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.
Voltage-dependent Ca2+ currents: Five types of voltage-dependent Ca2+ currents (L-, N-, P/Q-, R- and T-types) occur in many cell types, including cultured Xenopus spinal neurons23,24 , and can be segregated according to their inhibition by specific antagonists and voltage-dependent properties22. Interestingly, the proportion of each channel type differs in different cellular compartments of the same neuron, as for example, in the soma and presynaptic varicosity14. Moreover, growth cone signaling events triggered by diffusible guidance molecules (e.g., netrin-1 and Sema3A) are largely limited to growth cones16,17,25 (Fig. 3). Therefore, unlike in the majority of neurons where synaptic activity can be measured in the soma, as far away as 300 to 500 μm from the activated synapses, in the case of Xenopus spinal neurons, growth cone electrical activity must be measured directly in the growth cone. We used Ringer’s solution containing 10 mM Ca2+ to measure Ca2+ inward currents, while the majority of other voltage-dependent cation (i.e., Na+ and K+) currents were inhibited by saxitoxin (STX, 10 nM) and tetraethylammonium (TEA, 35 mM), in the bath. To measure L-type currents, the growth cone membrane potential was held at –40 mV to inhibit other currents by voltage-dependent inactivation8,16: The inactivation time constant for N-, P/Q-, R- and T-types at –40 mV are 345, 133, 201 and 68 ms, respectively (our computational prediction, see ref. 17). Voltage steps to +50 mV were delivered for 100-ms duration with 10-mV increments through recording electrodes at 0.067 Hz to trigger L-type inward Ca2+ currents16. With this procedure, ca. 80% of the evoked inward currents were sensitive to the bath-applied L-type Ca2+ channel antagonist, nimodipine (20 μM), confirming their identity16. For the measurement of other voltage activated (except the L-type) currents24, a holding potential of –50 mV was used to cause voltage-dependent inactivation of low-voltage activated (i.e., T-type) currents while L-type and residual T-type currents were excluded by nimodipine in the bath17. A holding potential of –80 mV was used to preclude total voltage-dependent inactivation. While holding at either –50 or –80 mV, the same voltage-steps were delivered to +50 mV as for the measurements of L-type currents. Currents were measured from more than 15 growth cones, either in the presence and absence of specific antagonists of each channel type: ω-conotoxin GVIA (1 μM), ω-agatoxin TK (100 nM), SNX-482 (1.75 μM) and pimozide (500 nM) for N-, P/Q-, R- and T-types, respectively, in the bath. Individual sets of measurements were ranked by the amplitude of their peak currents, which normally occurred during voltage-steps from the holding potentials to –10 ~ +10 mV. Then the currents evoked by each voltage-step in the absence of a specific antagonist were subtracted from those in its presence to determine the specific whole-cell Ca2+ current type. Developmental stage-dependent changes in each voltage-dependent Ca2+ channel (VDCC) component are discussed further in ANTICIPATED RESULTS.
Leak currents: Several diffusible guidance molecules evoke repulsive or attractive growth cone turning, depending on the level of growth cone intracellular Ca2+ ([Ca2+]i) increase they induce15,25,26. Non-voltage gated cation channels may both affect neural activity (such by depolarizing growth cones) and contribute to growth-cone [Ca2+]i increase as a result of their high Ca2+ conductivity27. Transient receptor potential canonical (TRPC)11 and cyclic nucleotide-gated cation (CNG)17 channels are such channels and are reported to mediate netrin-1 attractive and Sema3A repulsive signals, respectively. Application of either sequential voltage-steps28 or a voltage-ramp11 from –120 –60 mV to +60 +120 mV while cells are initially held at ~ 0 mV evokes these leak currents. A cocktail of VDCC blockers, in addition to those that block voltage-gated Na+ and K+ channels, is used to exclude contamination by leak currents11,28. However, many VDCC blockers also affect the amplitudes of leak currents through CNG (ref. 29) and Cl– (ref. 30) channels. We, therefore, performed computational analyses to design an inversed voltage-ramp protocol (ramp from +120 mV down to –120 mV, see ref. 17 for details) to minimize the use of VDCC blockers. This protocol takes advantage of the common voltage-dependent properties22 of VDCCs, inactivation of which shows a much longer time constant than that of activation, and occurs at higher positive potentials than does activation. In the presence of bath-applied STX (10 nM) and TEA (35 mM), as described above (voltage-dependent Ca2+ currents), or nimodipine (20 μM), due to the weak voltage-dependent inactivation of L-type Ca2+ currents, the growth cone potential was initially held at –10 mV, stepped up to +60 mV for 300 ms to establish the voltage-dependent inactivation of VDCC currents and followed by an inversed voltage ramp from +120 mV to –120 mV for 1 sec. During the inversed voltage ramp, VDCCs remain inactive and are prevented from reactivation. A shorter duration for the inversed voltage ramp is preferable. However, because up to 50%, compensation of series resistance can be achieved in this preparation, which is relatively poor compared with that of usual whole-cell recordings, we chose voltage ramps of one second duration to avoid distortion of leak current profiles. We monitored leak currents at 0.067 Hz and took the average of between four and ten consecutive evoked currents. For the measurement of TRPC currents, Ringer’s solution containing 10 mM Ca2+ was used to exclude contamination from currents through CNG channels (CNGCs)31. Leak currents were measured at more than 15 growth cones in the presence and absence of a TRPC blocker, 2-aminoethoxydiphenyl borate (2-APB, 50 μM), in the bath. 2-APB-sensitive TRPC currents were obtained as differential currents, similarly as described above (voltage-dependent Ca2+ currents). For the measurement of CNG currents, Ringer’s solution, containing either 1 mM Ca2+ or Ca2+-free (achieved with 0.5 mM EGTA, see below) to avoid CNGC blocking by external Ca2+ (ref. 31), was used. Differential CNG currents were obtained by subtracting currents in the presence of the bath-applied CNGC blocker L-cis diltiazem (25 μM) from those in its absence (i.e., L-cis diltiazem-sensitive currents).
Membrane potentials: Growth-cone membrane depolarization caused by the diffusible guidance molecule netrin-1 requires functional TRPC1s (ref. 11). The perforated patch recordings that were used in this study maintain the physiological intracellular ion milieu due to the slow perfusion rate of the internal recording solution into the cell and the robust homeostatic regulation (through ion channels and transporters) of the cellular ion concentration. Na+, K+ and Cl– (but not Ca2+) fluxes are the predominant contributors to membrane potential shifts, depending on their reversal potentials relative to the cell resting membrane potentials; Na+ influx and Cl– efflux (largely for immature neurons) result in depolarization, and K+ efflux and Cl– influx (largely function for shunting in mature neurons because the Cl- reversal potential is close to the cell resting potentials) result in hyperpolarization27. The conventional whole-cell patch method is advantageous compared with the perforated patch method for the determination of which ion fluxes contribute to membrane potential shifts, such that hyperpolarization would be caused by either increased K+ efflux or Cl– influx (alternatively decreased Na+ influx) and depolarization would be caused by either increased Na+ influx or Cl– efflux (alternatively decreased K+ efflux), since the whole-cell configuration can control intracellular ion concentrations15. For example, when Ringer’s solution containing 5 mM K+ and 149 mM Cl– is used as the external recording solution, an internal recording solution containing 140 mM K+ and 3 mM (or lower) Cl– sets the theoretical resting membrane potential to a K+ reversal potential of –84 mV or more hyperpolarized according to the Goldman-Hodgkin-Katz current equation27, and if the cell is hyperpolarized it depends solely on Cl– influx. Similarly, an internal recording solution containing 140 mM K+ and 12 mM (or higher) Cl– can be used to set the resting potential to a Cl– reversal potential of –60 mV (in 12 mM Cl–) or more depolarized, and if the cell is hyperpolarized it depends solely on K+ efflux. Using these types of manipulations, we recently were able to determine that an increased Cl– influx is responsible for the growth cone membrane hyperpolarization induced by the repulsive guidance molecules semaphorin 3A (Sema3A) and Slit 2, whereas an increased K+ efflux is responsible for the hyperpolarization induced by a repulsive netrin-1 (in ectopic UNC5B expressing growth cones) signal15. Another advantage of the whole-cell patch over the perforated patch is that with it hydrophilic agonists and antagonists can be administrated into growth cones through recording electrodes. The resultant membrane potential was determined as the potential difference between that during the whole-cell configuration and that after the electrode was removed. Liquid junction potentials (–16.2 mV and –13.4 mV for 3 mM and 30 mM Cl– containing internal recording solutions, respectively) were adjusted offline. With this procedure, growth cone membrane potentials can be measured stably for as long as 10 min. The downside of either the perforated or whole-cell patch is that the amplitudes and kinetics (i.e., time constant) can be artificially altered by the electrophysiological manipulations during measurements. Therefore, optical imaging of a VSD, which does not perturb the intracellular ion milieu, can be combined to exclude the artifacts of the whole-cell patch method (see below for the detail).