Authors
Makoto Nishiyama, Kazunobu Togashi, and Kyonsoo Hong
Summary
The small size, fragility and high motility of growth cones preclude sensitive detection of growth cone electrical activity, which is essential for an elucidation of the early signaling events, such as those triggered by extracellular signaling molecules (e.g., guidance molecules)1, that govern growth cone migration. Growth cone electrical activity was first detected using voltage-sensitive dyes (VSD)2,3, which visualized somatic Ca2+ spikes propagating along neurites to growth cones in cultured neuroblastoma cells (N1E-115). However, the low sensitivity of VSD (ΔF/F: 1% for 100 mV, ref. 4), allowed an assessment of only relatively large growth cone membrane potential changes (> 50 mV) caused by propagating spikes. Subsequently, conventional patch clamp techniques were employed to monitor action potentials and voltage-gated macroscopic or single channel currents in large growth cones (>30 μm in diameter) in cultured Helisoma5 and Aplysia6,7 ganglion neurons. Continued efforts achieved the recording of electrical activity from small growth cones (<10 μm in diameter)8–11 including those of presynaptic varicosities12–14. Cell attached8,10 and perforated patchs9,11–14 have been used to measure, respectively, single channel currents and macroscopic currents or membrane potentials.
However, the fragility and dynamic movements of growth cones have prevented application of the whole-cell patch methodology to small growth cones. Conventional whole-cell patch clamp recording has the advantage that membrane impermeable agonists and antagonists can be administrated in a relatively short time period (2–3 min)15 with good control of the intracellular ion milieu and commanding potentials without significantly sacrificing growth cone integrity (Fig. 1). Here, we describe procedures for the use of whole-cell patch configurations to measure voltage-dependent Ca2+ currents evoked by voltage-steps16 and voltage-independent leak currents evoked by inversed voltage ramps17, as well as membrane potentials15 in growth cones of cultured Xenopus spinal neurons. Improvements in optics and sensors have increased the sensitivity of measurements of neuronal activity by optical imaging of VSD, which allows the detection of field excitatory postsynaptic potentials (fEPSPs)18–20 of ca. 1 mV amplitude, and their long-term modulation21 (Fig. 2) in rat hippocampal slice preparations. Recently, we succeeded in monitoring slow kinetic membrane potential changes of ca. 15 mV using a VSD from a single cultured Xenopus spinal neuron growth cone15. Therefore, we will also describe a procedure for the measurement of membrane potential shifts induced by diffusible guidance molecules15 using this methodology.
Further details
The protocol was published on Protocol Exchange in 2008. To see the entire protocol, click on the source link.
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