Na⁺ current in presynaptic terminals of the crayfish opener cannot initiate action potentials

Abstract

Action potential (AP) propagation in presynaptic axons of the crayfish opener neuromuscular junction (NMJ) was investigated by simultaneously recording from a terminal varicosity and a proximal branch. Although orthodromically conducting APs could be recorded in terminals with amplitudes up to 70 mV, depolarizing steps in terminals to −20 mV or higher failed to fire APs. Patch-clamp recordings did detect Na⁺ current (I_Na) in most terminals. The I_Na exhibited a high threshold and fast activation rate. Local perfusion of Na⁺-free saline showed that terminal I_Na contributed to AP waveform by slightly accelerating the rising phase and increasing the peak amplitude. These findings suggest that terminal I_Na functions to “touch up” but not to generate APs.

presynaptic terminals are not universally excitable. Electrotonically compact terminals such as the calyx of Held (Leao et al. 2005) and some vertebrate neuromuscular junctions (NMJs) exhibit low excitability (Brigant and Mallart 1982). The Na⁺ current (I_Na) density in the calyx of Held was found to be low, and Na⁺ influx at the heminode was assumed to provide the current for full-height APs at release sites (Leao et al. 2005). Although the vertebrate NMJ exhibits considerable variation in morphology across species and locations, a significant proportion of motor end plates are morphologically compact, with 2–4 orders of branching within a space of 20 × 60 μm (Kang et al. 2014). Compact NMJ terminals are either not excitable or have low excitability (Brigant and Mallart 1982; Krzemien et al. 2000). By contrast, the axons with varicosities of CA3 mossy fibers (MF), the posterior pituitary and fast spiking interneurons, are thin and long, extending for 500-1,000 μm, and can fire APs (Geiger and Jonas 2000; Hu and Jonas 2014; Jackson and Zhang 1995). High excitability is essential for action potential to reach the distal ends of such axons. These studies suggest a general trend that, whereas terminals with low excitability need to be electrotonically compact to function, axons with long, thin arborizations are typically highly excitable so that AP can propagate to distal terminals.

Since terminals of the crayfish opener NMJ are organized in a “beads on a string” configuration and can extend for hundreds of micrometers (Florey and Cahill 1982), it is surprising that previous studies have hinted at low excitability for these terminals. Graded stimulations delivered by macropatch pipettes evoked all-or-none transmitter release in proximal varicosities but only graded release in distal ones (Dudel 1983). The latter behavior suggests the presence of nonspiking terminals. A recent study using a voltage indicator to monitor AP in terminals of the same preparation reached a similar conclusion (Lin 2013). In the present report, direct recordings from terminals were used to investigate the role of I_Na in terminals with low excitability.

METHODS

Crayfish (Procambarus clarkii), 5 cm head to tail, of both sexes were used. Treating a dissected opener of the first walking leg with protease XIV (1 mg/ml; Sigma-Aldrich) for 15 min “unsheathed” some glia cells surrounding varicosities. Terminals were identified by the fluorescence marker Alexa 568 injected before protease treatment. Patch clamp of terminals was performed on both excitors and inhibitors. Only inhibitors were used for simultaneous axon and terminal current-clamp studies because AP firing above 20 Hz in the excitor triggered muscle contraction and would tear a preparation weakened by the enzyme.

The preparation was maintained in crayfish saline containing (in mM) 195 NaCl, 5.4 KCl, 13.5 CaCl₂, 2.6 MgCl₂, and 10 HEPES (pH = 7.4). Sharp electrodes (1 M K-methanesulfonate; ∼40 MΩ) were used in two-electrode current clamp (CC) at the primary (1°) branching point (BP; IE-210, Warner Instrument). Patch clamp was performed with Axopatch 200B. Patch pipette (∼10 MΩ) capacitance was minimized by wrapping parafilm around the pipette shank, to ∼3 mm from the electrode tip. A K⁺-based solution used for current clamp contained (in mM) 180 K-gluconate, 12 KCl, 0.2 CaCl₂, 1 Na-EGTA, 10 Na-HEPES (pH = 7.4), 5 Mg-ATP, 0.3 K-GTP, 20 Tris-phosphocreatine. K-gluconate was replaced by Cs-methanesulfonate for I_Na recording. Liquid junction potential was not corrected. I_Na, 10-kHz filtered and 15-μs sampled, was activated by 20-ms steps and conditioned by a 100-ms prepulse to −100 mV. Capacitive and leak currents were subtracted with the P/n procedure. Calcium current was not detectable in outside-out patches.

When Axopatch 200B was used in CC (I-Clamp Fast) mode, electrode capacitance was compensated by complete cancelation of the capacitive current after gigaohm seals had formed. The bridge balance was achieved by dialing the correct electrode resistance first and, after switching to CC mode, setting series resistance compensation to 100%.

Since using the patch-clamp amplifier in CC mode has been reported to distort AP shapes under certain conditions (Magistretti et al. 1998), CC recordings were also performed with a voltage follower-type amplifier (Warner IE-210), with a custom-modified head stage for low current injection. The distributions of AP amplitude, duration, and afterhyperpolarization recorded with Warner IE-210 (n = 8) overlapped with those recorded with Axopatch 200B (n = 15). (See scatter plots on AP coupling ratio in Fig. 1D for example.)

Fig. 1.

Presynaptic terminals of the crayfish opener neuromuscular junction (NMJ) are not excitable. A1: on-cell patch from a terminal detects biphasic transients (red) corresponding to action potentials (APs) evoked by a 14-nA step at the primary (1°) branching point (BP; blue). A2: averages of on-cell transients (red) and APs (blue) from the traces in A1. B1: whole cell current of the same terminal exhibits a mixture of Na⁺ (I_Na) and K⁺ current (I_K), activated by voltage steps starting at −50 mV with 20-mV increments. B2: capacitive and leak currents activated by a −10-mV step. The black line represents a double exponential fit to the initial 5 ms of the transient. C1: Simultaneous current-clamp recordings from the 1° BP (blue) and the terminal (red). Current steps delivered at the 1° BP were 0, 10, and 12 nA. C2, top: recording configuration; bottom, APs marked by the rectangle in C1, aligned from their baselines. C3: a 200-pA step at the terminal depolarizes membrane potential (V_m) to −35 mV but does not evoke AP (thick red trace) and does not spread to the 1° BP (thick blue trace). Current steps injected at the 1° BP, simultaneously with the 200-pA step in the terminal, evoke subthreshold depolarization (thin dashed traces) and an AP train (solid thin traces). C4, top: hyperpolarization generated at 1° BP (blue) spreads to the terminal (red) with little attenuation; bottom, aligned APs corresponding to those identified by the rectangle in C3. D: AP coupling ratio (CR) plotted against passive CR from 23 preparations. CR was calculated as the ratio of amplitudes recorded at a patched terminal to that at the 1° BP. Filled circles represent data recorded from Axopatch 200B (n = 15) and open circles (n = 8) from Warner IE-210. Except for the top panel of C4, all voltage traces share the vertical scale in A1. A1, C1, and C3 share the same timescale. Bottom panels in C2 and C4 share the same timescale. All traces were obtained from the same terminal with K⁺-based pipette solution and with Axopatch 200B.

The series resistance compensation was switched off in voltage-clamp mode. Errors resulting from series resistance should be minor because 1) I_Na recorded in outside-out configuration was small, <100 pA, and 2) only experiments with an access resistance of <40 MΩ were used for analysis. All averaged data values are expressed as means ± SE.

Local perfusion of Na⁺-free saline was delivered by pressure ejection through a micropipette (tip opening ≈15 μm). The NaCl in control saline was replaced by choline chloride in Na⁺-free saline. Outflow of Na⁺-free saline was confirmed by the appearance of fluorescence (1 μM Alexa 568) at the tip of the perfusion pipette. I_Na recorded from a varicosity was eliminated after local perfusion was switched on (see Fig. 3, A and C). Perfusion was assumed to be localized because 1) the perfusion pipette tip was within 20 μm of patched terminals, and 2) local perfusion outflow (∼0.5 μl/min) was ∼2,000 times slower than the bath perfusion rate (∼1 ml/min). The localized nature of perfusion was supported by the observation that APs recorded at the 1° BP, ∼50–200 μm from perfused terminals, were never affected by perfusion.

Fig. 3.

Contribution of terminal I_Na to AP waveform. A: whole cell I_Na and I_K recorded from a terminal. B: voltage-clamp recording of invading, charging current (top trace) evoked by an AP train (bottom trace) initiated at the 1° BP. C: perfusion of Na⁺-free saline eliminates the I_Na and Na⁺-activated K⁺ current [I_K(Na)] shown in A. D: local perfusion of Na⁺-free saline does not alter AP shape at the 1° BP (axon) but slows and attenuates APs (gray dashed lines) in the terminal. All traces shown were obtained from the same terminal with K⁺-based pipette solution and with Axopatch 200B.

RESULTS

An AP train initiated at the 1° BP (Fig. 1A1, blue) evoked corresponding transients in the on-cell patch of a terminal (Fig. 1A1, red). The shape of on-cell transients (Fig. 1A2) represents a combination of inversion and the time derivative of intracellular counterparts (Sasaki et al. 2012). Whole cell access to the terminal, with a holding potential of −80 mV, gave rise to a holding current of −250 pA. The capacitive current evoked by a −10-mV command decayed with two exponential components, with time constants (τ) of 0.06 and 0.8 ms in the example shown in Fig. 1B2. Voltage steps uncovered a mixture of early I_Na and late K⁺ current (I_K; Fig. 1B1) with the K⁺-based pipette solution.

Capacitive transients compiled from 18 terminals gave rise to averaged fast and slow τ of 0.13 ± 0.01 and 1.33 ± 0.16 ms, respectively. Assuming that the fast component was attributable to the capacitance of the patched varicosities (Geiger and Jonas 2000) and a specific membrane capacitance of 1μF/cm², an averaged varicosity corresponds to a sphere with a radius of 3.0 ± 0.18 μm. This dimension is similar to the size of opener terminals reconstructed from serial electron microscopic sections (Cooper et al. 1995). The input resistance of crayfish varicosities, measured from the direct current (DC) evoked by a −10-mV step, was 168.5 ± 46.8 MΩ (n = 18) and significantly lower than that of MF boutons (>1 GΩ) of comparable size (∼2 μm) (Engel and Jonas 2005; Geiger and Jonas 2000).

Under current clamp, resting membrane potential (V_m) at the terminal was −58.4 ± 2.1 mV, whereas that at the 1° BP was −74.9 ± 1.5 mV (n = 15). Subthreshold depolarization at the 1° BP (Fig. 1C1, blue dashed trace) resulted in a similar potential at the terminal (red dashed trace). (See Fig. 1C2, top, for recording configuration.) APs evoked at the 1° BP spread to the terminal (Fig. 1C1, thin traces), but their amplitude was reduced to half (Fig. 1C2, bottom). The reduction in AP amplitude in the terminal despite strong DC coupling suggests that these terminals may not be active. Supporting this possibility, depolarizing the terminal up to −35 mV failed to fire any APs (Fig. 1C3, red thick trace); subthreshold depolarization initiated at the 1° BP delivered simultaneously with a 200-pA step in the terminal further depolarized V_m to −25 mV and still failed to fire AP (Fig. 1C3, red dashed trace). APs fired at the 1° BP spread to the depolarized terminal with slightly more attenuation than that observed without terminal depolarization (Fig. 1, C2 and C4, bottom).

Electrotonic coupling of passive signals between the 1° BP and terminals was asymmetrical. Whereas a 30-mV depolarization in the terminal (Fig. 1C3, red thick trace) failed to induce any change at the 1° BP (Fig. 1C3, blue thick trace), an 18-mV hyperpolarization at the 1° BP (Fig. 1C4, top, blue trace) caused a 17-mV potential in the terminal (Fig. 1C4, top, red trace). When the orthodromic coupling ratios of action potentials were plotted against those of passive signals initiated with a −20-nA and 20-ms step at the 1° BP, the majority of data points fell below the identity line (Fig. 1D). Open and filled circles represent data recorded from Warner IE-210 and Axopatch 200B, respectively. This distribution pattern is consistent with the expectation that passive cables attenuate fast transients more readily than slow signals.

In 54 terminals studied under current clamp, none fired an AP over a depolarization range of −35 to +5 mV. In 15 preparations where simultaneous BP and terminal recordings were made, AP amplitude at the two locations was 73.2 ± 2.8 and 41.5 ± 5.3 mV, respectively. In 19 of these terminals, voltage clamp was also performed. All 19 terminals exhibited I_K, and 16 of them showed I_Na. However, the whole cell I_Na potentially could have come from connecting axons, neighboring varicosities, or nearby tertiary branches. Outside-out patch was used to determine whether I_Na was present in the patched varicosities.

The whole cell I_Na in Fig. 2A exhibited unclamped spikes when voltage steps were above +10 mV. The poorly space-clamped I_Na were more frequently observed when the Cs⁺-based pipette solution was in use. The inset in Fig. 2A2 shows capacitive current recorded before (black trace) and after (gray trace) withdrawal of the patch pipette. Holding current was reduced from 263 to 8 pA. The capacitive transient evoked by a 10-mV step dropped from 246 to 42 pA and exhibited a single exponential decay. In 12 terminals with detectable whole cell I_Na, 3 showed no I_Na after pipette withdrawal. In the remaining nine endings, I_Na was reduced to 28.7 ± 9.6% of the whole terminal level, from 887 ± 387 to 85 ± 31 pA. Thus a significant fraction of terminal varicosities have I_Na channels. Figure 2A2 illustrates I_Na recorded after pipette withdrawal from the terminal used in A1. I_Na recorded from the excised patch exhibited rapid activation and gave rise to a current-voltage (I-V) curve with an extrapolated reversal potential (+75 mV) consistent with E_Na (Fig. 2A3, open circle). Also included in Fig. 2A3 are data from eight additional preparations (with different symbols) and the averaged results (filled circles; n = 9). I_Na in terminals shows a threshold at −40 mV and peaks around 0 mV. The conductance-voltage (G-V) curve calculated from the same data shows 50% activation at −11 mV (Fig. 2C).

Fig. 2.

Kinetics of I_Na recorded from opener terminals. A1: whole cell currents of a terminal activated by voltage steps from −80 to +40 mV with a 10-mV increment. A2: I_Na activated by the indicated voltage steps of an outside-out patch excised from the terminal used in A1. Inset: capacitive and leak currents, evoked by a 10-mV step, recorded before (black) and after (gray) patch excision. A3: normalized I-V curve recorded from 9 preparations (indicated by different symbols). Averaged data with error bars are indicated by filled circles. The gray curve was calculated from the Boltzmann fit of the activation curve (C) and E_Na = 75 mV. B1: I_Na inactivation mediated by 100-ms conditioning steps and tested with a 20-ms step to 0 mV. B2: normalized inactivation responses from 9 preparations (indicated by different symbols). Averages are indicated by filled circles. Averaged resting V_m at terminals is indicated. C: normalized activation curve calculated from the I-V curve shown in A3 with Boltzmann fit (gray). All traces and data points shown were obtained with Cs⁺-based pipette solution.

Steady-state inactivation was investigated with 100-ms conditioning pulses and tested with a step to 0 mV. Complete inactivation of I_Na occurred when the conditioning step was −30 mV (Fig. 2B1). Data compiled from the same preparations used in Fig. 2A3 show that 50% inactivation occurred at −51 mV (Fig. 2B2), close to the averaged resting V_m. The voltage dependence of both activation and inactivation is right-shifted by ∼10 mV compared with that recorded from the medial giant axon of the crayfish ventral nerve cord (Shrager 1974).

Although the activation time constant has not been curve-fitted for Hodgkin-Huxley modeling, the I_Na had a time to peak rise time of 0.21 ± 0.03 ms at 0 mV (n = 9), comparable to rise times for I_Na found in axons or terminals capable of firing AP in the mammalian central nervous system (CNS) (Engel and Jonas 2005; Hu and Jonas 2014). The rapid kinetics of terminal I_Na suggests that it should contribute to AP waveform. To evaluate this contribution, the charging current associated with an invading AP train was compared with the amplitude of I_Na in the same terminal. A charging current of ∼2 nA was recorded when the terminal was held at −80 mV (Fig. 3B). A voltage-step series in the same terminal evoked a mixture of I_Na and I_K, with a maximal I_Na of 80 pA (Fig. 3A). Since amplitude measurements of both whole cell I_Na and charging current are of limited accuracy, due to issues related to space clamp, lack of I_K block, and uncorrected series resistance for large charging currents, these parameters were not statistically compared. Nevertheless, the large difference between the two currents and the failure of large terminal depolarization to induce APs suggest that APs in terminals are mainly created by charging current and that local I_Na may shape AP waveform but cannot generate an AP. This hypothesis was further tested by comparing AP waveform before and after removal of local Na⁺. Onset of local Na⁺-free saline perfusion immediately eliminated I_Na recorded from a varicosity (Fig. 3, A and C). (The concomitant reduction of outward current was in part due to removal of Na⁺-activated K⁺ current.) Local perfusion had no impact on APs initiated and recorded at the 1° BP (Fig. 3D, axon) but slightly slowed the rising phase and reduced the peak amplitude of APs in the terminal (Fig. 3D, term). Similar effects were found in six of eight terminals tested. In the remaining two terminals, Na⁺-free saline had no effect on terminal APs. Since the precise area perfused by Na⁺ saline could not be defined because of the opener shell disrupting laminar flow of saline, effects shown here could be due to elimination of I_Na from more than one varicosity. Nevertheless, these observations are consistent with the hypothesis that local I_Na contributes to the shaping of invading AP in opener terminals.

DISCUSSION

This report shows that APs in varicosities of the opener axon were mainly generated by charging current from proximal branches and that terminals cannot generate APs. However, a small I_Na with a high threshold and fast activation rate was present in most terminals. The fast kinetics and small amplitude of I_Na suggested an auxiliary role, supported by the observation that local perfusion of Na⁺-free saline slightly decelerated AP rising phase and reduced AP amplitude. This report demonstrates a subtle and “quantitative” role for I_Na in presynaptic terminals with low excitability.

Motor nerve endings could potentially experience large fluctuations in resting V_m. The large mass of skeletal muscle means that vigorous contraction can raise extracellular K⁺ concentration locally and depolarize terminals. Furthermore, motor nerve terminals have a variety of presynaptic receptors that can shift resting V_m (Beaumont and Zucker 2000; Darabid et al. 2014; Wojtowicz and Atwood 1985). Presynaptic terminals therefore need to operate over a range of V_m without backfiring. Low I_Na magnitude reported presently could be a contributing factor toward minimizing backfiring. Another potentially important parameter is the large leak current and low input resistance recorded at resting V_m. The input resistance reported presently was ∼50% of that in MF boutons, after correcting for size differences (Engel and Jonas 2005). Finally, low I_Na could minimize the metabolic load on terminals (Alle et al. 2009). These considerations may also apply to the mammalian CNS, where local ionic and neurotransmitter concentrations can fluctuate due to restricted extracellular space.

Studies derived from mammalian CNS axons with strings of varicosities have consistently shown that they have a high Na⁺ channel density and can fire APs (Engel and Jonas 2005; Hu and Jonas 2014; Jackson and Zhang 1995). The low excitability of the crayfish opener axon, despite its long and “beads on a string” morphology, is surprising and raises the question of how APs may reach the distal varicosities. The unique adaptation of the crayfish opener axons may lie in the large diameter ratio between proximal (∼30 μm) to distal (<1 μm) branches (Florey and Cahill 1982). Small I_Na in terminals could play a boosting role in aiding AP spreading. Although a diameter ratio comparable to that of crayfish opener axons is not present in the mammalian nervous systems, a gradual reduction in internodal length has been suggested as a mechanism for accumulating charging current to depolarize motor end plates (Lindgren and Moore 1989; Quick et al. 1979). Morphological analyses of dorsal horn interneurons have reported thin, myelinated axons in close proximity to unmyelinated daughter branches with varicosities (Szucs et al. 2013). The myelinated compartment could potentially play a role in providing charging current for distal varicosities with low excitability. Thus terminal varicosities with low excitability could function, with advantages outlined above, in the mammalian CNS.

The data presented in this article suggest a dominant role for proximal branches of crayfish opener axons in generating terminal APs. However, the interactions between proximal and distal compartments need not be unidirectional, because the large number of terminals could collectively influence the V_m of proximal branches. The possibility of bidirectional interaction between axonal compartments and the roles I_Na may play in these interactions remain to be further explored.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author.

AUTHOR CONTRIBUTIONS

J.-W.L. conception and design of research; J.-W.L. performed experiments; J.-W.L. analyzed data; J.-W.L. interpreted results of experiments; J.-W.L. prepared figures; J.-W.L. drafted manuscript; J.-W.L. edited and revised manuscript; J.-W.L. approved final version of manuscript.