Basic Neuroscience
Short communication
Optopatcher—An electrode holder for simultaneous intracellular patch-clamp recording and optical manipulation

https://doi.org/10.1016/j.jneumeth.2013.01.017Get rights and content

Abstract

Optogenetics has rapidly become a standard method in neuroscience research. Although significant progress has been made in the development of molecular tools, refined techniques for combined light delivery and recording in vivo are still lacking. For example, simultaneous intracellular recording and light stimulation have only been possible by using two separate positioning systems. To overcome this limitation, we have developed a glass pipette holder which contains an additional port for the insertion of an optical fiber into the pipette. This device, which we called “optopatcher” allows whole cell patch-clamp recording simultaneously with direct projection of light from the recording pipette. The holder spares the use of an additional manipulator and, importantly, enables accurate, stable and reproducible illumination. In addition, replacement of standard pipettes is done as easily as with the available commercial holders. Here we used the optopatcher in vivo to record the membrane potential of neurons from different cortical layers in the motor cortex of transgenic mice expressing channelrhodopsin-2 under the Thy1 promoter. We demonstrate the utility of the optopatcher by recording LFP and intracellular responses to light stimulation.

Highlights

► The optopatcher: a new holder for simultaneous patch-clamp recording and light stimulation. ► We used the optopatcher for in vivo cortical patch-clamp recording and optogenetic activation. ► The holder can be used in multiple platforms whenever a glass pipette is used.

Introduction

Enormous advances have been made in optogenetics since it was introduced only several years ago (Zemelman et al., 2002, Boyden et al., 2005). New hardware technologies have been introduced in order to facilitate the efficiency and accuracy of viral vector delivery into the brain and in order to improve the precision of light-based manipulation of neural activity (Gradinaru et al., 2007). Simultaneous extra-cellular recording and optical stimulation can be achieved using commercial silicone optrodes (Zhang et al., 2009). Additional methods for combined optical manipulation and extracellular recordings have been developed by several laboratories (Stühmer and Almers, 1982, Diester et al., 2011, LeChasseur et al., 2011), but few studies have been performed with combined patch-clamp recordings and light stimulation in vivo (Cardin et al., 2009, Mateo et al., 2011). Light delivery in these studies was performed either by stimulation of superficial cortical layers through the microscope or using a separate optical fiber for stimulation. Using an optical fiber requires a second positioning system and poses a challenge in delivering reproducible amounts of light to the recorded cells, a parameter which could greatly affect the reliability of activation and the latencies to spike, both important quantitative parameters in many experiments. Importantly, simultaneous recording and illumination using a single device allows high repeatability and accuracy and can significantly reduce tissue damage caused by the positioning of an optical fiber within the tissue. However, a similar solution for light stimulation and intracellular patch-clamp recording is currently not available. Here we introduce a new device for combined whole cell patch-clamp recording and light stimulation and demonstrate recordings made using this device in transgenic mice expressing channelrhodopsin-2 (ChR2) in cortical neurons.

Section snippets

Animal preparation for recording

All surgical and experimental procedures were approved by the Weizmann Institute Animal Care and Use Committee. Two Thy1-COP4 mice (6 weeks old, B6.Cg-Tg(Thy1-COP4/EYFP)18Gfng/J, The Jackson Laboratories, Bar Harbor, ME) were initially anaesthetized with ketamine (i.p., 100 mg kg−1; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine mixture (Eurovet, Bladel, The Netherlands). The animals were placed in a standard stereotaxic device using modified zygomatic ear-bars. Lidocaine (2%) was applied

Results and discussion

Our goal was to optogenetically stimulate the neuronal network in the vicinity of the intracellularly recorded cell. This can be achieved by carefully positioning an optical fiber using a second micromanipulator. However, this approach can cause tissue damage, is more costly and less precise since it requires a very accurate positioning of the fiber. These problems become more severe when recordings are made in deep brain structures such as the thalamus (Brecht and Sakmann, 2002) or the

Conclusions

In summary, we designed an electrode holder for simultaneous intracellular patch-clamp recording and optical stimulation, and showed examples of recorded cortical neurons in anesthetized mice. The optopatcher prevents the need for a second manipulator and for insertion of the optical fiber into the tissue. It can be also used for any other type of recordings that make use of glass capillaries, such as LFP recording and single unit recording. Without any modifications, the optopatcher can be

Conflict of interest

None.

Acknowledgements

We thank Benny Pasmantirer and Oz Diner for designing the Optopatcher. We thank Patricia Sprysch for technical assistance. We thank Boaz Mohar and Inbal Meir for help in the preparation of the manuscript. This work was funded by Deutsche Forschungsgemeinschaft (SFB 889, TP C3), ERA-Net Neuron, grants 1160/11, 1565/10 and 1351/12 from the Israel Science Foundation and by the Israeli Center of Research Excellence (I-CORE) in Cognition (I-CORE Program 51/11).

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