A Computer- assisted Multi- electrode Patch- clamp System. 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. Pipette Offset Patch Clamp ElectrodeHere 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. Keywords: Neuroscience, Issue 8. Patch- clamp, automatic positioning, whole- cell, neuronal recording, in vitro, multi- electrode. 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 1. 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 otherwise. Moreover, precise stimulation of numerous cells also allows the quantification of recruitment of postsynaptic cells. Protocol. 1. Equipment Preparation. Control manipulators from a computer. Connect each micromanipulator controller box to a computer through serial ports (RS- 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. 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. Position the microscope at its central position 2 mm above the specimen focal plane (coordinates . This is the initial reference point for each electrode. 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. Voltage clamp techniques JAMES V. VE-2 Voltage Clamp User's Guide. Comprehensive protocol for effective patch clamp analysis. Set the amplifier to voltage-clamp and correct the pipette offset so the currents measured at that point are. Measuring Cardiac Electrical Activity. The tip of the recording pipette is filled with gramicidin. Run the Membrane Test Protocol and compensate for recording pipette offset. Pipette Offset Patch Clamp TechniqueA simple way to accomplish this is: Bring the tip of an electrode to the center of the field of view. Store the position on each axis of the manipulator and each axis of the microscope. Execute with the x- axis of the manipulator a relatively large movement (1 mm). Locate the tip once more moving only the microscope. 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. Repeat steps 2. 3 - 2. 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: 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: Using the initial reference point and the cosine matrix determine the position of each electrode in microscope coordinates. 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 4. Repeat steps 1. 2. Enable storing the positions of relevant features in the tissue, such as cells or anatomical reference points, in microscope coordinates. Acquire video and overlay relevant information. Mechanically align the x- axis of motion of the microscope with the horizontal axis of the microscope camera. Install on the computer a framegrabber with live video and overlay capability and a software development kit (SDK). Implement live video display operation with the SDK. Convert the microscope coordinate system to the camera reference system by the appropriate translation and scaling. Draw the relevant features in camera coordinates and overlay on the live video the resulting image at regular intervals of about 4. Figure 3). Control Amplifiers. Use the amplifier software to control the amplifier settings from the interface. The patch pipette comes into contact with the bath solution. Most patch clamp experiments need to correct for liquid. Use the pipette offset potentiometer to zero the volt-. Pipette Offset The Axopatch 200B amplifier provides . Instruments patch-clamp amplifier incorporating the innovative Capacitor-Feedback technology for single-channel recording, and resistive. Patch-Clamp Technique in ESC-Derived Cardiomyocytes. Patch-clamp configurations and electrical connection of pipette, biological membrane, and patch-clamp measuring instruments. Pipette offset adjustment. Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared. Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation. Control oscilloscopes. Connect the oscilloscopes to the PC using serial ports. Determine the oscilloscope scale, coupling and temporal resolution for the different steps of the patch- clamp procedure in voltage- clamp (e. Use a 1. 2 V/5 V power supply to supply each electronic component appropriately. Connect the output of one membrane pump to the positive pressure buffer (a 1. Connect the input of one membrane pump to the negative pressure buffer (a 1. 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. Connect each of the pressure buffers to a valve connected to the main pressure compartment. Connect a valve between the main pressure compartment and the atmosphere. Connect a pressure sensor to the main pressure compartment. Connect a pressure sensor to each buffer. Connect the pressure control system to a data acquisition board. Connect each pressure sensor to one analog input. Connect each valve to a digital output. Remove atmospheric pressure offset from all sensors by opening all valves except those connecting to the membrane pumps and subtracting the measured pressure. Implement pressure control Define in the interface a control to activate or de- activate positive pressure control for each pipette. Define a minimal positive pressure for the pipettes of about 7. Periodically (every 0. Upon threshold crossing open the positive pressure buffer to the main pressure compartment and this compartment towards the pipette in question during brief periods (2. Close all other valves. De- activate further pressure control as the final approach towards a cell of interest is initiated. Apply negative pressure for seal formation 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. Centralize commands onto a human interface device. Connect a commercially available wireless gamepad to the PC. Implement readout of joystick status. For instance, use the Direct. X libraries for C/C++ to perform readouts every 5 msec. Compare current status with previous status to detect which buttons have been pressed, released or kept pressed since last time step. Assign functions to each button in the gamepad. An example of this mapping is shown in Figure 5. Patch- clamp Procedure. Prepare the brain slices of the region of interest. Place a brain slice of interest, with the region of interest in the center of the microscope's displacement range. Cell Selection. 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. 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#. Attribution of cells to pipettes. 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'. 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. Prepare the Pipettes. Fill the pipettes (6 - 8 M. Place the headstages in their fixations but do not slide them forward to avoid touching the bath with the pipette tip. Enable the positive pressure control and select all pipettes to ensure that the tips will remain clean. Gently slide each headstage in place. Locating the pipette tips.
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