that diffuse into the patch pipette following membrane rupture to achieve the whole-cell configuration. In amperometric mode, IPE detects total catechols, whereas in cyclic voltammetric mode it provides more specific information on the nature of the detected molecules and may selectively quantify catecholamines, providing a direct approach to determine cytosolic concentrations of catecholaminergic transmitters and their metabolites. Here we provide detailed instructions on setting up PA and IPE, performing experiments and analyzing the data.

Keywords: Patch amperometry, IPE, cyclic voltammetry, electrochemistry, exocytosis, synaptic vesicle, fusion pore, catecholamine, cytosolic

1. Introduction

Release of neurotransmitters and neuropeptides from neurons and release of hormones from neuroendocrine cells occurs by exocytosis: a specialized mechanism of secretion in which secretory vesicles fuse with the plasma membrane. Fusion of a single vesicle with the plasma membrane increases the plasma membrane area by the area of the vesicle membrane and produces a stepwise change in membrane capacitance. For large vesicles these capacitance steps can be measured in whole-cell patch clamp capacitance measurements [1]. Vesicle fusion with the plasma membrane begins with the formation of a narrow fusion pore [2]. As for ion channels, the biophysical properties of fusion pores were initially characterized mainly by determining the pore conductance and its fluctuations. The first measurements of fusion pore conductance were performed with whole-cell patch clamp recordings from beige mouse mast cells with extremely large (>2 μm diameter) secretory vesicles [3].

When a vesicle fuses with the plasma membrane in a whole-cell patch clamp recording under voltage clamp, the added vesicle capacitance is charged rapidly to the voltage clamp potential. For very large vesicles, the associated charging current can be measured as a brief current transient, which can be analyzed to reveal the fusion pore conductance during the first ~100 μs of its existence [3,[4,[5]. Once the charging transient ceases, the fusion pore conductance can still be monitored by analyzing the currents generated by time-varying voltages applied to the cell in voltage clamp because the vesicle membrane is charged and discharged via the narrow fusion pore [3,[6]. These early studies showed that the average initial fusion pore conductance is ~200–300 pS and that the fusion pore conductance subsequently fluctuates until it either increases to an unmeasurably large value, indicating full fusion with the plasma membrane, or returns to zero, indicating fusion pore closure in a transient fusion event.

When a vesicle releases its contents, the release of oxidizable molecules can be detected with a CFE because the released molecules transfer electrons to the CFE when they make contact with its surface [7]. The resulting amperometric current indicates directly the rate at which the molecules arrive at the CFE surface [8]. To determine the relation between fusion pore conductance and flux of transmitter, experiments were performed using beige mouse mast cells loaded with serotonin. Whole cell capacitance measurements were performed while a CFE recorded simultaneously the release of serotonin (Fig. 1a). These experiments revealed that in mast cells the release flux was proportional to fusion pore conductance [9]. These pioneering and ground-breaking experiments could not be repeated in the same way to study fusion pore conductance and release from neurons or neuroendocrine cells because neurosecretory vesicles are very small. Even in bovine chromaffin cells the dense core vesicles have a typical diameter of ~250 nm, corresponding to a vesicle membrane capacitance of only ~2 fF, which is difficult to resolve as a distinct capacitance step in whole cell patch clamp capacitance measurements.

Fig. 1.

Fig. 1

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Different modes of recordings. (a) Simultaneous whole-cell capacitance measurement and amperometric recording with a carbon fiber electrode (CFE). The equivalent circuit of the whole-cell patch clamp configuration with a fusing vesicle is shown in the box. (b) Patch amperometry (PA) in a cell-attached mode. The CFE and the ground electrode are both located inside the patch pipette. The sine wave for capacitance measurement is applied to the bath electrode. (c1) Equations for calculating fusion pore conductance (GP) and vesicle capacitance (CV) from the changes in real (Re) and imaginary (Im) parts of the current output of the lock-in amplifier. Note that Re and Im denote the changes relative to the respective baselines for each individual fusion event. (c2) Alternatively, the time course of GP can be calculated from Im and the final value of the Im change after vesicle full fusion (Imfinal). (d) Intracellular patch electrochemistry (IPE). After attaining a whole-cell mode, substances diffusing from the cytosol into the patch pipette are observed as a slow wave of oxidation current. CFE is used either in amperometric mode where it detects total catechols (a sum of catecholamines and their metabolites), or in cyclic voltammetric mode where it preferentially measures catecholamines.

Such small capacitance steps can, however, be well resolved in cell-attached patch clamp capacitance measurements [10,[11], which have very low noise [12]. The low noise comes from the much smaller patch membrane conductance and capacitance compared to the whole cell membrane. Although analysis of the rapid initial charging currents to determine the initial fusion pore conductance as in [3] has so far not been possible in cell-attached mode (see Note 1), the determination of fusion pore conductance from the real and imaginary part of the currents in a capacitance measurement using a lock-in amplifier is feasible [11] and the optimization of the recording parameters to determine fusion pore conductance has been analyzed [12].

Patch amperometry (PA) enables simultaneous measurements of vesicle fusion and transmitter release.

The dense core vesicles of chromaffin cells release catecholamines, which are oxidizable molecules and release from single vesicles can be well resolved using a CFE [7]. To combine cell-attached capacitance measurements with amperometric detection of release, the CFE needs to be mounted inside the patch pipette since release is now directed into the pipette tip. This led to the development of the patch amperometry technique (Fig. 1b), which has been described in considerable technical detail [13,[14]. PA recordings from bovine chromaffin cells revealed capacitance steps associated with amperometric spikes, demonstrating that these capacitance steps do indeed reflect exocytosis of single catecholaminergic dense core vesicles [15](see example on Fig 5a). Since the capacitance step size and the amperometric charge can be used to obtain the vesicle volume and the amount of catecholamines inside the same vesicle, correspondingly, the vesicular catecholamine concentration can be calculated for full fusion events [15,[16].

Fig. 5.

Fig. 5

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Examples of PA recordings. (a) Exocytotic events can be identified by the synchronous appearance of an amperometric spike (green trace), and a step in the Im (Y2, red trace) proportional to membrane area of fused vesicles. A transient increase in Re (Y1, blue trace ) associated with a capacitance step indicates formation and expansion of a narrow fusion pore (data from PMID: 12944522). (b) Exocytotic event with exceptionally long foot signal. From top to bottom: Amperometric current (IAmp, green trace), ΔCm (Im/ω, red trace), Re (blue trace), and fusion pore conductance GP (black trace) with superimposed foot current on expanded scale (IAmp, green trace). Note synchronous fluctuations in IAmp and GP (data from PMID: 9333242)..

Amperometric spikes are frequently preceded by a so-called “foot signal” [17]. And it was suspected that this foot signal or “pre spike feature” may reflect the slow leakage of transmitter through a narrow fusion pore. This hypothesis was confirmed using PA. It was found that the rate of catecholamine release fluctuates in parallel with the fusion pore conductance determined by sine wave-based admittance analysis (see example on Fig 5b), indicating that during the foot signal the rate of catecholamine release is controlled by the fusion pore [15,[18]. The initial fusion pore conductance is variable, as in other cell types, with a mean value of ~300 pS in cell-attached as well as excised inside-out patches [19]. Once the fusion pore opened, its conductance fluctuates until fusion goes to completion or until it closes again.

Intracellular Patch Electrochemistry (IPE) measures cytosolic oxidizable metabolites.

As PA experiments often conclude with the rupture of the plasma membrane, it was noticed that attaining the whole-cell is followed by a slow wave of amperometric current (see example on Fig 6a). Further analysis demonstrated that in chromaffin cells [20], PC12 cells [21] and primary cultures of mouse dopaminergic neurons [22,[23,[24] this current mostly represents oxidation of cytosolic catechols – the sum of catecholaminergic transmitters, such as DA, NE and Ep, and their metabolites, such as L-DOPA, DOPAC, DOPEG, etc. To provide specificity to the measurements of cytosolic molecules, CFE can also be used in cyclic voltammetric (CV) mode of detection, which has previously been employed to detect DA release kinetics from acute striatal slices [25]. As CFE in CV mode produces 10–20-fold higher oxidation peaks for DA, EP, and NE than for other catechol metabolites [20], a combination of amperometric and CV measurements yields information about both the cytosolic pools of the transmitters that have been shown to be cytotoxic, and about the rate of their turnover. Overall, to perform IPE recordings, the rig is modified, removing the lock-in amplifier and adding a capability to perform electrochemical recordings in the CV mode (Fig. 1d).

Fig. 6.

Fig. 6

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Example of IPE recordings. (a) Example of IPE recording in an amperometric mode. Cell membrane rupture characterized by a large increase in membrane capacitance (arrowhead) is followed by a slow wave of amperometric current that represents oxidation of cytosolic metabolites. In the case of chromaffin cells and primary mouse neurons, IPE in amperometric mode detects the sum of catecholamines and their metabolites. Notice that vesicle fusion events can be observed both before and after attaining the whole-cell mode. (b) In the CV mode, triangular voltage ramps at of 250 mV/msec are applied at 100 msec intervals (top), producing capacitive and Faradic currents (bottom). (c) Faradic component of the CV current presented as a preudo-3D plot where the intensity of color indicates oxidative current at a given voltage and time. Horizontal cross-section of the plot at a fixed voltage (pink) produces the time course of catecholamine diffusion into the tip of the pipette (d), whereas vertical cross-section at fixed time (green) gives a voltammogram of catecholamines (e). (f) Dilution of cytosolic catecholamines at the tip of the patch pipette is calculated as (Vcell+VPP)/Vcell. Cell volume Vcell = S*4/3*π*(Rcell)3, where S is 0.5, 0.75, or 1 depending on the shape of the cell under the microscope. Pipette volume VPP=π*h/3*(R12+ R22 +R1*R2), where h = 2 * h’. (adopted from PMID: 12843288)

2. Materials

All solutions should be prepared using ultrapure water and analytical grade reagents; store all reagents at room temperature. Follow all waste disposal and animal welfare regulations.

2.1. Solutions and small parts

  1. Cells to be studied need to be plated such that they are easily accessible on an inverted microscope (see Note 2).

  2. Pipette and bath solutions for cell type and experimental conditions to be studied. For chromaffin cells and primary murine dopaminergic neurons:
    1. Bath solution: 140 mM NaCl, 5 mM KCl, 2–5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES/NaOH, 10 mM glucose, pH 7.3.
    2. Pipette solution for PA: 50 mM NaCl, 100 mM TEA-Cl, 5 mM KCl, 10 mM CaCl2, 1 mM MgCl2, 10 mM HEPES/NaOH, pH 7.3.
    3. Pipette solution for IP: 140 mM KGlu, 2 mM MgCl2, 0.1 mM CaCl2, 1 mM EGTA, 10 mM HEPES, 2 mM ATP, 0.1 mM GTP, pH 7.3

  3. Teflon-coated silver wire.

  4. Borosilicate glass capillaries, outer diameter 2.0 mm, inner diameter 1.4 mm, length 85 mm.

  5. Carbon fibers (5 μm diameter).

  6. Polyethylene (PE)-tubing outer diameter 0.8 mm, inner diameter 0.4 mm.

  7. Patch pipette filling needle.

  8. 1 ml plastic syringes and 0.22 μm syringe filters.

  9. 3M KCl.

  10. 20 ml beaker with ethanol

  11. Two pairs of #5 fine forceps with tips protected by 0.5 cm of polyethylene tubing.

  12. Sticky wax.

  13. RG58C/U type coaxial cables with 50 Ω impedance.

2.2. Equipment

  • 1

    Inverted microscope on a vibration-isolation table with micromanipulators and microscope-mounted video camera (see Note 3).

  • 2

    Patch clamp amplifier such as the HEKA EPC-7, EPC-10, or Axopatch 200B (see Note 4).

  • 3

    Electrochemistry amplifier with grounded headstage connector shield (see Note 5).

  • 4

    Analog-to-digital (A/D) converter (see Note 6).

  • 5

    Computer for controlling the A/D board and data recording (see Note 7).

  • 6

    Pipette puller.

  • 7

    CFE-Pulling device (see Step 3.4.2).

  • 8

    Stereo microscope.

  • 9

    Patch amperometry pipette holder. Schematics and assembly instructions for a holder that fits the Axopatch 200B [20] are given below (see Step 3.5). STL files for printing the holder can be downloaded at https://github.com/DSulzerLab. We recommended using a stereolithography 3D printer such as FormLabs as it produces small electrode holder parts at sufficient accuracy and precision. See below for the assembly instructions.

2.3. Equipment for PA only

  • 10

    Lock-in amplifier (SR 830; Stanford Research Instruments, Sunnyvale, CA, USA) with a 10kΩ:100kΩ 1:10 voltage divider at the signal input [12].

  • 11

    Computer controlled analog signal adder. This device adds the sine wave from the lock-in to the holding potential and pulses as generated by the D/A converter. A TTL signal switches addition of the sine wave on and off (see Note 4).

  • 12

    Microforge for pipette tip conditioning (such as CPM-2; ALA Scientific Instruments Inc., Westbury, NY, USA) mounted on a suitable microscope.

  • 13

    Hotplate to melt sticky wax for pipette coating.

3. Method

3.1. Rig setup for PA

  1. Make connections on the rig as shown on Fig. 2a. Configure the data acquisition system to continuously record for at least 30 min 5 traces (Y1, Y2, I, V, A) at a sampling rate ≥1kHz. The digital-to-analog (D/A) output controls the holding potential and provides pulses to monitor seal formation. A second D/A or TTL output provides a trigger signal to synchronize the oscilloscopes with the pulses. A third digital output provides a TTL signal to switch the sine wave adder.

  2. The headstage of the amperometric amplifier is mounted on a micromanipulator to patch the cells and the headstage of the patch-clamp amplifier is mounted near the recording chamber. An Ag|AgCl wire immersed into the bath is connected to the input pin of the patch-clamp amplifier head stage. Do not make any connections to the ground pin of the headstage of the patch-clamp amplifier since the ground electrode will be connected to the amperometric amplifier shield.

  3. Image acquisition capabilities are optional to store images of the pipette tip on the cell for later determination of the distance between the patch membrane and the tip of the CFE. An inexpensive USB or firewire camera is sufficient. Alternatively, the distance can be measured and noted during the experiment with an eyepiece micrometer in the microscope.

  4. Set the lock-in amplifier SR830 as indicated in Table 1. Set EPC-7 STIM SCALING to 0.1, which produces a 50 mV rms pipette voltage from the 0.5 V sine wave provided by the lock-in amplifier. Note that the scaling factors for the Axopatch 200B differ for the front panel and back panel switched inputs. If the front panel switched input is used, then the scaling factor is 0.02 and the lock-in output voltage must be set to 2.5 V to obtain a 50 mV rms pipette voltage signal. For the EPC-7 built-in FILTER 1 is set to10 kHz to avoid clipping since a 20 kHz sine wave is used. The current monitor output is fed into the lock-in amplifier input via the 1:10 voltage divider because the output range of the EPC-7 is ±10 V, whereas the maximum input range of the lock-in amplifier is only ± 1 V. Since the whole-cell mode of the Axopatch employs a smaller feedback resistor lower gain may be used.

  5. At correct phase setting, the lock-in amplifier output Y1 is proportional to the membrane conductance and the output Y2 is proportional to the membrane capacitance [10]. The approximate phase setting for the lock-in amplifier can be found by slightly varying the C-slow compensation of the EPC-7. This may be done with open headstage. Set EPC-7 to voltage clamp mode, gain to 50 mV/pA; C-slow compensation to 10 pF range, C-slow to 0.2 pF, G-series to 0.2 μS, series resistance compensation off; filter1 to 10 kHz, filter2 to 3kHz; stimulus scaling to 0.1, TR to 2 μs. Apply sine wave to stimulus input and adjust C-fast and τ-fast to null the current. Now slightly vary C-slow making sure the EPC-7 is not saturated (not “clipping”) and change the phase setting of the lock-in amplifier. At the correct phase, changes in C-slow compensation appear only in the CH2 output (Y2, imaginary part of pipette current) with no change in CH1 output (Y1, real part). Note that an increase in C-slow compensation must produce a decrease in Y2 (capacitance decrease). This phase setting changes if any filters are changed or the gain is changed to the low gain range. Always use 50 mV/pA gain for low noise recording. The procedure is analogous for the Axopach 200B in the “whole cell β=1 mode” choosing the corresponding settings for SERIES RESISTANCE and WHOLE CELL CAP.

  6. Using the calibrated C-slow compensation, a defined change such as 200 fF can be applied. However, this produces a large capacitive current saturating the amplifier. Thus, for calibration the stimulus scaling must be switched from 0.1 to 0.01 giving a 10 times smaller signal. This does not affect the phase setting. Now the change of the CH2 output is recorded for the 200 fF C-slow change. Since the actual recordings from cells will be done with stimulus scaling set to 0.1, this CH2 output change corresponds to a 20 fF change in a cell recording (see Note 8). In the PA_Acquire program (see Note 7) a calibration value is entered to convert the data into capacitance and conductance units.

  7. The pipette current (I) is filtered with the built-in 3 kHz FILTER 2 to suppress the 20 kHz sine wave, and acquired by the A/D converter. An additional 500 Hz low pass filter (optional) is recommended to filter the pipette current. The stimulus voltage (V) is filtered at 500 Hz and acquired to monitor holding potential changes. Set the voltage on the amperometric amplifier to +700 mV and gain to 10–40 mV/pA and acquire the amperometric current (A) filtered at 500 Hz.

Fig 2.

Fig 2

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Schematics for the connections between different electronic components for PA (a) and IPE (b).

Table 1.

SR830 lock-in amplifier settings

Time constant 1 ms, 24 dB
Signal Input Input: A, Couple: AC, Ground: FLOAT
Sensitivity 1 V
Reserve LOW NOISE
Filters LINE & 2xLINE
Channel 1 Output X, OFFSET Off, RATIO Off, Expand Off
Channel 2 Output Y, OFFSET Off, RATIO Off, Expand Off
Interface RS232, 8, 9600, none
Reference Phase +120*, Freq 20 kHz, Ampl 0.5 V, Harm # 1, Trig SINE, Source INTERNAL
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*

The phase setting needs to be determined as described below.

3.2. Rig setup for IPE

  1. Make connections on the rig according to Fig. 2b. One D/A output controls the holding potential for the patch pipette and provides pulses for seal formation. Another D/A controls voltage applied to the CFE in amperometric and cyclic voltammetry (CV) modes. We used two Axopatch 200B amplifiers for the rig (see Notes 9 and 10).

  2. A detailed description of CV can be found elsewhere [26]. Data acquisition and analysis for IPE in both CV and amperometric modes were performed using a subroutine locally written in Igor Pro (see Note 7).

  3. Image acquisition is required for the calculation the dilution of cytosolic transmitter inside the tip of the patch pipette. An inexpensive USB or firewire camera is sufficient.

3.3. Pipette preparation and assembly

  1. Patch pipettes should have a tip diameter of 2 to 3 μm, a taper angle at the very tip of at least 20°, and a taper length of about 5 ± 0.5 mm. From 85 mm capillary, two pipettes of ~47 mm are yielded. A large opening angle is necessary to bring the tip of the CFE close to the pipette tip (see Note 11). Inspect each pipette after pulling for proper geometry.

  2. For PA only: It is essential to coat pipettes with wax or Sylgard as previously described to reduce and stabilize pipette capacitance [27]. The coating must cover a sufficient part of the tip such that it protrudes well beyond the bath surface to minimize stray capacitance and bath-pipette capacitance fluctuations. Dipping the pipette tip in melted wax to reduce stray capacitance is the quicker method [13]. Right before use, fire polish pipettes with a microforge: Approach a heated wire with the pipettes tip such that the wax flows out and the tip narrows slightly.

3.4. Carbon fiber electrode (CFE) fabrication

  1. Cut a ~12 cm long piece of polyethylene (PE) tubing, immerse one end in a beaker with ethanol, such that the entire tube fills by capillary action. Take a carbon fiber of at least 5 cm length with the PE-tube protected tweezers and insert it into the PE tubing. Wick away ethanol by tapping tube on filter paper. Evaporate remaining ethanol at 50 °C for 30 min (see Note 12).

  2. Bend a Pt/Ir wire of ~5 cm length and a diameter of 0.4 mm into a loop of ~6 mm diameter. Connect wire via a foot switch to a variable power supply (12V, > 3A). Place loop vertically under a dissection microscope. Adjust the heat of the wire loop by holding a piece of PE-tubing in the center of the wire loop (wire resistance of about 1 ohm, current about 2 A). After the heat is switched on, it should soften within 10–15 s, and melt within 20–25 s.

  3. With the loop placed horizontally, hold a carbon fiber filled tube with both hands in the center of the heated wire loop, melt PE, and pull very gently. Pull on one side (about 1 cm) and push on the other side about half as much. This creates a tapered electrode that will fit easily in the patch pipette. Let PE melt around carbon fiber as a thin layer (see Note 13). Do not let both parts separate yet. Take a fresh scalpel blade and separate both parts on a clean glass surface.

  4. Approach heated wire with carbon fiber tip such that the thin layer of PE melts back and forms a small bead. In a quick movement, switch off heat of wire, touch wire with the small PE-bead and pull CFE away from wire quickly. This forms an insulating layer of PE about 0.1 μm thickness around the carbon fiber Fig. 3. A description on how to set up a programmable CFE puller is available as Supplementary Note 1 at https://www.nature.com/articles/nmeth0905-699

  5. Before use, re-cut tip to expose a freshly cut surface. Backfill CFE with 3 M KCl with a syringe via a pipette-filling needle. Cut CFE to a total length of ~90 mm.

  6. Test CFE in amperometric and CV modes before electrode assembly.
    1. In amperometric mode: Directly after applying the +700 mV, potential the amperometric current should decrease slowly, reaching a level that represents a CFE resistance of 35–140 GΩ within 1–2 min (20–5 pA at +700 mV stimulation voltage). The noise of the amperometric current should be below 1 pA rms.
    2. In CV mode (for IPE only): Applying CV voltage ramps to the CFE produces large capacitive current the amplitude of which should stabilizes within 10–20 min. Add 1 μM dopamine to the bath solution and you should see a clear increase of the current that corresponds to DA oxidation potential (typically 350–400 mV). At the same time, the difference voltammogram should show the typical DA oxidation profile [26].

Fig 3.

Fig 3

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Example of a carbon fiber electrode prepared by asymmetrical pulling. Lower image is a magnification of the box on the upper image. Note that the PE tubing tappers smoothly onto the CF and its tip lacks any beads of melted PE. Such a CFE can be easily inserted into the glass patch pipette and its tip can be moved very close to the pipette tip. Scale bar is 100 μm.

3.5. Electrode and holder assembly

  1. The close distance between the CFE and the patch membrane is important to reduce diffusional broadening of the amperometric spikes [8] and diffusional low-pass filtering of amperometric current fluctuations associated with fluctuations of fusion pore conductance. To achieve this, pipette holders were developed that contain the CFE in fixed position while the part that holds the pipette is connected to the part holding the CFE with an adjustable thread. With this design, the distance between pipette tip and CFE tip can be varied before the cell is actually patched. For the positioning a compromise needs to be made such that the CFE tip is close enough to detect catecholamines released through the fusion pore with minimal diffusion time, but far enough not to interfere with the seal formation.

  2. An electrode holder design and assembly for the Axopatch 200B are shown on Fig 4. There are two silver wires. The CFE wire is connected to the center pin of the BNC-plug of the holder and will go inside the CFE tubing. The ground wire is connected to the side pin and will be inside the patch pipette but outside the CFE tubing. Make sure to clean off Teflon coating from silver wire ends.

  3. Cut the ground wire to be shorter than the CFE wire so that the two will be distinguishable when the holder is assembled. Chlorinate both wires after cutting.

  4. After assembly, only tighten threads T1 and T2 (Fig 4b). Turn the fine threaded cap (T4) until about 1/2–2/3 of the thread is exposed. Housing and cap should not move or tilt significantly against each other; if this happens, apply vacuum grease to the thread.

  5. Make sure thread T3 is not tightened and slide the 3M KCl-filled CFE all the way onto the longer silver wire making sure the latter touches the KCl inside the PE tubing but is not pushed all the way to the tip of it. After tightening T3, the CFE should be fixed inside the holder.

  6. Make sure thread T5 is not tightened and carefully insert the CFE tip into a glass pipette filled with pipette solution, preferably under a dissection microscope or a magnifying glass. Take care that the CFE does neither bend backwards nor break nor poke through the pipette tip. Unscrew T4 if CFE is too long and does not fit inside the pipette - if necessary, remove the CFE from the holder by loosening T3, cut it from the back and start over from step 4.

  7. Once the glass pipette is in place and the CFE is visible inside, tighten T5.

  8. Under a stereomicroscope, turn the fine threaded cap (T4) to bring the tip of the CFE close to the tip of the pipette. Be careful not to push through the pipette tip with the fiber as this will not only destroy the pipette, but also scrape off the PE insulation from the CFE. Don’t try to push the CFE too far as it’s optimal position will be determined later.

  9. Attach the electrode holder to the headstage with T6 on the collar. Use a crocodile clip to connect the ground pin of the holder to headstage’s ground.

  10. Add about 100 μl of bath solution and immerse the tip of the patch clamp electrode in the bath.

  11. If the CFE is too close to the tip of the pipette, it’s PE coating will start blocking the tip also acting as a resistor that will interfere with catecholamine detection and capacitance measurements. This can be monitored as a change in the height and the shape of the square test pulses applied to the patch clamp amplifier or CV current applied to the electrochemistry amplifier. Start test pulses and/or CV pulses and carefully turn T4 of the holder to approach the pipette tip with the CFE tip until they are 5–10 μm apart (1–2 times the CFE diameter). Again, be careful not to poke the CFE through the pipette tip as this might scratch off the thin insulation layer on the CFE. Continue moving the CFE closer, noting the change in the pulse current or CV current. If current transients decrease or changes shape, stop and unscrew T4 (thus moving CFE away from the tip of the patch pipette) till currents are undistorted and look the same as before (see Note 14).

  12. At final CFE position, acquire two images for further measurement of catecholamine diffusional distance (Y). Take one image focusing on the tip of the patch pipette and another one focusing on the tip of the CFE. Superimpose the two images and measure the distance h’ of the vertical projection of h. As the holder is typically at 45° to the plane of the dish, h can be approximated as 1.41*h’ (see Fig. 6).

Fig. 4.

Fig. 4

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PA/IPE Electrode holder design and assembly. Schematic of the assembled electrode holder (a), 3D model of its parts (b) and individual parts of the holder (c). Connecting threads (T1–6) are shown on b.

3.6. Patching and recording exocytotic events with PA

  1. For general patch clamp instructions follow the excellent description provided by Penner [28]. Use low patch clamp amplifier gain and give test pulses. Put slight positive pressure on the pipette and approach a cell using the micromanipulators. Upon touching a cell, the pipette resistance will slightly increase. Release positive pressure and apply suction until pipette resistance reaches several GΩ.

  2. Increase gain of the EPC-7 to 50 mV/pA and set stimulus scaling to 0.1. Adjust C-fast and τ-fast compensation. Switch off test pulses, switch on sine wave and start data acquisition. Readjust C-fast and τ-fast to compensate remaining capacitive currents until the sine wave current has minimal amplitude on the oscilloscope. Give some capacitance calibration pulses (if necessary, at reduced stimulus scaling; see Step 3.1.5). Try to complete these adjustments within a few seconds after achieving a gigaseal as cells release most releasable vesicles at the beginning of the recording due to mechanical stimulus of patching.

  3. Exocytotic events can be identified by the synchronous appearance of an amperometric spike and a step in the Y2 trace proportional to membrane area increment, possibly associated with a transient increase in Y1 indicating a narrow fusion pore (Fig 5a). Amperometric spikes without a change in Y2 trace indicate a transient breakage of the cell during sealing and freely floating vesicles outside the cell or clipping of the patch clamp amplifier. Observe the sine wave amplitude of the patch clamp current monitor during the experiment. Make sure it does not exceed ±10 V, even if the clipping indicator might not be on. Readjust C-fast and τ-fast compensation to decrease the current amplitude. Steps in Y2 and a transient in Y1 without amperometric spike might indicate that the CFE is too far from the patch, the CFE is broken or the vesicle was empty [29]. If capacitance steps are not confined to the Y2 trace but have a projection in Y1 either the EPC-7 is clipping (check!) or the phase of the patch is different from the pre-set phase. This may be corrected off-line (while clipping cannot be corrected for). To test the phase setting gentle suction pulses may be applied which produce capacitance changes. They will likely have the same projection in Y1 as the capacitance steps from exocytotic events.

  4. The end of an experiment is marked by either losing the seal or by the cell going to the whole cell configuration. In the latter case the cytosolic catecholamines diffuse out of the cell and cause a rather slow but fairly big wave in the oxidation current [20] (you are doing IPE now!).

  5. Event analysis addresses quantal analysis, vesicular concentrations, considerations for patch capacitance measurements and fusion pore analysis [11,[12,[15,[16,[18,[19,[29,[30] as well amperometric spike analysis [8,[17,[31,[32,[33,[34,[35] and relationship between cell electrical activity and fusion. A detailed description of fusion pore analysis for cell-attached capacitance measurements and consideration for choosing the optimal sine wave frequency depending on vesicle size to be studied has been provided elsewhere [12].

  6. Vesicle capacitance (CV) and fusion pore conductance (GP) can be calculated from the real and imaginary components of the lock-in amplifier current as shown in Fig 1c (see Note 15). An Igor Pro experiment to view the recordings and to perform analysis, can be downloaded as Supplementary Software from https://www.nature.com/articles/nmeth0905-699

3.7. Measuring cytosolic catecholamines with IPE

  1. Acquire images of the pipette (see 3.5.11) and the cell body. Make sure to record image scaling. These will be used during analysis to estimate dilution of cytosolic catecholamines inside the tip of the patch pipette.

  2. After attaining high resistance seal (see 3.6.1), apply sharp suction to the patch pipette. When the plasma membrane is ruptured, substances diffusing from the cytosol into the patch pipette are observed as a slow wave of oxidation current. In amperometric mode, IPE detects total catechols (a sum of catecholamines and their metabolites), whereas in cyclic voltammetric mode, it preferentially measures catecholamines [20].

  3. In the amperometric mode, the CFE potential is held at +700 mV. After baseline subtraction, the total area, the charge (Q), of the oxidation current wave is used to estimate cytosolic catechol concentration as C = Q / (n * F * V), where n=2 is the number of electrons transferred from one molecule to the CFE, F=96,485 C/mol is Faraday’s constant, and Vcell is the cytosolic volume (Fig. 6).

  4. In CV mode, triangular voltage ramps from a holding −450 mV potential to +800 mV and then back to −450 mV over 10 msec (scan rate, 250 mV/msec) are applied at 100 msec intervals (Fig. 6). During the ramps, catecholamines produce a unique oxidation–reduction profile (voltammogram) allowing to differentiate them from other oxidizable cytosolic metabolites.

  5. Start CV pulses with patch pipette submerged in the bath saline. Let pulses run till the amplitude of CV current transients (and current sampled at the oxidation potential for catecholamines; 300–450 mV) is stable.

  6. After recording the wave of cytosolic transmitter entering the patch pipette, measure catecholamine-specific oxidation at the maximum of the current wave using subtraction voltammogram.

  7. The height of the catecholamine oxidation peak can then be converted to catecholamine concentration using calibration curves. After accounting for differences in the active CFE area and the dilution of catecholamines inside the patch pipette (Fig. 6e), concentration in the cytosol can be calculated (see Note 16). This is described in detail in [20] and is incorporated into Igor Pro routine for the analysis of IPE data (see Note 7).

Supplementary Material

PA/IPE electrode holder

Acknowledgements

The preparation of this chapter was supported by NIH grant R35GM139608 to M.L. and a JPB grant (PI: D. Sulzer) for E.V.M.

4 Notes

Analysis of the rapid charging transients to determine the initial fusion pore conductance has so far not been possible in cell-attached because for a vesicle capacitance CV=2 fF, even for an extremely small fusion pore with a conductance GP as low as 10 pS, the time constant to charge the vesicle τ=CV/GP would be as short as 200 μs. The total charge to change the vesicle membrane potential by 100 mV is 0.2 fC or about 1,250 elementary charges. While this charge may in principle be detectable, so far it has not been possible to resolve the time course of such small transients in a cell-attached recording, which would be required to obtain a fusion pore conductance estimate.

Since the electrode configuration is reversed (the sine wave voltage is applied to the bath electrode) the stray capacitance between the bath solution and the microscope needs to be minimized to compensate most of it and to reduce noise. We use custom-made dishes in which a 10 mm hole is punched at the center of the tissue culture dishes and a 12 mm cover slip is glued underneath with Sylgard®. After poly-d-lysine and laminin coating, any type of cells can be plated. For the experiment, fill solution only into the inner well (typically <100 μL). Do not spill solution onto the surrounding plastic since overflowing and evaporation of solution gives rise to cycles of large stray capacitance, making PA recordings extremely difficult.

It is important to use an objective with long working distance to minimize stray capacitance. We use a Zeiss 20x/0.5 or 40x/0.75 Plan-Neofluar objectives. An immersion objective on an upright microscope would produce a very high stray capacitance that cannot be compensated.

For PA, the head stage to which the pipette holder is connected must provide ground potential to the pipette holder shield. This is important to consider because some patch clamp amplifier head stages normally drive the head stage housing and pipette holder shield with the pipette potential to reduce stray capacitance. In this case a modified headstage must be used where the housing is grounded. We obtained best results with the EPC-7. Similarly, the Axopatch 200B can be used in “whole cell β=1” mode for both PA and IPE. As the Axopatch 200B has two command signal inputs that are added internally - one being controlled by a front panel switch - the signal adder is not necessary, although the sine wave must then be switched on and off manually.

Axopatch 200B can be used as the electrochemistry amplifier for both PA and IPE. Additionally, a modified version of VA10 (NPI Electronic) with grounded headstage shield can be employed. The HEKA EPC-10 is a dual amplifier that may be very practical. However, for the PA, a modified EPC-10 headstage must be used due to the fact that probe housing in the standard EPC-10 configuration is connected to “Vref”. This standard headstage thus cannot be used since the reference electrode in the holder is connected to the shield. A modified headstage is available from HEKA where the housing and BNC connector shield are changed to “GND”.

For PA, an A/D board with at least 5 channels and a sampling rate >1 kHz is required; for IPE, only 2 channels are needed. We used National Instruments PCI-MIO-16XE-10 board for PA and InstruTECH ITC-18 board for IPE.

For IPE data acquisition and analysis, we use home-written macros in Igor Pro (Wavemetrics) available for download at https://github.com/DSulzerLab. Igor Pro macros for PA acquisition and analysis are available from https://www.nature.com/articles/nmeth0905-699.

For convenience we modified the patch clamp amplifier by inserting a switchable 20 Ω resistor in series with the C-slow potentiometer of the EPC-7. This capacitance dither applies capacitance compensation changes of 20 fF for calibration. In the Axopatch 200B the built-in 100 fF capacitance dither may be used but make sure to avoid saturation of the amplifier. It is possible to change the dithering circuit to provide a 10 or 20 fF signal following guidelines available upon request from Axon Instruments.

During CV, voltages up to 1V are applied to the CFE. As Axopatch 200B amplifier has a 1/10 divider on its command input, a D/A converter capable of generating an output voltage up to 10V is required. We used an ITC-18 D/A for this purpose, but there might be other options that we did not test.

Patch pipette command voltage is only needed for the control of seal formation between the plasma membrane and the glass pipette. We therefore do not routinely record the current output from the patch pipette amplifier and are using amplifier’s own controls to transition from cell-attached to whole-cell configurations.

Although puller settings need to be found experimentally, the following might serve as a guideline: The Sutter puller P-97 is set up with a box filament of 3 × 3 × 3 mm. Air pressure was set to 500, the air before pull and the air after pull was 5 seconds each. The ramp test for the capillaries was 655. A single line program looped four times and had the parameters heat 615, pull 0, velocity 50, and time 200. For larger taper angles the velocity might be reduced slightly without increasing the tip diameter substantially. Pipette resistance in the bath should be 1–3 MΩ.

An alternative method is to use vacuum to suck the CF inside the PE tubing. On a piece of white paper, separate exactly one CF fiber (>10 cm) from a fiber bundle (this can be done under a dissection microscope. Holding it with a finger on one side, approach the other side with a 12–15 cm piece of PE tubing connected to a vacuum line. Once the CF is sucked inside the tubing, disconnect from vacuum, then carefully let go of the finger. Cut excess CF from both sides.

Pulling of PE tubing requires some practice. Pull just PE tubing without carbon fibers first until desired shape and taper are achieved. When pulling with a carbon fiber, make sure that PE melts around the fiber and does not just form a very thin tube.

A longer distance will broaden and flatten amperometric signals due to diffusion. It is difficult to record good amperometric signals at distances > 30 μm. At too short a distance, besides possible interference with electrophysiological and electrochemical measurements, the CFE might interfere with seal formation as the patch of the cell is sucked into the pipette during sealing.

When a fusion pore is formed and if this is the only change during the respective time interval, CV and GP can be calculated for the time course of Im and Re using the equations of Fig. 1c1. It is, however, possible that during the time interval of interest ion channels open or close or the seal resistance fluctuates. These phenomena produce additional changes in the Re trace such that the equations of Fig.1c1 will not provide accurate results. In this case, the equations of Fig.1c2 may be used calculating GP from Im and the final capacitance step size. With this method, the predicted and measured time courses should be compared to determine if the recording is consistent with the properties expected of a fusion pore opening.

As IPE in CV mode detects catecholamine concentration at the tip of the CFE, which is a few micrometers away from the cell, it is necessary to account for the dilution of molecules inside the patch pipette. A simple “geometric” approach can be used where cellular volume and the volume of the patch pipette are estimated from photographs taken before each recording, and the concentration of catecholamines at the CFE is multiplied by the calculated dilution factor (Fig 6f). However, this method underestimates the true cytosolic catecholamine concentrations for two reasons: (1) there is a loss of molecules from the diffusion of catecholamines around the CFE into the shaft of the pipette, and (2) the actual free volume of the cytosol can be smaller than the total cell volume as a portion of the cytosol is occupied by vesicles and other organelles. Therefore, as an alternative approach, we used a random walk simulation [36] to fit experimentally recorded sampled current traces and calculate the initial concentration of catecholamines inside the cell. Based on the comparison of the two approaches, a simpler geometric method was employed and then multiplied by the factor of 1.65 for a more accurate representation of cytosolic transmitter concentration.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

PA/IPE electrode holder
NIHMS1849708-supplement-PA_IPE_electrode_holder.zip (7.5MB, zip)

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