Abstract
Electrophysiological measurements of single synapses are challenging given the size of a single synapse relative to a patch pipette. In addition, one has to take into account the limitations of microscopes in that they need to provide acceptable visualization of a single synapse for patching. However, despite these limitations, researchers have successfully measured single synaptic function along dendrites. The purpose of this chapter is to introduce the techniques that can be implemented to measure single synaptic function. Included in this chapter are such techniques as localized perfusion, localized electrical stimulation, photostimulation, and imaging. These techniques are designed with the assumption that multiple excitatory synapses do not contact a single spine, but rather only one synapse per spine. Whereas this assumption is supported by some empirical data [1], other data suggest otherwise [2], meaning that a complete understanding of the anatomical region is necessary before beginning single synapse experiments.
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Notes
- 1.
A loose patch (Fig. 4) is formed in the absence of the conventional gigaseal, which is necessary for all other patch clamp techniques (e.g., cell-attached, inside out, outside out, whole cell). Instead, in a loose patch clamp, a loose seal is formed with a low electrical resistance (2–11 MΩ). Typically, the micropipettes used for patch-clamp experiments have resistances of 2–5 MΩ. Therefore, watching the resistance of the micropipette while approaching the membrane can indicate when a loose patch is forming (the resistance will increase). As the distance between the micropipette and the membrane decreases, the resistance increases. By observing the changes in resistance, the experimenter can avoid coming too close to the membrane and thus forming a seal. Instead, the micropipette should stay far enough away from the membrane so that a gigaseal is not formed leaving the membrane intact (no suction is used). Since the loose patch method is a noninvasive patch, the EPSCs are visualized as outward and not inward currents. This patch-clamp technique is useful in that the micropipette can be removed from the membrane sampling multiple locations without any damage to the cell.
Fig. 4 
A loose patch. A micropipette is positioned just above the cell membrane so that a gigaseal is not formed. Notice that the membrane is not deformed in a loose patch as is the case in a cell-attached patch in which negative pressure elicits a gigaseal (see Fig. 1.2)
- 2.
Two-photon laser scanning microscopy is a fluorescent imaging technique that enables deep tissue penetration, reduced light diffraction in the tissue and limits phototoxicity [16]. It works by exciting a fluorophore (a fluorescent chemical compound that reemits light when excited) using two photons of low energy (i.e., two photons with half of the wavelength needed to excite the fluorophore). The simultaneous absorption of the two photons by the fluorophore results in fluorescence emission. What gives this imaging technique the advantages listed above is that the probability of two photons simultaneously exciting a fluorophore is very low. Therefore, the two photons must be concentrated spatially and temporally at the focal plane. Only then at the focal plane are fluorophores excited, thus drastically reducing background noise.
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Graziane, N., Dong, Y. (2016). Measurement of a Single Synapse. In: Electrophysiological Analysis of Synaptic Transmission. Neuromethods, vol 112. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3274-0_18
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DOI: https://doi.org/10.1007/978-1-4939-3274-0_18
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