Patch-clamp recording from mossy fiber terminals in hippocampal slices

Abstract

Rigorous analysis of synaptic transmission in the central nervous system requires access to presynaptic terminals. However, cortical terminals have been largely inaccessible to presynaptic patch-clamp recording, due to their small size. Using improved patch-clamp techniques in brain slices, we recorded from mossy fiber terminals in the CA3 region of the hippocampus, which have a diameter of 2–5 μm. The major steps of improvement were the enhanced visibility provided by high-numerical aperture objectives and infrared illumination, the development of vibratomes with minimal vertical blade vibrations and the use of sucrose-based solutions for storage and cutting. Based on these improvements, we describe a protocol that allows us to routinely record from hippocampal mossy fiber boutons. Presynaptic recordings can be obtained in slices from both rats and mice. Presynaptic recordings can be also obtained in slices from transgenic mice in which terminals are labeled with enhanced green fluorescent protein.

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Figure 1: Schematic illustration of the “magic cut”.
Figure 2: IR-difference interference contrast images of mossy fiber terminals in stratum lucidum of the hippocampal CA3 region under experimental conditions.
Figure 3: Functional criteria for unequivocal identification of hippocampal mossy fiber boutons during recording.
Figure 4: Morphological criteria for unequivocal identification of hippocampal mossy fiber boutons.

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Acknowledgements

We thank K. Winterhalter, S. Becherer and M. Northemann for technical assistance and Dr. G. Stocca for biocytin filling of the bouton shown in Figure 4. We also thank Dr. P. Caroni for providing the Thy1-EGFP mice. This work was supported by the Deutsche Forschungsgemeinschaft.

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Correspondence to Peter Jonas.

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Competing interests

We have an industrial collaboration with Leica Microsystems, Nussloch, Germany, to commercialize our custom-made slicer and our device for measuring vertical vibrations of the cutting blade (Geiger et al., 2002). The Institute of Physiology receives royalties of 7.5% of the net income from the Vibroslicer VT 1200 and the measuring device Vibrocheck, with a minimum of 30,000 per year.

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Bischofberger, J., Engel, D., Li, L. et al. Patch-clamp recording from mossy fiber terminals in hippocampal slices. Nat Protoc 1, 2075–2081 (2006). https://doi.org/10.1038/nprot.2006.312

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