Fast Drug Application 1

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Fast Drug ApplicationManfred Heckmann and Stefan Hallermann

1. What Do We Gain from Fast Drug Application?“The determination of the three-dimensional structures of pro-

tein molecules showed for the first time in detail the construction of the molecular ‘machines’ of the life cycle. If we want to learn how these ‘machines’ work, it is not sufficient only to know their construction. We actually have to see them at work, and this requires dynamical studies, i.e. penetration into the dimension of time” (Eigen, 1968).

The dimension of time is of particular relevance at synapses. Synaptic events are usually short, on the order of milliseconds, and their exact duration and timing are of prime relevance. “For many synaptic receptors/channels, the ‘natural’ mode of transmitter application is a short, steep pulse, and many of these systems show rapid desensitisation” (Dudel et al., 1992). This quotation is from the first review of a technique that allows very fast application of drugs to receptor channels in outside-out patches. This chapter provides practical tips and additional information for setting up a fast drug application system. For further information about this technique, see Jonas (1995) and Sachs (1999).

2. Material and EquipmentChapters 1 to 5 provided general information about the patch-

clamp equipment. This chapter focuses on the aspects that are rel-evant for fast drug application. We describe our system, consisting of a piezo, a monoluminal application pipette, and an application chamber.

From: Neuromethods, Vol. 38: Patch-Clamp Analysis: Advanced Techniques, Second EditionEdited by: W. Walz @ Humana Press Inc., Totowa, NJ

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2.1. The Microscope

In principle, the technique of fast drug application does not require a microscope. However, a microscope is helpful for patch-clamping in most preparations. We tried different types of micro-scopes and found upright microscopes more convenient than inverted microscopes. Differential interference contrast (DIC) optics facilitate visualizing the liquid filament, and a video camera with a monitor is also helpful. We use upright microscopes with a fixedstage (Fig. 1A). The micromanipulator is mounted on the table. If, instead, a microscope with a mobile stage is used, the micromanipu-lator should be mounted on the stage. With respect to the micro-manipulator, it is important to recognize that one needs to cover relatively long distances (up to 10 mm) with the electrode during an experiment with fast drug application. Because electrodes need to be replaced frequently, mounting the head-stage or the whole micro-manipulator on a hinge might help. In the course of an experiment, a patch electrode is lowered onto the preparation, an outside-out patch is excised (Hamill et al., 1981), and, with a fixed stage and the micromanipulator mounted on the table (Fig. 1A), the stage is moved relative to the electrode to reach the application chamber. Thereby the electrode tip remains in the optical field.

Fig. 1. A piezo-driven application system on the stage of an upright microscope. (A) The photograph and the schematic drawing show com-ponents of the system: the piezo device (1), which can be fixed in its frame (2) with a screw (3), and its power supply (4); a platelet (5) to hold the application pipette (6) connected to a tube (7); in- (8) and out-flow (9) of the application chamber (10); in- (11) and out-flow (12) of the bath (13); objective (14); head-stage of an Axopatch 200A amplifier (15); patch-clamp electrode (16); fixed stage (17) of an upright microscope. (B) The applica-tion pipette is made from borosilicate glass with an outer diameter of 0.5 mm and an inner diameter of about 0.3 mm. To obtain the desired angle, a longer piece of glass (5 to 10 cm long) is heated in the flame of a common lighter. A scratch with a diamond knife on the convex side of the bend facilitates breaking the glass as close to the bend as desired. The open end of the application pipette can then be fire-polished in the lighter flame. (C) The application pipette is glued into a fiberglass platelet. A tube whose inner diameter fits tightly the outer diameter of the pipette is then put over the other end. Finally, the tool is mounted to the piezo with a screw.

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2.2. The Piezo

The piezo (1 in Fig. 1A) is contained in a metal tube and pre-stressed by a spring. We use the low-voltage minitranslator P810.30 element from Physik Instrumente (Waldbronn, Germany). It length-ens by 45 μm on application of 100 V. It needs to be driven with sufficient power to overcome its capacitive load. Suitable power supplies for this piezo element are available from Physik Instru-mente. To fix the piezo device on the stage, we use a frame (2 in Fig. 1A), held by strong magnets. The piezo can be rotated within the frame to allow easy access to the application pipette. A plastic screw (3 in Fig. 1A) facilitates fixing the piezo.

2.3. Application Pipets and Tubing

Our application pipettes are made from glass tubes. We found borosilicate glass from Hilgenberg (Malsfeld, Germany), with an outer diameter of 0.5 mm and an inner diameter of about 0.3 mm suitable. An important constraint is that the opening of the applica-tion pipette needs to fit under the objective of the microscope. Usually we work with a 20× LD Zeiss objective (14 in Fig. 1A). Therefore, the application pipette needs to be bent to fit below the objective (Fig. 1B). To obtain the desired angle, we heat a longer piece of glass (5 to 10 cm long) in the flame of a common lighter. A scratch with a diamond knife on the convex side of the bend facili-tates breaking the glass as close to the bend as desired. One end of the application pipette is then fire-polished in the lighter flame and the application pipette is glued into a fiberglass platelet. A tube whose inner diameter fits tightly the outer diameter of the pipette is placed over its other end. Silicon might help to seal this con-nection. Finally, the platelet is mounted to the piezo with a screw. The tube is connected to a six-port valve (HVX 86915 Hamilton, Darmstadt, Germany) that facilitates changing the solution flowingto the application pipette rapidly. Syringes are used as solution reservoirs. The syringes are sealed airtight and connected to com-pressed air with a pressure regulator.

2.4. Application Chamber

The tip of the application pipette is placed in an application chamber (10 in Fig. 1A) that is separated from the rest of the bath. We make our application chamber from a Kimax glass tube (outer diameter 3 mm). An opening of about 120 degrees and about 1 cm


in length is made in the tube. This opening is the access to the chamber for both the application pipette and the patch electrode. The application chamber is glued into the bath chamber. The ends of the glass tube, that is, the application chamber, are then con-nected to a solution reservoir and a pump. It is important to obtain a very steady flow of the solution in the application chamber. We use the hydrostatic pressure depending on the height of the solu-tion reservoir and an adjustable pump to fine-tune the flow as precisely as possible. If the pump is not ideal, an air chamber (i.e., a large syringe, half full with water and connected parallel to the tube) may help to steady the flow. We adjust the pump so that a little solution is always sucked from the bath to avoid contamina-tion of the bath with the solution in the application chamber.

2.5. Patch Electrodes

Many electrodes are used in experiments with outside-out patches and a rapid application system. We prefer using electrodes with a resistance of about 10 MΩ when filled with a standard intra-cellular saline. We produce these electrodes on-line during the experiments, which has the advantage that the tip size and the geometry of the electrode can always be fine-tuned as desired. For example, by adjusting the tip diameter, one can try to increase or decrease the numbers of channels in a patch. We found the DMZ Universal Puller by Zeitz Instruments (München, Germany), suit-able, but other commercial pullers may also be appropriate. Zeitz Instruments also offers a quartz-glass puller for low-noise record-ings (Dudel et al., 2000; Hallermann et al., 2005).

2.6. Computers and Software

We use commercially available computers and software for the recording and evaluation of our data. A pulse generator like the Master8 from AMPI (Jerusalem, Israel), or the Max21 from Zeitz Instruments are useful to control the piezo device. Usually, the recording software triggers the pulse generator. Short current traces (100 to 3000 ms) are recorded at intervals of 0.01 to 60 seconds directly onto the hard drive of the computer. To make sure that periodic noise sources are likely to be out of phase, we use an interval of, say, 1.001 second instead of exactly 1 second. The most important functions of the data analysis program are averaging, subtracting, and fitting current traces. Jitter or imprecise timing of


the traces (inevitable in earlier times with videotapes or date-recorders) precludes precise subtraction. On good days up to 2,000 sweeps have to be processed per patch, which can be a cumber-some procedure with some programs. We found the ISO2 software (MFK, Taunusstein, Germany) more useful for this purpose than PClamp from Axon or PulseFit from HEKA. However, using ISO2 is more and more difficult on new computers due to outdated hardware requirements. To facilitate data evaluation, the processor hardware must perform optimally.

3. Procedures, Applications, and Results3.1. Testing the System

After the equipment has been set up, it needs to be tuned. The first step is to optimize the solution flow from the application pipette. One needs a steady stream of solution with a sharp inter-face (Fig. 2A). This can easily be judged with the microscope and the video monitor if there is a difference in osmolarity between the solutions. Turn on the solution flow, focus on the liquid filament,and check that it remains steadily in focus for, say, half an hour or the period you want to record from a patch. To measure the time course of the solution exchange, monitor the liquid junction poten-tial. A reasonable exchange rate is shown in Fig. 2C and D. Keep in mind that inevitably the exchange at an intact patch is somewhat slower than at an open pipette.

The position of the patch electrode relative to the liquid filamentis relevant for the solution exchange (Fig. 2E,F). It is important to map out the best position and to practice how to find it. With a real patch, there is no time for searching for the position, and the process is more difficult than with an open pipette. It is advisable to control the performance of the system routinely after experiments (use pressure to blow away the patch).

3.2. Working with Real Patches

Artifacts that superimpose with the currents of interest arise when the piezo is turned on and off. To illustrate the problem, we picked an extreme example (Fig. 3). We cannot give definitiveadvice about how to reduce these artifacts; you have to try several approaches to find the one that works. Small changes in the setup often have large effects. Particularly important are the mounting of the piezo on the stage, the size of the application pipette, its fixation


to the piezo, and the type of electrode glass. We have the impres-sion that quartz glass electrodes, which have otherwise superior noise performance, tend to ring more than usual borosilicate elec-trodes in our rapid application system. Some artifact will always

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Fig. 2. Testing the performance of the system. (A) The liquid filamentwith reduced osmolarity close to a patch-clamp electrode is visible in dif-ferential interference contrast. (B) In response to a voltage pulse (thin lines above the current traces), the liquid filament moves 45 μm toward the electrode. After the pulse, the liquid filament returns to its initial position (dotted lines). Recording the liquid junction current during such a move-ment with an open patch electrode facilitates measuring the speed of the solution exchange (C). In this case a 12.5-ms pulse was applied to the piezo. (D) The rise and decay phase of the trace shown in C are shown on an expended time scale. The 10% to 90% rise time is about 30 μs, and the decay of the current is fitted with a monoexponential function with a time constant of 44 μs. (E,F) Effects with less favorable positions of the same patch-clamp electrode. Due to unavoidable vibrations of the system, the tip is not necessarily always immersed for the whole pulse duration.


remain, which can be eliminated by subtraction of blank traces (Fig. 3C). Usually it is possible to start off with a much smaller vibration artifact than the one shown in Fig. 3 in a real recording, and it is then often possible to obtain clean average currents even at a very high gain (see, for example, the trace with 0.2 mM glutamate in Fig. 4 of Heckmann and Dudel, 1997).

3.3. Data Analysis

We review here the data for one particular receptor channel (Heckmann et al., 1996), to briefly illustrate how we obtain kinetic information from average current responses with a fast drug appli-cation system. The interested reader should also refer to Colquhoun et al. (1992), Dilger and Brett (1990), Dudel et al. (1990), and Franke et al. (1991).

Figure 4A shows data from a recording with an outside-out patch. In this case recombinant kainate receptors were exposed to various glutamate concentrations. A first step might be to measure

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Fig. 3. Vibration artifacts. (A) Average current response from an outside-out patch from a CA3 pyramidal neuron of a rat hippocampal slice. The patch was held at −80 mV, and 10 mM glutamate was applied for 50 ms. Large artifacts occur at the beginning and the end of the pulse (thin lines above the current traces). This is a common problem, but an extreme example is shown here. (B) Average current response from the same outside-out patch recorded a few seconds earlier without glutamate flowing through the application pipette. (C) Subtraction of the trace shown in B from the one in A purges the current.


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Fig. 4. From current traces to the rate constants of channel activation. The data shown here and in Fig. 6 are from outside-out patches with recombinant, homomeric kainate receptors (GluR6), expressed in HEK 293 cells. (A) Averaged current traces from a patch, which was held at −60 mV, are shown superimposed. The peak current amplitude depends on the glutamate concentration. Each trace is the average of three to seven single responses to 0.03, 0.3, 1, or 10 mM glutamate. The horizontal bar above the traces indicates the application (pulse duration 50 ms; interval 5 s). (B) Double logarithmic plot of the peak current î versus the glutamate concentration. î was normalized to the î value with 10 mM glutamate. The data from five patches are marked by different symbols. The gray line is a fit of the data with the kinetic mechanism and the rate constants given in Fig. 5. The peak current amplitude is half-maximal with about 0.5 mM (ratio of k−/k+). For glutamate concentrations Km, the slope is about 1.3, giving a lower limit of 2 for the number of binding steps. (C) Dependence of current rise time on glutamate concentration. The rising phase of the traces in A are shown normalized. In addition, the response of the open electrode recorded at the end of the experiment is shown. (D) Double logarithmic plot of the rise time (10–90%) versus the glutamate concentra-tion. The data from five patches are shown. The gray line is a fit of the data with the kinetic mechanism.


the affinity of the receptors. High densities of receptors are prefer-able in these experiments because one needs to measure current responses also at low agonist concentrations. Dose–response curves for the peak current amplitude and the current rise times are shown in Fig. 4B and D. The half-maximal activating concentration (Km) is about 0.5 mM (Fig. 4B). For glutamate concentrations well below the Km, the logarithmic slope is above 1.3 for these receptors. This provides a lower limit of 2 for the number of agonist binding steps (Fig. 5). To estimate the k+ and k− rate constants and the channel opening (β) and closing rate constants (α), we fitted the scheme to the two dose-response curves (Fig. 5). Commercial programs like ChanneLab from Synaptosoft Inc. (http://www.synaptosoft.com)or Berkeley Madonna (http://www.berkeleymadonna.com) are user-friendly environments in which to do these simulations. More information about the shape of the rise time curves is found in Franke et al. (1991), Heckmann et al. (1996), and von Beckerath et al. (1995).

As is apparent in Fig. 4A, the current decays in the presence of glutamate. This is due to desensitization of the receptors, and it demands at least one desensitized stated (A2D in Fig. 5) that is reached either from A2R or A2O. Experiments like the ones shown in Fig. 6C and D provide more information about the kinetics of desensitization. The experiments shown in Fig. 6A and B measure

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Fig. 6. Measuring desensitization. (A) Time course of recovery from desensitization. As shown in Fig. 4A, the currents desensitize. To measure the recovery from desensitization, here a control pulse was followed by a test pulse after 0.1, 0.3, 0.5, 1, 2, 3, and 5 s. Averaged currents of three to five single responses are shown. (B) Plot of the relative current amplitudes with 0.3 and 10 mM glutamate versus pulse interval. The solid and dashed gray lines are the results of computer simulations of these experiments for the two concentrations. (C) To quantify steady-state desensitization by low glutamate concentrations, responses to test pulses with a saturating glutamate concentration were recorded as controls. The other responses were recorded in the presence of 0.1 to 10 μM glutamate, as indicated on the left side of the traces. The traces are averages of four to seven single responses from one patch. (D) Double logarithmic plot of the relative peak current versus steady-state glutamate concentration. The data from four patches are shown. The gray line is the result of a computer simulation of this experiment with the model given in Fig. 5.


recovery from desensitization. Simulations facilitate testing reac-tion schemes (Fig. 5) and estimating the relevant rate constants.

In addition to the purely agonist elicited responses, the tech-nique of fast drug application can be used to study the mechanism of competitive inhibition, ion-channel block, and allosteric modification.

4. PerspectivesFast drug application allows one to obtain information about

many aspects of receptor channel function that cannot be studied in the steady state. The technique described here is very useful for basic research, characterization of new pharmacological com-pounds (Krampfl et al., 2006) or mutations in receptor channel subunits (Krampfl et al., 2005; Maljevic et al., 2006; Wyllie et al., 2006). With the modification described by Tour et al. (1995), even more complex pulse protocols are possible. The main limitation in experiments with fast drug application is the stability and quality of the recording, which requires training. A challenge for the future is to advance with fast drug application from average current traces to low-noise single-channel recordings (Hallermann et al., 2005).

AcknowledgmentsThis work is supported by the Deutsche Forschungsgemeins-

chaft.

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