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.
Subscribe to Journal
Get full journal access for 1 year
only $41.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Katz, B. The Release of Neural Transmitter Substances (Liverpool University Press, Liverpool, 1969).
Llinás, R.R The Squid Giant Synapse (Oxford University Press, New York, 1999).
von Gersdorff, H. & Borst, J.G.G. Short-term plasticity at the calyx of Held. Nat. Rev. Neurosci. 3, 53–64 (2002).
Forsythe, I.D. Direct patch recording from identified presynaptic terminals mediating glutamatergic EPSCs in the rat CNS, in vitro . J. Physiol. (Lond.) 479, 381–387 (1994).
Borst, J.G.G., Helmchen, F. & Sakmann, B. Pre- and postsynaptic whole-cell recordings in the medial nucleus of the trapezoid body of the rat. J. Physiol. (Lond.) 489, 825–840 (1995).
Bollmann, J.H., Sakmann, B. & Borst, J.G.G. Calcium sensitivity of glutamate release in a calyx-type terminal. Science 289, 953–957 (2000).
Schneggenburger, R. & Neher, E. Intracellular calcium dependence of transmitter release rates at a fast central synapse. Nature 406, 889–893 (2000).
Meinrenken, C.J., Borst, J.G.G. & Sakmann, B. Calcium secretion coupling at calyx of Held governed by nonuniform channel-vesicle topography. J. Neurosci. 22, 1648–1667 (2002).
Sun, J.Y. & Wu, L.G. Fast kinetics of exocytosis revealed by simultaneous measurements of presynaptic capacitance and postsynaptic currents at a central synapse. Neuron 30, 171–182 (2001).
Malenka, R.C. & Nicoll, R.A. Long-term potentiation—a decade of progress? Science 285, 1870–1874 (1999).
Bischofberger, J., Engel, D., Frotscher, M. & Jonas, P. Mechanisms underlying the efficacy of transmitter release at mossy fiber synapses in the hippocampal network. Pflügers Arch. 453, 361–372 (2006). 10.1007/S00424-006-0093-2
Claiborne, B.J., Amaral, D.G. & Cowan, W.M. A light and electron microscopic analysis of the mossy fibers of the rat dentate gyrus. J. Comp. Neurol. 246, 435–458 (1986).
Chicurel, M.E. & Harris, K.M. Three-dimensional analysis of the structure and composition of CA3 branched dendritic spines and their synaptic relationships with mossy fiber boutons in the rat hippocampus. J. Comp. Neurol. 325, 169–182 (1992).
Acsády, L., Kamondi, A., Sík, A., Freund, T. & Buzsáki, G. GABAergic cells are the major postsynaptic targets of mossy fibers in the rat hippocampus. J. Neurosci. 18, 3386–3403 (1998).
Geiger, J.R.P. & Jonas, P. Dynamic control of presynaptic Ca2+ inflow by fast-inactivating K+ channels in hippocampal mossy fiber boutons. Neuron 28, 927–939 (2000).
Alle, H. & Geiger, J.R.P. Combined analog and action potential coding in hippocampal mossy fibers. Science 311, 1290–1293 (2006).
Dodt, H.U. & Zieglgänsberger, W. Visualizing unstained neurons in living brain slices by infrared DIC-videomicroscopy. Brain Res. 537, 333–336 (1990).
Stuart, G.J., Dodt, H.U. & Sakmann, B. Patch-clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy. Pflügers Arch. 423, 511–518 (1993).
Geiger, J.R.P. et al. Patch-clamp recording in brain slices with improved slicer technology. Pflügers Arch. 443, 491–501 (2002).
Salin, P.A., Scanziani, M., Malenka, R.C. & Nicoll, R.A. Distinct short-term plasticity at two excitatory synapses in the hippocampus. Proc. Natl. Acad. Sci. USA 93, 13304–13309 (1996).
Edwards, F.A., Konnerth, A., Sakmann, B. & Takahashi, T. A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system. Pflügers Arch. 414, 600–612 (1989).
Sakmann, B. & Stuart, G. Patch-pipette recordings from the soma, dendrites, and axon of neurons in brain slices. In Single-channel Recording (eds. Sakmann, B. & Neher, E.) 199–211 (Plenum, New York, 2nd edn. 1995).
Paxinos, G. & Watson, C. The Rat Brain in Stereotaxic Coordinates 2nd edn. (Academic press, San Diego, 1986).
Paxinos, G. & Franklin, K.B.J. The Mouse Brain in Stereotaxic Coordinates 2nd edn. (Academic press, San Diego, 2001).
Swanson, L.W. Brain Maps: Structure of the Rat Brain 2nd edn. (Elsevier, Amsterdam, 1998).
Castaneda-Castellanos, D.R., Flint, A.C. & Kriegstein, A.R. Blind patch clamp recordings in embryonic and adult mammalian brain slices. Nat. Protocols 1, 532–542 (2006).
Davie, J.T. et al. Dendritic patch-clamp recording. Nat. Protocols 1, 1235–1247 (2006).
Palmer, M.J., Taschenberger, H., Hull, C., Tremere, L. & von Gersdorff, H. Synaptic activation of presynaptic glutamate transporter currents in nerve terminals. J. Neurosci. 23, 4831–4841 (2003).
Bischofberger, J., Geiger, J.R.P. & Jonas, P. Timing and efficacy of Ca2+ channel activation in hippocampal mossy fiber boutons. J. Neurosci. 22, 10593–10602 (2002).
Spruston, N., Lübke, J. & Frotscher, M. Interneurons in the stratum lucidum of the rat hippocampus: an anatomical and electrophysiological characterization. J. Comp. Neurol. 385, 427–440 (1997).
Schmitz, D. et al. Axo-axonal coupling: a novel mechanism for ultrafast neuronal communication. Neuron 31, 831–840 (2001).
Galimberti, I. et al. Long-term rearrangements of hippocampal mossy fiber terminal connectivity in the adult regulated by experience. Neuron 50, 749–763 (2006).
Engel, D. & Jonas, P. Presynaptic action potential amplification by voltage-gated Na+ channels in hippocampal mossy fiber boutons. Neuron 45, 405–417 (2005).
Hallermann, S., Pawlu, C., Jonas, P. & Heckmann, M. A large pool of releasable vesicles in a cortical glutamatergic synapse. Proc. Natl. Acad. Sci. USA 100, 8975–8980 (2003).
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.
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.
About this article
Cite this article
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
CUL3 Deficiency Causes Social Deficits and Anxiety-like Behaviors by Impairing Excitation-Inhibition Balance through the Promotion of Cap-Dependent Translation
Functional Electron Microscopy (“Flash and Freeze”) of Identified Cortical Synapses in Acute Brain Slices
P2Y 13 receptors regulate microglial morphology, surveillance, and resting levels of interleukin 1β release