We would like to thank Gilad Silberberg, Michele Pignatelli, Thomas K. Berger, Luca Gambazzi, and Sonia Garcia for valuable advice on improvements for the patch-clamp procedure automation. We thank Rajnish Ranjan for valuable advice and assistance with software implementation. This work was funded in part by the EU Synapse project&#160;and partly by the Human Frontiers Science Program.

1. Equipment Preparation
<ol>
	<li>Control manipulators from a computer
	<ol>
		<li>Connect each micromanipulator controller box to a computer through serial ports (RS-232).</li>
		<li>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.</li>
		<li>Standardize the reference system of the manipulators so that zero is the closest possible position with respect...

<ol>
	<li>Perin, R., Berger, T. K., Markram, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synaptic+organizing+principle+for+cortical+neuronal+groups.">A synaptic organizing principle for cortical neuronal groups.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 5419-5424 (2011).</li><li>Berger, T. 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The authors declare that they have no competing financial interests.

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...

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.

<table border="1">
	<tbody>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td>40X Immersion Objective</td>
		</tr>
		<tr>
			<td>Manipulators</td>
			<td>Luigs &#38; Neumann</td>
			<td>SM-5</td>
			<td>Serial protocol used</td>
		</tr>
		<tr>
			<td>Amplifiers</td>
			<td>Axon Instruments</td>
			<td>MultiClamp 700B</td>
			<td>SDK used</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Till Photonics</td>
			<td>VS 55</td>
			<td>BNC analog output</td>
		</tr>
		<tr>
			<td>Framegrabber</td>
			<td>Data Translation</td>
			<td>DT3120</td>
			<td>SDK used</td>
		</tr>
		<tr>
			<td>Oscilloscopes</td>
			<td>Tektronix</td>
			<td>TDS 2014</td>
			<td>Serial communication</td>
		</tr>
		<tr>
			<td>Data acquisition</td>
			<td>InstruTECH</td>
			<td>ITC 1600</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition</td>
			<td>National Instruments</td>
			<td>PCI-6221</td>
			<td>Library used (.dll)</td>
		</tr>
		<tr>
			<td>Pressure valve</td>
			<td>SMC</td>
			<td>SMC070C-6BG-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure sensor</td>
			<td>Honeywell</td>
			<td>24PCDFA6G</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane pump</td>
			<td>Schego</td>
			<td>Optimal</td>
			<td></td>
		</tr>
	</tbody>
</table>

a computer-assisted multi-electrode patch-clamp system

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.

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...

Watch this Scientific Journal Video about A Computer-assisted Multi-electrode Patch-clamp System at JoVE.com

A Computer-assisted Multi-electrode Patch-clamp System

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.

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

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Neuroscience

13.6K vistas.  Ecole Polytechnique Federale de Lausanne.  De múltiples electrodos patch-clamp grabaciones constituyen una tarea compleja. Aquí se muestra cómo, mediante la automatización de muchas de las medidas experimentales, es posible acelerar el proceso que conduce a una mejora cualitativa en el rendimiento y el número de grabaciones. 