Voltage-Dependent Enhancement of Electrical Coupling by a Subthreshold Sodium Current

Kinetics and time dependence of depolarization-enhanced responses suggests the activation of a subthreshold conductance

Although the waveform of the depolarization-enhanced AD coupling is remarkably similar to that observed at resting and hyperpolarized potentials, detailed analysis of its kinetics revealed differences that are consistent with the progressive activation of a subthreshold voltage-dependent membrane conductance. Three lines of evidence support this conclusion. (1) The time-to-peak of the AD coupling potential, measured about the threshold of the afferent, was increased when compared with that obtained at resting potential (Fig. 2A), averaging 1.13 msec (±0.06 SEM; n = 35) and 0.72 msec (±0.03 SEM; n = 38), respectively (p < 0.0001). Furthermore, both the amplitude of the AD coupling and its time-to-peak were proportionally affected by increasing membrane depolarization (Fig. 2A, inset). (2) The time constant of the decay of the depolarization-enhanced response was found to be increased by almost twofold (Fig. 2B). The estimated time constants of the decays averaged 1.38 msec (±0.16 SEM) and 0.73 (±0.05 SEM), for the responses obtained at depolarized and resting potentials, respectively (n = 35; p < 0.0002). (3) The depolarization-enhanced AD coupling fluctuated in amplitude (Fig. 2C). Statistical analysis of the waveform of the AD coupling obtained at depolarized potentials showed a marked increase in the SD at the falling phase of the response when compared with those obtained at resting potential (Fig. 2D). Because in contrast to chemical synapses electrical transmission is not probabilistic in nature, the observed fluctuations could represent the involvement of a voltage-dependent amplifying mechanism.

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Figure 2.

Kinetics of the voltage-dependent enhancement of electrical coupling. A, Depolarization not only increases the amplitude of the response but also its time-to-peak. Superimposed traces show the antidromic coupling potential recorded at resting potential (-73 mV) and at near the threshold of the cell (-66 mV). Note the increased time-to-peak of the depolarization-enhanced response. Inset, Relationship between the amplitude of the AD coupling (abscissa) and its time-to-peak (ordinate) obtained at different membrane potentials for the same fiber. Depolarization increased proportionally both the amplitude and the time-to-peak of AD coupling. B, Depolarization also slows the time course of the decay of the coupling potential. Superimposed traces represent the normalized decays of the recordings illustrated in A. There is almost a twofold increase in the estimated time constant of the decay at depolarized potentials (0.6 vs 1.1 msec). C, Depolarization-enhanced responses fluctuate in amplitude. Superimposed traces represent single consecutive responses obtained at -66 mV; note the variability in the amplitudes of the AD couplings. D, Statistical analysis of amplitude fluctuation of the AD coupling at depolarized membrane potentials. Superimposed traces illustrate the averaged (mean; calibration, 4 mV) responses with its SDs (SD; calibration, 1 mV) obtained at resting potential (top) and -66 mV (bottom) in the same experiment. Note the increase of the SD in the falling phase of the depolarization-enhanced response.

We next examined the time dependence of the voltage-dependent enhancement of AD coupling by measuring the amplitude of this potential at different intervals after the onset of a 50 msec depolarizing pulse (Fig. 3A). Although never reaching the values observed at resting potential, the amplitude of the AD coupling decreased with interval duration and reached plateau for intervals >15 msec (Fig. 3A,B). Strikingly, the voltage dependence of the AD coupling at the 40 msec interval showed a linear relationship with proportional changes at both sides of the resting potential, lacking the characteristic steep enhancement between the resting potential and the threshold of the cell observed at the onset of depolarizing pulses (Fig. 3C). When fit with a straight line, the slope of this relationship averaged 0.09 (±0.015 SEM; n = 12) and was indistinguishable (p > 0.6) from the estimated linear component (k₁) of the voltage dependence at the onset of the same depolarizing pulse (Fig. 3C). Thus, the time dependence of the component represented by the k₂ constant confirms the existence of the two independent mechanisms proposed by quantitative analysis of the voltage dependence and suggests the involvement of an amplifying mechanism that inactivates-deactivates over time. Consistent with this interpretation, the enhanced responses at the onset of the depolarizing pulse exhibited different kinetics than those observed at both the 40 msec interval and the resting potential. This property is illustrated in Figure 3D, in which the averaged amplitudes of the AD coupling obtained at the onset, 40 msec, and resting potential [4.11 ± 0.24 SEM (n = 35), 2.43 ± 0.22 SEM (n = 38), and 1.57 ± 0.12 SEM (n = 38) mV, respectively] are plotted against the time constants of their respective decays [1.38 ± 0.16 SEM (n = 35), 0.56 ± 0.04 SEM (n = 38), and 0.73 ± 0.05 SEM (n = 18) msec, respectively].

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Figure 3.

Time dependence of the voltage-dependent enhancement of AD coupling. A, Averaged responses, obtained at different intervals after the onset of a 50 msec depolarizing pulse, are superimposed. B, The graph summarizes the quantitative relationship between the amplitude of the antidromic coupling potential (AD Coup. Ampl., ordinate) and the interval after the onset of the pulse (Interval, abscissa) for five different experiments. The amplitude of the recorded responses decreased gradually with interval duration and reached plateau for intervals >15 msec. C, Voltage dependence of AD coupling at the end of a 50 msec depolarizing pulse (48 msec interval). AD coupling still exhibits voltage dependence (left). Quantitative relationship (right, Voltage-dep. Late; filled circles) between the amplitude of the antidromic coupling and the membrane potential of the fiber at the end of the 50 msec current pulses showed a linear relationship at both sides of the resting potential, with characteristics similar to the k₁ component calculated from the voltage dependence at the onset of the pulse (k₁ early). Also superimposed is the calculated k₂ component (k₂ early). D, The enhanced AD couplings obtained at the onset and at the end of 50 msec pulses exhibit different kinetics. The amplitudes of the antidromic coupling (ordinate) obtained at the onset (Early), at the late portion of the depolarizing pulse (Late), and at resting potential (RP) are plotted versus the time constants (abscissa) of the their respective decays (n = 5; bars represent SEM).

Voltage-dependent enhancement of AD coupling is sensitive to QX-314 and TTX

Both the voltage sensitivity and membrane potential range of the time-sensitive (k₂) component are consistent with those of a subthreshold Na⁺ current (Crill, 1996). Therefore, we tested the effect of intracellularly injected QX-314 (which blocks Na⁺ channels) on the amplitude of the AD coupling obtained at different membrane potentials (Fig. 4A). The amplitude of the depolarization-enhanced AD coupling was reduced after the injection of QX-314 (∼60% in the example of Fig. 4A), whereas that recorded at the resting potential remained, in this case, unaffected. The amplitude of the AD coupling obtained at depolarized and resting potentials after QX-314 injection averaged 46% (±4 SEM) and 81.3% (±10 SEM) of those obtained at the same potential before QX-314, respectively (n = 14). These changes occurred within a time window in which the fast action potentials of the afferents remained essentially unaffected (Fig. 4B, inset). The fact that depolarization-enhanced potentials were differentially affected by QX-314 excludes the possibility that the observed reduction was caused either by a decrease in gap junctional conductance or by diffusion of this drug to the M-cell to affect the antidromic spike generation, the source of the coupling. This finding was confirmed by performing simultaneous extracellular recordings of the M-cell antidromic spike during QX-314 injections (data not shown). Thus, although the diffusion of small or ineffective amounts of QX-314 to the M-cells cannot be ruled out, our data are consistent with previous reports (Logan et al., 1996; Mann-Metzer and Yarom, 1999) indicating that QX-314 is impermeable through neuronal gap junctions.

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Figure 4.

Voltage-dependent enhancement of AD coupling is blocked by both QX-314 and TTX. A, Superimposed traces obtained at resting potential (-67 mV) and -58 mV recorded 5 min (control) and 25 min (QX-314) after the penetration of the cell with an electrode containing 50 mm QX-314. Note the contrast between the marked reduction in the amplitude of the coupling recorded at the depolarized potential and the lack of change of the response obtained at resting potential. B, Quantitative relationship between the AD coupling and the membrane potential in the same fiber 5 min (control, filled circles) and 25 min after the penetration with the QX-314-containing electrode (QX-314, empty circles). The relationship observed after QX-314 is similar to the k₁ component calculated early in the recording (k₁ control). Inset, Suprathreshold depolarizing pulses obtained before and after intracellular injection of QX-314. Blockade of the voltage-dependent enhancement of AD coupling occurs, whereas spikes are still not significantly affected by QX-314. C, Plot of the difference between the quantitative relationships obtained at control and after the QX-314 effect in the same experiment. The component removed by QX-314 was quantitatively similar, when fitted with an exponential function, to the k₂ component calculated earlier in the recording (k₂ = 0.21 mV^-1). D, TTX blocks the voltage-dependent enhancement of AD coupling. Superimposed traces obtained at resting potential (RP, -71 mV) and near threshold (-65) in control and after topical application of TTX (1 μm) in the VIIIth nerve. As with QX-314, note the marked reduction in the amplitude of the coupling recorded at the depolarized potential, with no significant change of the response obtained at resting potential. E, The antidromic action potential of the M-cell remained unchanged at the time that the voltage-dependent enhancement of AD coupling was already suppressed. Traces illustrate the M-cell antidromic action potential recorded simultaneously with the afferent before and 10 min after the application of TTX. F, Plot summarizes the data obtained for the QX-314 and TTX experiments. The mean amplitude of the AD coupling potential at resting potential (RP) and -60/-65 mV (Depo) are plotted before and after QX-314 (left) and TTX (right) as percentages of their amplitude at resting potential during control conditions (bars represent SEM; not visible for responses at resting potential).

The slope of the relationship between the AD coupling amplitude and the membrane potential after QX-314 lacked the characteristic steep voltage dependence observed between the resting potential and threshold of the afferent (Fig. 4B). When fitted with a straight line, the slope of the relationship obtained after QX-314 resembled that of the k₁ linear component of the relationship obtained earlier in the recording, during control conditions (Fig. 4B). The slope averaged 0.086 (±0.014 SEM; n = 5), and it was not significantly different from the k₁ in control conditions (p > 0.8), suggesting that QX-314 selectively targeted the time-dependent supralinear component of the voltage dependence. Accordingly, the difference between the values in control and after QX-314 (Fig. 4C) could be fit with an exponential function with a constant that averaged 0.24 mV^-1 (±0.033 SEM; n = 5), a value that was not significantly different from the k₂ component estimated in control conditions (p > 0.2).

Because QX-314 has been reported to have nonspecific actions on other than Na⁺ channels, we tested the effect of extracellular application of TTX, which specifically blocks Na⁺ channels, on the amplitude of the AD coupling obtained at different membrane potentials. Because the application of TTX would ultimately lead to a reduction of the antidromic spike of the M-cell (the source of the coupling), these experiments were performed while simultaneously recording from this neuron (Fig. 4E). As with QX-314, we observed a large decrease in the amplitude of the AD coupling at depolarized potentials, whereas those obtained at resting potential remained essentially unaffected (Fig. 4D). Overall, the reduction of the amplitude of the AD coupling obtained at depolarized and resting potential after TTX was quite similar to those observed after QX-314, averaging 50% (±6 SEM) and 93% (±9 SEM) of their values in control conditions, respectively (n = 8; p < 0.0005). The M-cell antidromic spike remained unaffected (Fig. 4E), averaging 96% of control (±7 SEM; n = 7; p > 0.6) at the time during which these changes of AD coupling amplitude were observed (we generally observed a delay of ∼30 min before the M-cell antidromic spike was affected because TTX was applied topically on the nerve entry point, and the somata of this cell lies deep within the medulla). As expected, the slope of the voltage-dependent relationship obtained after TTX was linear and similar to that obtained after QX-314 and lacked the characteristic steep voltage dependence observed between the resting potential and threshold of the afferent (data not shown).

Figure 4F summarizes the data obtained for the QX-314 and TTX experiments; the amplitudes of the AD coupling at resting and depolarized potentials before and after the action of these drugs are plotted as percentages of the amplitude at resting potential during control conditions. Neither QX-314 nor TTX reduced the amplitude of the depolarization-enhanced AD couplings to the values observed at resting potential, consistent with the existence of an additional mechanism (k₁ component) that is responsible for ∼20% of the amplification observed during depolarization. Finally, both intracellular QX-314 and extracellular TTX ultimately led to a profound reduction of the amplitude of the action potential of the afferents (∼70% decrease) that was identical to that observed when Na⁺ was replaced by tetramethyl ammonium in the extracellular perfusion solution (data not shown), confirming that both drugs targeted Na⁺ channels.

Slow-inactivating TTX-sensitive membrane conductance underlies voltage dependence of AD coupling

To test for the existence of a subthreshold voltage-sensitive Na⁺ conductance, the membrane properties of the afferents were examined by measuring their current-voltage relations without superimposed coupling potentials. The membrane responses to current steps were similar in all studied fibers (n = 16), and one set of these responses is illustrated in Figure 5A. For a given size depolarizing current pulse, we found a nonlinear voltage response that was maximal within ∼2 msec of the pulse onset and then rapidly decayed to a steady-state value. The magnitude of this initial response was not proportional to the injected current and was more obvious for near-threshold depolarizations (Fig. 5A). The steady-state late response was a linear function of the polarizing current for depolarizing and hyperpolarizing pulses (Fig. 5B, filled circles), but the early responses exhibited the previously mentioned nonlinearity in the depolarizing direction, taking the form of an apparent increase in membrane slope resistance (Fig. 5B, open circles). This initial nonlinear response to depolarizing pulses was reduced by TTX application (Fig. 5C), averaging 31% of control (±13 SEM; n = 4). As a result of TTX application, the I-V relationship became linear, undistinguishable from that obtained at steady state (Fig. 5D). To demonstrate that this TTX-sensitive subthreshold membrane response is the mechanism responsible for the amplification of AD coupling during depolarization, we compared the time courses of both phenomena. As shown superimposed in Figure 5E, the decay of the membrane response to a near-threshold depolarizing pulse closely followed the time course of the above-mentioned time dependence of AD coupling amplitude (see above). Accordingly, the amplitude of AD coupling at the 40 msec interval of a 50 msec depolarizing pulse was not significantly affected by QX-314, averaging 80% of control (±13 SEM; n = 10; p = 0.3). Such a small (although not significant) reduction in amplitude after QX-314 at the 40 msec interval is ascribable to a parallel reduction in this coupling observed at resting potential (see above; it averaged 81.3%, ±10 SEM, of control; n = 14; difference not significant; p = 0.33). This observation suggests that large AD couplings could activate at resting potential a significant portion of this amplifying current (see next section).

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Figure 5.

TTX-sensitive nonlinear membrane conductance contributes to the voltage dependence of AD coupling. A, Voltage responses to seven current pulses (50 msec duration, -1 to +2.4 nA; below). Note the progressively larger transient membrane response (open circle) at the pulse onset. B, Voltage (ΔV; ordinate)-current (abscissa) relation obtained in the same afferent fiber, measured at 2 (open circles) and 48 msec (filled circles). The voltage-current relation is the same at both times for hyperpolarizations, and the membrane behaves linearly in the steady state (voltage responses at ∼50 msec were fit with a straight-line function; solid line). At pulse onset, however, the V-I relationship exhibits a nonlinearity in the form of an apparent increase in membrane slope resistance with depolarization. C, Effect of 10 μm TTX on the membrane voltage responses. Superimposed traces represent the voltage responses to a pulse of +2.4 nA (below), before (Control) and 15 min after application of TTX. D, Voltage (ΔV)-current relation obtained in the same afferent fiber at the onset of the pulse before (Control, filled circles) and after (empty circles) TTX application. Voltage responses after TTX were fit with a straight-line function (solid line). Note the disappearance of the nonlinearity observed at the onset of the pulse. E, Nonlinear membrane properties match the time dependence of the voltage-dependent enhancement of AD coupling. The decay of the membrane voltage response to a +2.4 nA/50 msec pulse is illustrated superimposed to the amplitudes of the AD coupling obtained at different intervals after the onset of a 50 msec depolarizing pulse of lesser magnitude (open circles; same data shown in Fig.3B). F, Voltage-dependent changes can be replicated on a brief depolarizing current pulse injected through the presynaptic recording electrode. The depolarization produced at resting potential by a brief current pulse (1 msec duration; 0.5 nA) was dramatically enhanced by depolarization and slightly decreased by hyperpolarization (±0.9 nA). Observe the increased rise time and decay of the depolarization-enhanced response, which resembled those observed for the AD coupling (compare with Figs. 1B, 2A).

If this subthreshold membrane conductance, but not the voltage-dependent properties of the gap junction channels, is responsible for most of the amplification of electrical coupling, the changes in the AD coupling should be replicated at different membrane potentials by injection of a current pulse through the afferent electrode. That is, depolarization and hyperpolarization of the afferent should then have the same effect on the potential produced by a constant current pulse as it does on the AD coupling potential. To directly demonstrate that this membrane property is responsible for amplification of the AD coupling, we asked whether voltage-dependent changes could be replicated on a brief depolarizing current pulse (1 msec duration) injected through the presynaptic recording electrode (Fig. 5F). We found that the depolarization produced at resting potential by the brief current pulse was dramatically enhanced by depolarization (pulse averaged 175.5% ± 9.4, SEM, of control; n = 5) and slightly decreased by hyperpolarization (pulse averaged 82.6% ± 9.4, SEM, of control; n = 5). Furthermore, depolarization-enhanced brief pulses had kinetic changes (increased rise time and decay) (Fig. 5F, right) that resembled those observed for the AD coupling and exhibited a similar time dependence (data not shown).

Subthreshold Na⁺ currents are slowly inactivating and generally opposed by repolarizing potassium (K⁺) conductance(s) (Crill, 1996). Both the time dependence of the amplification and kinetic characteristics of the depolarization-enhanced AD coupling potentials, which are generally followed by a slight hyperpolarization (∼0.5-1 mV at -65 mV membrane potential) (Fig. 6A, arrow), suggested the involvement of a subthreshold repolarizing response. Therefore, we tested the effect of the intracellular injection of a combination of K⁺ channel blockers (0.5/1.0 m TEA-Cl, 0.075/0.15 m 4-aminopyridine, 1/2 m CsCl; see Materials and Methods) on the AD coupling and membrane responses to depolarizing steps. We found that the injection of K⁺ channel blockers prolonged the decay of the AD coupling (Fig. 6B) and the initial voltage response to a depolarizing pulse (Fig. 6C). Remarkably, the time dependence of the AD coupling amplification was no longer observed (Fig. 6D). Figure 6E illustrates the amplitude of AD coupling potentials obtained at different intervals after the onset of a 50 msec depolarizing pulse, in the absence (filled circles) and presence (empty circles) of K⁺ channel blockers in the recording electrode for five and seven experiments, respectively. In contrast with that observed in control conditions (Fig. 5B), the V-I relationship after injection of K⁺ channel blockers exhibited at the end of 50 msec pulses a nonlinearity in the depolarizing direction in the form of an apparent increase in membrane slope resistance (Fig. 7A,B, filled circles). This nonlinearity was sensitive to extracellular application of TTX (10 μm) (Fig. 7B), which linearized the membrane response (Fig. 7C, empty circles). Thus, the experiments using K⁺ channel blockers revealed the presence of a slowly inactivating subthreshold Na⁺ conductance that is counterbalanced by a delayed repolarizing potassium conductance(s), which is responsible for the observed time dependence of the voltage-dependent amplification of the AD coupling.

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Figure 6.

K⁺ channel blockers abolished the time dependence of voltage-dependent enhancement of AD coupling. A, Superimposed traces illustrate the antidromic coupling potential obtained at resting potential (RP, -73 mV) and at -64 mV, at the onset of a depolarizing pulse. The depolarization-enhanced AD coupling is generally followed by a small hyperpolarization (arrow), suggesting the involvement of repolarizing conductances. B, Superimposed traces illustrate the amplitude and time course of the AD coupling during a depolarization produced by current injection (0.4 nA) 1 min (Control) and 15 min after the penetration of the afferent with a recording solution containing a combination of K⁺ channel blockers (1.0 m TEA-Cl, 0.15 m 4-aminopyridine, 2 m CsCl). C, Voltage responses to depolarizing current steps of 0.8 nA, immediately (control) and 15 min after (K⁺ blockers) penetration of the fiber with an electrode containing a combination of K⁺ channels blockers. D, K⁺ channel blockers eliminate the time dependence of the voltage-dependent enhancement of AD coupling. Superimposed traces show the AD coupling at different intervals from the onset of a 0.5 nA depolarizing pulse. E, Graph summarizes the amplitude of the AD coupling (ordinate) at different intervals (abscissa) after the onset of a 50 msec depolarizing pulse for seven different fibers (open circles). The amplitudes of AD couplings obtained in the absence of K⁺ channel blockers in the recording solution are superimposed (filled circles; same data as illustrated in Fig. 3B). Bars represent ± SEM.

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Figure 7.

Involvement of a persistent Na⁺ current in the voltage-dependent enhancement of the AD coupling. A, Voltage responses to depolarizing and hyperpolarizing current pulses (50 msec duration; -1 to +1 nA) after intracellular injection of K⁺ channels blockers. Note the late apparent increase in the resistance of the cell (inward rectification) with progressively stronger depolarizing pulses (filled circles). B, Voltage responses to depolarizing and hyperpolarizing current pulses (±1 nA) obtained in the same afferent before and after the application of TTX (10 μm). TTX blocks the apparent increase in membrane resistance observed at the end of the depolarizing pulse induced by the injection of K⁺ channels blockers. C, Voltage (ΔV_m, ordinate)-current (Current, abscissa) relationship obtained in the same afferent before (filled circle) and after (open circle) the application of TTX. The apparent increase in input resistance (late inward rectification) is abolished by topical application of TTX. D, Enhancement of AD coupling results from the interplay between a subthreshold Na⁺ current and repolarizing K⁺ conductances. Superimposed traces illustrate the AD coupling potential obtained at resting potential (RP; -73 mV) and at a depolarized potential (-66; same experiment illustrated in Fig. 2A). Depolarization progressively activates a subthreshold Na⁺ current (graded area) that is deactivated by contrasting voltage-sensitive K⁺ conductance(s) (darker graded area).

Taken together, our results indicate that a subthreshold TTX-sensitive noninactivating or slowly inactivating Na⁺ conductance is responsible to a large extent for the amplification of AD coupling and is opposed by delayed repolarizing K⁺ conductance(s), and this interplay closely reproduces the waveform of the AD coupling obtained at the resting potential (Fig. 7D). Such remarkable resemblance with the waveform of the AD coupling at resting potential is demonstrated by the fact that the changes in amplitude and area of the depolarization-enhanced response for the example illustrated in Figure 7D were very similar (3.5- and 4.5-fold increase, respectively).

Nonlinear amplification of electrical coupling at resting potential

We showed that because of the activation of a subthreshold Na⁺ current the coupling coefficient for antidromic impulses from the M-cell to each club ending is enhanced by depolarization of the terminal of that ending. Such nonlinearity of the AD coupling coefficient could also exist at resting membrane potential if this signal were large enough to activate a significant fraction of the amplifying current, which could occur when gap junctional conductances were greater. To test this hypothesis we took advantage of the fact that the amplitude of the AD coupling potential between neighboring club endings dramatically varies in size and asked whether larger potentials were differentially affected by intracellular injection of QX-314. The variability in amplitude of AD coupling potentials reflects the large variation in gap junctional conductance observed among these terminals (Smith and Pereda, 2003) that is dynamically regulated by the activity of their colocalized glutamatergic synapses (Yang et al., 1990; Pereda and Faber, 1996; Pereda et al., 1998, 2003b; Smith and Pereda, 2003). By recording presynaptically from these afferents, we found that the amplitude of the AD coupling at resting potential was reduced by QX-314 injection and that the magnitude of the reduction was positively correlated with the amplitude of the coupling potential in control conditions (Fig. 8A) (n = 12). The differential susceptibility of larger AD coupling potentials to QX-314 can account for the slight overall reduction of this signal observed at resting potential (see above) (Fig. 4F).

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Figure 8.

Nonlinear amplification of retrograde coupling at resting potential. A, Graph plots the amplitude of the AD coupling potentials at resting potential in control conditions (abscissa) versus their respective reduction after intracellular injection of QX-314 (ordinate) expressed as percentage of amplitude in control (n = 13). B, Simultaneous presynaptic and postsynaptic recordings between club endings and the M-cell lateral dendrite (top) allow correlation between orthodromic and antidromic coupling potentials in the same terminal. Graph (bottom) illustrates the relationship between the amplitude of orthodromic (abscissa) and antidromic (ordinates) coupling potentials in six terminals, obtained while recording from the same dendrite. The dotted line represents the linear relationship between orthodromic and antidromic couplings estimated using the three terminals that exhibit small orthodromic coupling potentials with, presumably, lower conducting junctions. Club endings that exhibit larger orthodromic potentials (presumably higher conducting junctions) showed AD couplings that were bigger than those expected from the relationship obtained in low-conducting terminals.

To demonstrate that larger, QX-314-sensitive AD coupling potentials corresponded to higher conducting junctions, we compared coupling coefficients between neighboring terminals within the same dendrite. For this, we obtained in one fish multiple paired recordings between single afferent fibers and the same M-cell dendrite and plotted the amplitude of the orthodromic coupling potentials (the potential recorded in the M-cell corresponding to the presynaptic spike) (Fig. 8B) as an indicator of the degree of conductance of the junctions in each particular terminal versus the amplitude of their corresponding AD couplings (Fig. 8B). We found that this relationship was not linear because afferents that showed larger orthodromic potentials (and presumably higher conducting junctions) exhibited AD couplings that were bigger than those expected from the projection of a linear relationship obtained using terminals that exhibited small orthodromic potentials (Fig. 8B, bottom, dotted line).

Amplification of retrograde coupling operates under physiological conditions

We used the AD coupling to explore the mechanisms underlying the voltage dependence of retrograde electrical transmission but argued that such mechanisms could operate during coactivation of the afferents, because signals would be large enough to activate a significant fraction of the subthreshold Na⁺ current in the terminals. To demonstrate that this amplification operates under these conditions, we tested the effect of QX-314 on the amplitude of the retrogradely transmitted coupling potential attributable to the dendritic depolarization produced by the activation of a population of these afferents; that is, the characteristic intradendritically recorded mixed, electrical, and chemical, synaptic potential in response to VIIIth nerve stimulation (Fig. 9A) (Furshpan, 1964) can be recorded simultaneously as a coupling potential in nearby terminals for which the stimulus strength is subthreshold (Fig. 9B, VIIIth N. coup) (Pereda et al., 1995). This coupling potential has been shown to have voltage-dependent characteristics identical to those of the AD coupling (Pereda et al., 1995) and is capable (when a sufficient number of afferents are recruited by adjusting VIIIth nerve stimulation) of eliciting an action potential in the recorded afferent (“backfiring”) (Pereda et al., 1995). For the experiments reported here, we adjusted the amplitude of the VIIIth nerve coupling to be at the threshold of the cell, where this depolarization was able to occasionally elicit spikes in the recorded afferent (Fig. 9B, inset); this allowed us to monitor the size of the coupling potential as well to test the potential role of its amplification in recruiting subthreshold afferents. We found that the amplitude of this response was greatly attenuated by intracellular injection of QX-314 (Fig. 9B), averaging 53.9% of the values obtained at control (±8% SEM; n = 5), and was no longer able to backfire the afferent, suggesting that amplification of retrograde transmission takes place during orthodromic activation of a population of afferent fibers. To demonstrate the existence of this amplification under more physiological conditions, we used natural stimulation in the form of brief sound clicks (Fig. 9A). These experiments were performed during “intraterminal” recordings (Pereda et al., 1998; Smith and Pereda, 2003) within a radius of 40-60 μm from the lateral dendrite, localized by an initial penetration of that process, while monitoring the responses to natural stimulation delivered by a speaker located near the animal's head (500 μsec duration click). This recoding site, in the vicinity of the lateral dendrite of the M-cell, is electrotonically distant from the hair cell side of the afferent and therefore rules out a potential contamination of the recordings with auditory evoked synaptic potentials. Figure 9C illustrates the intradendritic response to this brief auditory stimuli (top) and the coupling, obtained in sequential recordings, in one neighboring terminal for which the stimulus strength was subthreshold (bottom). This coupling was also suppressed by QX-314 and averaged 59.6% of control (range, 58.1-62.2%; n = 4), indicating that amplification of retrograde coupling by the subthreshold Na⁺ current occurs under physiological conditions. Unfortunately, we were not able to backfire subthreshold recorded terminals with this sound-evoked coupling potential because of the impossibility of recruiting enough afferents to generate a sizable coupling potential without directly activating the recorded one. This is attributable to the fact that sound stimulation depends on the physiological characteristics of the afferents, and therefore, unlike electrical stimulation, the number of activated afferents by auditory stimulation cannot be finely adjusted. Nevertheless, on the basis of the properties of the VIIIth nerve coupling (also produced by the orthodromic activation of auditory afferents), it is reasonable to assume that afferents might be similarly recruited by natural stimulation.

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Figure 9.

Voltage-dependent enhancement of retrograde electrical coupling operates under physiological conditions. A, Dendritic synaptic potentials (Mauthner cell) evoked by suprathreshold electrical or natural stimulation of VIIIth nerve afferents (B, C, top traces; dark orange afferents; VIIIth Nerve stim. and Sound click) can be recorded as coupling potential, in neighboring, subthreshold terminals (yellow terminals, club ending; bottom traces in B and C, respectively). I_Na+P = persistent (subthreshold) Na⁺ current in the club endings. B, Mixed synaptic response (electrical and chemical) produced by extracellular electrical stimulation of the posterior branch of the VIIIth nerve (VIIIth Nerve stim.) evokes a retrograde coupling potential in a subthreshold terminal (bottom; VIIIth Nerve coup.). Inset, Amplitude of the VIIIth nerve coupling (asterisk) was adjusted to be at the threshold of the presynaptic afferent (truncated spike, 100 mV). Superimposed traces in the bottom panel represent the amplitude of this retrograde response obtained right after (control) and 15 min after the penetration of the terminal with an electrode containing a 50 mm solution of QX-314 (A, QX-314). C, Intradendritically recorded sound-evoked synaptic potential (Sound click; 500 μsec pulse) can also be recorded, intracranially, as a coupling potential in neighboring inactive terminals (bottom; Sound coup.). Superimposed traces represent the retrograde responses obtained right after (Control) and 5 min after the penetration of the synaptic terminal with an electrode containing QX-314. Note the reduction in amplitude of both retrograde coupling potentials observed after injection of QX-314 (red traces). D, Depolarization-enhanced retrograde coupling can recruit subthreshold afferents. The AD coupling was paired with a presynaptic depolarization (blue trace; compare the enhanced response with that obtained at resting potential; black trace; RP) at a membrane potential in which it was able to occasionally elicit action potentials in the presynaptic afferent (black trace; truncated spike, 100 mV). Intracellular injection of QX-314 (red trace) removes the amplification of AD coupling (along with a small reduction in the depolarization produced in the current pulse), which is not longer capable of eliciting action potentials in the afferent. Inset, Effects of QX-314 occurred within a time window in which the fast action potentials of the afferents remained largely unaffected. Superimposed recordings show the amplitude of the action potential of the recorded afferent in control (black trace) and after QX-314 (red trace) at the time in which the changes in the AD coupling amplitude were observed.

These data suggest that amplification of retrograde transmission by this current might play an essential physiological role in promoting the recruitment of subthreshold afferents during the activation of a population of these fibers. Because of its voltage dependence, such integrative property of this amplifying mechanism would be particularly relevant in afferents that were subliminally activated by the stimulus (i.e., depolarization did not reach threshold). To directly demonstrate this property, we paired the AD coupling with a presynaptic depolarization before and after the intracellular injection of QX-314 (the AD coupling was used instead of the VIIIth nerve coupling to avoid possible contamination by local responses produced by changes in axonal excitability after the nerve stimulation). The presynaptic depolarization (a current pulse delivered through the recording electrode) was adjusted for the AD coupling to be at the firing threshold of the cell (Fig. 9D); again, this allowed us to both visualize the coupling potential and test the potential role of its amplification in recruiting subthreshold afferents. We found that although under control conditions the depolarization-amplified AD coupling was able to elicit action potentials in the presynaptic afferent, this recruitment was blocked by QX-314, which dramatically reduced its amplitude (Fig. 9D, red trace). This phenomenon was observed in all of the explored fibers (n = 14) and occurred within a time window in which the fast action potentials of the afferents remained essentially unaffected (Fig. 9D, inset).

Intrinsic membrane properties contribute to bi-directional electrical communication

Recent work shows that QX-314-sensitive currents, presumably mediated by Na⁺ channels, are involved in the amplification of electrical coupling between cerebellar inhibitory interneurons (Mann-Metzer and Yarom, 1999) and hippocampal pyramidal cells (Schmitz et al., 2001). Both computer modeling (Traub, 1995) and experimental data (Mann-Metzer and Yarom, 1999) suggest that subthreshold amplification of electrical coupling by intrinsic membrane properties plays a pivotal role in promoting the synchronization of weakly coupled neuronal networks. Our results support the view of these authors that electrical synapses and intrinsic membrane properties interact to create a highly modifiable communication pathway (Mann-Metzer and Yarom, 1999).

In the case of the club endings, intrinsic membrane properties act unilaterally to ensure efficient retrograde communication from the M-cells to these presynaptic terminals. Electrical coupling in this system occurs between two heterologous cell types of dramatically different size and electrical properties. Despite the fact that electrical transmission would result, enhanced by the higher input resistance of the presynaptic endings, retrograde transmission is challenged by the geometrical and electrical properties of the M-cell that favor the spread of depolarizations from the lateral dendrite (where these terminals are segregated) toward the soma of this cell (Fig. 9A). Voltage-clamp analysis of the M-cell dendritic resting conductances revealed the presence of an inward K⁺ rectifier that by increasing dendritic resistance during depolarizations acts to boost excitatory inputs (Faber and Korn, 1986). Although the boosting of dendritic synaptic inputs would result in some enhancement of retrograde communication, it has been proposed that the key functional role of this current is to facilitate the spread of excitatory currents to the remarkably low impedance soma (∼115 kΩ) (Fukami et al., 1964) by increasing the dendrite to soma transmission during depolarizations (Faber and Korn, 1986). The presence of the voltage-dependent amplifying mechanism in the presynaptic club endings described here would ensure the detection of relevant postsynaptic signals, compensating for the challenging electrical and geometrical conditions.

Possible contribution of the junctional membrane

Although the mechanism underlying the weaker voltage dependence (represented by the k₁ component) is not the focus of the present paper, we speculate that it likely reflects a voltage-dependent property of the junctional membrane, given the absence of other presynaptic conductances that could underline this behavior (Pereda et al., 1995). An adequate study of the observed phenomenon would require simultaneous recordings under voltage-clamp conditions. This behavior could be transjunctional in origin, because a difference of ∼10 mV exists between the resting potentials of both cells (-70 mV in the terminal and -80 in the M-cell dendrite). In the crayfish, fast rectification of electrical coupling from the lateral and medial giant axons to the motor giant axon (Furshpan and Potter, 1959) is believed to be caused by a transjunctional voltage dependence maintained by a difference between the resting potentials of the involved cell, and the removal of this voltage difference restores electrical bi-directionality (Giaume and Korn, 1983). Coupling of cells expressing dissimilar connexins that form hemichannels of different gating polarities is one way to establish rectifying junctions (Barrio et al., 1991; Bennett and Verselis, 1992; Verselis et al., 1994), and in some cases asymmetry can be strongly modulated by small changes in difference of holding potentials (Bukauskas et al., 2002). Interestingly, connexin35 (O'Brien et al., 1998), the fish ortholog of the neuron-specific mammalian gap junction forming protein connexin36 (Condorelli et al., 1998), has been reported to be abundantly present at both sides in the gap junction plaques between club endings and the M-cell, suggesting that electrical transmission is mediated via homotypic gap junction channels (Pereda et al., 2003a). Although the presence of additional neuronal connexins cannot be ruled out, the nonlinear behavior of these junctions suggests that hemichannels of identical connexin composition could exhibit subtle functional differences on each side of the junction, as a result, for example, of posttranslational modifications. Indeed, it has been proposed that only postsynaptic hemichannels (M-cell side) are the target of regulation by protein kinases (Pereda et al., 1994, 1998).

Functional significance

Amplification of retrograde coupling allows club endings to be electrically coupled to each other through the lateral dendrite of the M-cell (Pereda et al., 1995). Such coupling may act as a mechanism of lateral excitation, enhancing (1) transmitter release when fibers are activated simultaneously (Smith and Pereda, 2003; Curti et al., 2004) and (2) the effectiveness of this input when only a portion of these fibers is active by synchronizing that afferent population and promoting the recruitment of new fibers that are close to threshold (Pereda et al., 1995). It is generally believed that patterns of lateral excitation play relevant physiological roles in amplifying the responses of sensory afferents tuned to qualitatively similar stimuli (Herberholz et al., 2002). In contrast to other examples, such as retinal cones (DeVries et al., 2002) and crayfish locomotor networks (el Manira et al., 1993), in which electrical synapses are located between the presynaptic neurons, club endings are electrically coupled to each other through the M-cell lateral dendrite. This coupling arrangement would explain why whereas in some systems junctional properties prevent the antidromic spread of postsynaptically originated currents (Furshpan and Potter, 1959; Auerbach and Bennett, 1969: Ringham, 1975; Edwards et al. 1991), physiological properties of the club endings actually promote this phenomenon. Finally, this phenomenon could also underlie other important sensory mechanisms, because backfiring of mormyromast afferents by retrograde electrical communication (also demonstrated in club endings) (Pereda et al., 1995) has been proposed to mediate a rapidly acting type of lateral inhibition (Slesinger and Bell, 1985). Interestingly, as in the Mauthner cell system, Herberholz et al. (2002) recently demonstrated that indirect coupling through the postsynaptic cell is important in recruiting unstimulated afferents in the crayfish escape circuit. The similarity of the two systems suggests that this mechanism is functionally relevant and might constitute a widespread strategy among circuits in which synchrony of afferents is important.

Thus, as a result of the presence of a subthreshold Na⁺ current, the coupling coefficient from the Mauthner cell to the club endings is voltage dependent. Such enhancement can be activated by depolarization of the presynaptic terminal, by an increase in junctional conductance, or during large dendritic depolarizations. Because all of these conditions coexist when a population of these afferents is activated by strong acoustic signals, this current is likely to play an important functional role. That is, the synchronization that it promotes would act to enhance the synaptic response, increasing the probability of initiating an escape response.

Similar asymmetric amplification of electrical coupling by intrinsic membrane properties might also occur between mammalian cells. Most of the electrical coupling in mammalian neurons takes place at remote dendritic sites (Llinás et al., 1974; Peinado et al., 1993; De Zeeuw et al., 1995; Fukuda and Kosaka, 2000, 2003) between dissimilar tortuous neuronal processes in which geometry, despite the presence of bi-directional gap junction channels, might challenge efficient bi-directional communication. Unilateral or asymmetric amplification of weaker electrical coupling by intrinsic membrane properties in one of the coupled cells might act to promote synchronized neuronal activity, by ensuring bidirectional communication between neuronal dendritic processes of different size and shape.