A Computer-assisted Multi-electrode Patch-clamp System

Summary

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

Abstract

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Introduction

The capacity to record and stimulate multiple sites with micrometer precision is extremely useful for experimentally achieving a better understanding of neuronal systems. Many techniques have been developed to this end but none allow the submillivolt resolution achieved by the patch-clamp technique, essential for studying subthreshold activity and individual postsynaptic potentials. Here we cover the development of a twelve-electrode computer-assisted patch-clamp system aimed at simultaneously recording and stimulating a large number of individual cells with sufficient precision for the study of neuronal connectivity. While many other applications can be conceived for such a system, it lends itself particularly well to the study of synaptic connectivity given that the number of possible connections within a group of neurons grows proportionally to the square of the number of neurons in question. Therefore, while a system with three electrodes allows testing the occurrence of up to six connections and most often recording a single one, recording twelve neurons allows testing the occurrence of up to 132 connections and frequently observing over one dozen (Figure 1). The observation of dozens of connections simultaneously makes it possible to analyze the organization of small networks and infer statistical properties of the network structure that cannot be probed otherwise¹. Moreover, precise stimulation of numerous cells also allows the quantification of recruitment of postsynaptic cells².

Protocol

1. Equipment Preparation

1. Control manipulators from a computer
   1. Connect each micromanipulator controller box to a computer through serial ports (RS-232).
   2. Implement the commands for positioning, querying and adjusting settings to be sent via the serial port. Given speed and hardware compatibility issues C/C++ is recommended as the programming language.
   3. Standardize the reference system of the manipulators so that zero is the closest possible position with respect to the motors and positive movement is directed away from the motors.
   4. Position the microscope at its central position 2 mm above the specimen focal plane (coordinates [0, 0, 2000]).
   5. Store, for each manipulator, the local coordinates that allow the tip of a pipette to be observed in the center of the field of view of the microscope. This is the initial reference point for each electrode.
2. Visualize electrode positions. It is very useful to be able to track the position of each electrode during an experiment. A graphical representation is the most intuitive way of accomplishing this. To that end the reference systems of each electrode and that of the microscope must be matched. A simple way to accomplish this is:
   1. Bring the tip of an electrode to the center of the field of view.
   2. Store the position on each axis of the manipulator and each axis of the microscope.
   3. Execute with the x-axis of the manipulator a relatively large movement (1 mm).
   4. Locate the tip once more moving only the microscope.
   5. Calculate the difference in the microscope position since it was last measured. These are the projections of the electrode axis movement onto the microscope axes.
   6. Repeat steps 2.3 - 2.5 for the Y and Z axes of the electrode manipulator. This allows a matrix of projections onto the microscope axes to be determined (Figure 2) also called cosine matrix:
      
   7. Invert this matrix to make it possible to calculate the movements, in three dimensions, required by the electrode manipulator to reach a given position in the microscope coordinate system:
      
   8. Using the initial reference point and the cosine matrix determine the position of each electrode in microscope coordinates.
   9. Display graphically, based on the microscope coordinates, the position of each electrode at regular intervals. We chose to use C/C++ drawing libraries (GDI+) to draw electrode positions every 40 msec.
   10. Repeat steps 1.2.1 - 1.2.7 every time manipulator angles are changed or if position changes of several millimeters are needed, for example, when the type of electrode is changed.
3. Enable storing the positions of relevant features in the tissue, such as cells or anatomical reference points, in microscope coordinates.
4. Acquire video and overlay relevant information.
   1. Mechanically align the x-axis of motion of the microscope with the horizontal axis of the microscope camera.
   2. Install on the computer a framegrabber with live video and overlay capability and a software development kit (SDK).
   3. Implement live video display operation with the SDK.
   4. Convert the microscope coordinate system to the camera reference system by the appropriate translation and scaling.
   5. Draw the relevant features in camera coordinates and overlay on the live video the resulting image at regular intervals of about 40 msec (Figure 3).
5. Control Amplifiers.
   1. Use the amplifier software to control the amplifier settings from the interface.
6. Control oscilloscopes.
   1. Connect the oscilloscopes to the PC using serial ports.
   2. Determine the oscilloscope scale, coupling and temporal resolution for the different steps of the patch-clamp procedure in voltage-clamp (e.g. electrode in bath, seal formation, whole-cell configuration) and current-clamp.
   3. Send the appropriate oscilloscope setting commands whenever amplifier commands are issued from the interface.
7. Control pipette pressure.
   1. Assemble a pressure control system according to Figure 4.
      1. Use a 12 V/5 V power supply to supply each electronic component appropriately.
      2. Connect the output of one membrane pump to the positive pressure buffer (a 100 ml container).
      3. Connect the input of one membrane pump to the negative pressure buffer (a 100 ml container).
      4. Connect the pipette holder tubing to a pressure sensor in the pressure control system and a pneumatic valve that connects to the main pressure compartment.
      5. Connect each of the pressure buffers to a valve connected to the main pressure compartment.
      6. Connect a valve between the main pressure compartment and the atmosphere.
      7. Connect a pressure sensor to the main pressure compartment.
      8. Connect a pressure sensor to each buffer.
      9. Connect the pressure control system to a data acquisition board.
      10. Connect each pressure sensor to one analog input.
      11. Connect each valve to a digital output.
   2. Remove atmospheric pressure offset from all sensors by opening all valves except those connecting to the membrane pumps and subtracting the measured pressure.
   3. Implement pressure control
      1. Define in the interface a control to activate or de-activate positive pressure control for each pipette.
      2. Define a minimal positive pressure for the pipettes of about 70 mbar.
      3. Periodically (every 0.5 sec) detect whenever pressure in pipettes under active pressure control drops below the set threshold.
      4. Upon threshold crossing open the positive pressure buffer to the main pressure compartment and this compartment towards the pipette in question during brief periods (20 msec) until pressure in the pipettes is above threshold. Close all other valves.
      5. De-activate further pressure control as the final approach towards a cell of interest is initiated.
   4. Apply negative pressure for seal formation
      1. Close all valves and open the negative pressure buffer valve towards the main pressure compartment and this compartment towards the pipette in question for the duration the experimenter requires by keeping a button pressed.
8. Centralize commands onto a human interface device.
   1. Connect a commercially available wireless gamepad to the PC.
   2. Implement readout of joystick status. For instance, use the DirectX libraries for C/C++ to perform readouts every 5 msec.
   3. Compare current status with previous status to detect which buttons have been pressed, released or kept pressed since last time step.
   4. Assign functions to each button in the gamepad. An example of this mapping is shown in Figure 5.

2. Patch-clamp Procedure

1. Prepare the brain slices of the region of interest.
2. Place a brain slice of interest, with the region of interest in the center of the microscope's displacement range.
3. Cell Selection
   1. Identify cells of interest by browsing with the microscope. Store the position of the cells based on the microscope coordinate system with a right mouse-click on the live video display on top of the cell of interest.
   2. The graphical interface will display the selected cells as well as the micropipettes for a global overview and software will add a marker on the cell position that will be overlaid on the images. Additionally an image of the cell is captured for future reference in the file 'Cell#.jpg'.
4. Attribution of cells to pipettes
   1. After selecting the cells of interest, assign which pipette will record each cell. The graphical interface provides assignment controls that should be set. Visualize a preview of the final configuration by selecting the checkbox 'Show Final Positions'.
   2. Select each pipette for which the final position preview is desired or check the 'Select All' checkbox. Disable the current position display for better visualization if needed.
5. Prepare the Pipettes
   1. Fill the pipettes (6 - 8 MΩ are usually best if many pipettes are used) with intracellular solution and load them into their holders. Place the headstages in their fixations but do not slide them forward to avoid touching the bath with the pipette tip.
   2. Enable the positive pressure control and select all pipettes to ensure that the tips will remain clean. Gently slide each headstage in place.
6. Locating the pipette tips
   1. Position the microscope to a central position with focus 3 mm above the slice by pressing button R2 while holding button 'A'. Use the corresponding position for each manipulator as stored from the previous experiment by pressing button L2 while holding button A.
      Note: At this point there should be either a distinctive shadow of a pipette in view or a small movement along the pipette's axis should be enough to observe it in most cases. Compensation for the slight differences in pipette shape has to be performed manually.
   2. Bring the pipette tip into focus. Place the tip on the red central dot of the video display without moving the microscope (it should still be at position [0, 0, 2000]).
   3. Inform the software that the pipette is at the center of the display by pressing button Z while holding button C. After each pipette tip is located, send that pipette backwards so that the following one can be located by pressing button L1 while holding button A.
7. Approaching the cells
   1. Once all pipette tips have been precisely located and each pipette is attributed to a cell, automatically position the pipettes close to their respective cells. Simply right-click in the center of the group of cells and select the option 'Cluster' on the popup menu. On the cluster options window that will appear, select all the pipettes that you want to position at this time and click on 'Go with all checked pipettes'. Repeat this operation for each cluster of cells of interest.
   2. Perform the final approach manually. The position of the pipettes relative to the cells can be specified differently but usually is simply 200 µm away from the cell in the axis of the pipette and 200 µm above it in the vertical direction, which is enough to keep the pipette's tip outside the tissue. Wait until the positioning of the pipettes is finished and move the microscope towards a pipette by pressing button R1 while holding button C.
   3. Recalibrate each pipette position by focusing the microscope on its tip anywhere in the video display and pressing button B while holding button C. A square grid should briefly appear indicating the identified position of the pipette. If the position is not correct, position the tip on the central dot of the video display and press button Z while holding button C.
8. Establish the cell-attached configuration
   1. Confirm that the cell of interest for the current pipette is correctly marked. Otherwise move the microscope to match the cell of interest with the central red dot. Mark the cell by pressing button Y while holding button C. Press button L2 while holding button C to position the pipette 200 µm away from its assigned cell, the microscope will automatically move to the corresponding position.
   2. Adjust the pipette position so it matches the red dot. Adjust the pipette offset by pressing button R1 while holding button X.
   3. Activate the test pulse by pressing button L1 while holding button X. Slowly approach the pipette to its attributed cell.
   4. Upon observing the formation of a dimple on the surface of the cell's membrane apply a brief pulse of negative pressure by pressing button Y while holding button Z to allow the applied pressure to reach the cell. A holding potential of about -65mV should established at this point by pressing the button L2 while holding button X.
9. Whole-cell configuration
   1. Once a Giga-seal is formed for each cell, start rupturing the membranes by applying negative pressure.
10. Perform recordings
    1. Use the stimulation/acquisition system to perform your recordings. Apply pulses or trains of pulses on one individual cell at a time and observe responses on the remaining cells to map connectivity among the recorded cells.
11. Recede pipettes
    1. Once recordings are finished, recede pipettes slowly from the tissue by right-clicking on the Table radio-button. Select 'Recede->Pipettes 500 µm' to have the pipettes recede a short distance along their axes. Observe the drift in potential of the cells (clearing the tips by applying some positive pressure may help).
    2. To recede pipettes all the way back, set the positioning speed to 'Fast' and repeat the same operation but choose the 'All' option. Remove the used pipettes by gently sliding out the headstages and unscrewing the pipettes from the holders. It is useful to avoid twisting the holders in their sockets since this might greatly interfere with the pipette tip position.

Results

Following the methods described above we succeeded in performing whole-cell recording of up to twelve neurons simultaneously, nearly doubling the largest number of neurons simultaneously patch-clamped thus far. Examples of networks of direct synaptic connections between Pyramidal Neurons recorded in Layer V of the somatosensory cortex of rats are shown in Figure 6.

The determination of connection probability profiles as a function of inter-somatic distance for a given cell-typ...

Discussion

An immediate question usually arises concerning the rate of success of the procedure we described. For high success rates preparation is essential. Pipettes must have tip openings that are adequate for the cells beings recorded. Filtering the intracellular solution to avoid clogged pipettes is also important. Extremely clean, freshly pulled pipettes are another requirement. A binomial distribution is the simplest model that can be used to understand how these issues affect the final yield. It is reasonable to expe...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Microscope	Olympus	BX51WI	40X Immersion Objective
Manipulators	Luigs Neumann	SM-5	Serial protocol used
Amplifiers	Axon Instruments	MultiClamp 700B	SDK used
Camera	Till Photonics	VS 55	BNC analog output
Framegrabber	Data Translation	DT3120	SDK used
Oscilloscopes	Tektronix	TDS 2014	Serial communication
Data acquisition	InstruTECH	ITC 1600
Data acquisition	National Instruments	PCI-6221	Library used (.dll)
Pressure valve	SMC	SMC070C-6BG-32
Pressure sensor	Honeywell	24PCDFA6G
Membrane pump	Schego	Optimal