Protein Kinase C Enhances Electrical Synaptic Transmission by Acting on Junctional and Postsynaptic Ca2+ Currents

Abstract

By synchronizing neuronal activity, electrical transmission influences the coordination, pattern, and/or frequency of firing. In the hemaphroditic marine-snail, Aplysia calfornica, the neuroendocrine bag cell neurons use electrical synapses to synchronize a 30 min afterdischarge of action potentials for the release of reproductive hormone. During the afterdischarge, protein kinase C (PKC) is activated, although its impact on bag cell neuron electrical transmission is unknown. This was investigated here by monitoring electrical synapses between paired cultured bag cell neurons using dual whole-cell recording. Voltage clamp revealed a largely voltage-independent junctional current, which was enhanced by treating with a PKC activator, PMA, before recording. We also examined the transfer of presynaptic action potential-like waveforms (generated in voltage clamp) to the postsynaptic cell (measured in current clamp). For control pairs, the presynaptic spike-like waveforms mainly evoked electrotonic potentials; however, when PKC was triggered, these stimuli consistently produced postsynaptic action potentials. To assess whether this involved changes to postsynaptic responsiveness, single bag cell neurons were injected with junctional-like current mimicking that evoked by a presynaptic action potential. Unlike control neurons, which were less likely to spike, cells in PMA always fired action potentials to the junctional-like current. Furthermore, PKC activation increased a postsynaptic voltage-gated Ca²⁺ current, which was recruited even by modest depolarization associated with an electrotonic potential. Whereas PKC inhibits gap junctions in most systems, bag cell neurons are rather unique, as the kinase potentiates the electrical synapse; in turn, this synergizes with augmented postsynaptic Ca²⁺ current to promote synchronous firing.SIGNIFICANCE STATEMENT Electrical coupling is a fundamental form of communication. For the bag cell neurons of Aplysia, electrical synapses coordinate a prolonged burst of action potentials known as the afterdischarge. We looked at how protein kinase C, which is upregulated with the afterdischarge, influences information transfer across the synapse. The kinase activation increased junctional current, a remarkable finding given that this enzyme is largely considered inhibitory for gap junctions. There was also an augmentation in the ability of a presynaptic neuron to provoke postsynaptic action potentials. This increased excitability was, in part, due to enhanced postsynaptic voltage-dependent Ca²⁺ current. Thus, protein kinase C improves the fidelity of electrotonic transmission and promotes synchronous firing by modulating both junctional and membrane conductances.

Keywords: action potential; afterdischarge; gap junction; junctional current; neuroendocrine cell; persistent Ca2+ current.

Figure 1.

Bag cell neurons are electrically coupled in culture and show a voltage-independent junctional current. A, Two electrically coupled cultured bag cell neurons under dual whole-cell current clamp using our standard K⁺-Asp-based intracellular saline in the pipettes and nASW in the bath. A 1 nA depolarizing current injection into neuron 1 evokes action potentials (APs) (V1; bottom), resulting in ETPs in neuron 2 (V2; top). Time-base applies to both traces. Top inset, Phase-contrast photomicrograph of paired bag cell neurons in soma–soma configuration after 2 d in culture. Large processes are visible in the upper FOV. The whole-cell pipettes (pip) are slightly of focus in the lower FOV. Bottom inset, Recording configuration with both cells in whole-cell mode. B, Dual whole-cell voltage clamp of coupled bag cell neurons also using K⁺-Asp-based intracellular saline and nASW. Both neurons are held at −60 mV, and neuron 1 is stepped from −90 to 60 mV in 200 ms, 10 mV intervals (V1; top). This evokes voltage-dependent membrane current in neuron 1 (I1; top-middle) and junctional current in neuron 2 (I2; bottom). Time-base applies to all panels. C, Plotting the average end-pulse current from neuron 2 as postsynaptic or junctional current (I-post) against the presynaptic voltage of neuron 1 (V-pre) shows a mainly linear, voltage-independent relationship. That stated, there is some modest reduction of the inward current at presynaptic voltages more positive than 0 mV. N value refers to number of pairs.

Figure 2.

Presynaptic stimulation with action potential-like waveforms shows electrotonic transmission between coupled bag cell neurons. With neuron 1 voltage-clamped and neuron 2 current-clamped in a pair of electrically coupled bag cell neurons in vitro (inset), delivery of action potential-like waveforms from specific phases of the afterdischarge (to neuron 1) evokes electronic responses in the coupled partner (neuron 2). A, A spike-like waveform delivered from −60 mV under voltage clamp, mimicking the input stimulus of the afterdischarge (V1; top), elicits an action current in neuron 1 (I1; middle) and evokes an ETP in neuron 2, which is first set to −60 mV under current clamp with bias current (V2; bottom). The action current initially presents rapid inward current (Ca²⁺ and Na⁺ current), which is quickly obscured by the large outward K⁺ current. B, C, Waveforms corresponding to the action potential during the fast phase (B) or slow phase (C) of the afterdischarge, both from a holding potential of −40 mV, again evoke action currents in neuron 1 as well as produce ETPs (essentially spikelets) in neuron 2, which is also originally set to −40 mV. The slow-phase ETP is slightly broader. A–C, Time-base applies to the top, middle, and bottom.

Figure 3.

Pharmacological block of junctional current and voltage transfer between coupled bag cell neurons. A, In an electrically coupled cultured pair, both bag cell neurons are held at −60 mV (top and bottom middle) under whole-cell voltage clamp. A 200 ms pulse to −90 mV in neuron 1 evokes membrane current in neuron 1 (I1; black trace, top-middle) and junctional current in neuron 2 (I2; black trace, bottom). Bath application of 100 μm NPPB to the same pair of neurons for 10 min markedly inhibits junctional but not membrane current (gray traces, top and bottom). Time-base applies to all traces. B, Summary graph show that both NPPB and NFA (100 μm) significantly reduce the end-pulse junctional current (NPPB, t₍₄₎ = 2.077, *p < 0.04, NFA, t₍₄₎ = 5.317, *p < 0.007; both paired Student's t test). Numbers in white columns are n values and indicate the number of pairs recorded before and after exposure to drug. C–E, Left, While voltage clamping neuron 1 and current-clamping neuron 2, delivering the input-phase (C), fast-phase (D), or slow-phase (E) stimulus (stim; at arrowhead) to neuron 1 elicits an ETP in neuron 2 (black traces). In all cases, the subsequent addition of 100 μm NPPB largely eliminates the ETP (gray traces). D, E, Control traces reproduced from Figure 2. Right, Summary graphs show that both NPPB and NFA significantly lower the peak response of all three types of ETP (NPPB input-phase, t₍₄₎ = 3.277, *p < 0.02; NPPB fast-phase, t₍₄₎ = 2.576, *p < 0.04; NPPB slow-phase, t₍₄₎ = 3.544, *p < 0.02; NFA input-phase, t₍₃₎ = 2.224, *p < 0.03; NFA fast-phase, t₍₄₎ = 2.379, *p < 0.04; NFA slow-phase, t₍₄₎ = 2.385, *p < 0.05; all paired Student's t test). The n values indicate the number of pairs recorded before and after exposure to drug.

Figure 4.

PKC activation increases junctional current between coupled bag cell neurons. A, Following a 20 min treatment with DMSO, both bag cell neurons in a cultured pair are held at −60 mV under voltage clamp (left inset). When a pulse to −90 mV is given to neuron 1 (right inset), it evokes a junctional current (I2) in neuron 2. B, In a different pair, exposed to 100 nm of the PKC activator, PMA for 20 min before recording, the junctional current is more than double that of control. Scale bars apply to both A and B. C, Neurons from another pair, previously exposed for 20 min to 100 nm of the inactive PMA-analog, 4-α-phorbol, are voltage clamped at −60 mV. Stepping neuron 1 from −90 to 0 mV in 200 ms, 10 mV intervals (V1; top), whereas keeping neuron 2 at −60 mV (V2; middle) results in range of small junctional currents (I2; bottom). D, When the same voltage-step protocol as per C is delivered to a bag cell neuron pair that has been treated with PMA for 20 min, the junctional current is larger at all test voltages. Scale bars apply to both C and D. E, Group data show that, compared with electrically coupled cells given the vehicle, DMSO, providing PMA significantly enhances the junctional current elicited by a presynaptic step to −90 mV (U = 22.0, *p < 0.02; Mann–Whitney U test). N values indicate number of pairs recorded following DMSO or PMA treatment. F, A summary plot of neuron 2 end-pulse current (I-post) versus the test potential in neuron 1 (V-pre) reveals that the junctional current produced by each presynaptic step is clearly greater for pairs incubated in PMA (close circles) compared with the inactive analog, 4-α-phorbol (open circles). At all given test potentials, the difference between the junctional currents is significant (−90 mV, t₍₃₅₎ = 3.673, p < 0.001; −80 mV, t₍₃₅₎ = 4.297, p < 0.0002; −70 mV, t₍₃₅₎ = 3.596, p < 0.002; −50 mV, t₍₃₅₎ = 3.609, p < 0.0002; −40 mV, t₍₃₅₎ = 4.383, p < 0.0002; −30 mV, t₍₃₅₎ = 4.392, p < 0.0002; −20 mV, t₍₃₅₎ = 3.834, p < 0.0006; −10 mV, t₍₃₅₎ = 4.174, p < 0.0003; 0 mV, t₍₃₅₎ = 4.258, p < 0.0002; all Student's t test with Welch correction). Consequently, the slope is more than two-fold greater when PKC is activated. N values indicate number of pairs recorded subsequent to 4-α-phorbol or PMA treatment.

Figure 5.

Activating PKC modulates electrical transmission between coupled bag cell neurons. A, After treating pairs of cultured bag cell neurons for 20–30 min with either DMSO or 100 nm PMA, neuron 1 is voltage-clamped at −40 mV and neuron 2 is current-clamped such that the membrane potential is initially set to −40 mV (left inset). Left, For DMSO, a fast-phase stimulus to neuron 1 (stim, at arrowhead; middle inset) typically evokes an ETP in neuron 2 (V2). Right, For a pair where PKC is activated by PMA, the most common response is a postsynaptic action potential in neuron 2 following a fast-phase stimulus to neuron 1. Scale bars apply to both traces. B, Similarly, delivering the slow-phase stimulus (right inset) to neuron 1 elicits an action potential in neuron 2 for pairs treated with PMA (right) but not DMSO (left). Scale bars apply to both traces. C, Group data reveal that PKC activation leads to a greater probability of postsynaptic spiking. Following a single fast- or slow-phase presynaptic stimulus, the frequency of action potential occurrence is significantly greater in pairs treated with PMA compared with DMSO (fast-phase RR = 3.85, *p = 0.03; slow-phase RR = 6.6, *p < 0.03; both Fisher's exact test). D, When neuron 1 receives dual slow-phase stimuli at 1 Hz, the presence of PMA results in neuron 2 firing a spike in response to both stimuli. E, Group data also show how PMA renders it significantly more likely that dual presynaptic slow-phase action potential-like waveforms, given at 1 Hz, produce two spikes in neuron 2 (RR = 6.6, *p < 0.03; Fisher's exact test); however, this is not the case for dual fast-phase waveforms given at 5 Hz (RR = 1.76, p > 0.05; Fisher's exact test). N values indicate number of pairs recorded following DMSO or PMA treatment. F, Left, When a pair is incubated for 20 min in 100 nm 4-α-phorbol, and neuron 1 is given the same fast-phase stimulus as per A, the response in neuron 2 (V2) is always an ETP. Right, However, if neurons are treated with PMA, the majority of cases see an action potential being brought about in neuron 2. Scale bars apply to both traces. G, Group data establish that, compared with 4-α-phorbol, PMA significantly increases the likelihood that a single fast- or slow-phase presynaptic stimulus will elicit a postsynaptic action potential (fast-phase RR = 6.933, *p = 0.0002; slow-phase RR = 2.889, *p = 0.0004; both Fisher's exact test). N values indicate number of pairs recorded following 4-α-phorbol or PMA treatment.

Figure 6.

Broadening of the input stimulus evokes larger electrotonic potentials when PKC is activated. A, Neuron 1 from an electrically coupled bag cell neuron pair is voltage-clamped at −60 mV, whereas the membrane potential of neuron 2 is initially set to −60 mV in current clamp (inset). Ten action potential waveforms resembling the input stimulus are applied to neuron 1 at a frequency of 5 Hz (V1; top). As the half-width of each successive stimulus is broadened by ∼1 ms (compare waveform 1 to 10), it evokes an ETP in neuron 2 (V2). A pair treated for 30 min with DMSO before recording presents an ETP that gradually increases in size during the 10 waveform stimulus train (middle). This is far more evident when a different pair is previously incubated with 100 nm PMA, yielding an ETP that clearly grows over time (bottom). Numbers indicate the ETP following waveforms 1–10. Scale bars apply to the middle and bottom panels; abscissa also applies to the top panel. B, Summary data for DMSO-treated (clear circles) and PMA-treated (black circles) pairs. The ETPs in PMA show a significant upward trend (F_(1,54) = 16.8537 p < 0.04, repeated-measures ANOVA; p = 0.0001, test for linear trend) compared with those in DMSO (F_(1,54) = 6.2154, p > 0.05, repeated-measures ANOVA). Top and bottom error bars for PMA and DMSO datasets, respectively, removed for clarity. N values indicate number of pairs recorded following DMSO or PMA treatment. C, Example of one of two additional pairs exposed to PMA, where the stimulus train eventually leads to action potentials in neuron 2 (V2).

Figure 7.

Following PKC activation, a fast-phase junctional current-like stimulus evokes action potentials more frequently. A, Under dual whole-cell voltage clamp (inset), both bag cell neurons of a cultured electrically coupled pair are held at −40 mV. A fast-phase action potential-like waveform stimulus applied to the presynaptic neuron (V1; top) induces an inward junctional current in the postsynaptic neuron (I2; bottom). Time-base also applies to top panel. B, Summary data show that the junctional current evoked by the fast-phase spike-like waveform has a peak amplitude of ∼−300 pA (left) and an area of ∼10 nA × ms (right). N values indicate number of pairs. C, Single bag cell neurons are current-clamped (inset) with the membrane potential initially set to −40 mV, and a depolarizing (positive) form of the fast-phase (top) junctional current is injected. In neurons given DMSO for 20–30 min before recording, the junction-like current injection induces an ETP (gray trace; middle) 56% of the time and an action potential (black trace; middle) 44% of the time. However, for a separate neuron, after 100 nm PMA, the stimulus always evokes a spike (bottom). Scale bars apply to the middle and bottom panels; abscissa also applies to the top panel. D, Summary data of action potential occurrence show that in PMA there is a significantly greater chance of spiking in response to the fast-phase current injection (RR = ∞, *p < 0.03; Fisher's exact test). N values indicate number of individual neurons recorded following DMSO or PMA. D, E, F, n values indicate number of individual neurons recorded following DMSO or PMA treatment. E, F, The average action potential amplitude (D) or area (E) is significantly greater when PKC is activated by PMA (amplitude, t₍₉₎ = 2.531, *p < 0.04; area, t₍₉₎ = 3.333, *p < 0.009; both unpaired Student's t test).

Figure 8.

Activation of PKC enhances bag cell neuron Ca²⁺ current evoked by a waveform that mimics an electrotonic potential. A, Single bag cell neurons are bathed in Ca²⁺-Cs⁺-TEA ASW and treated with either DMSO or 100 nm PMA for 20–30 min before whole-cell voltage clamp using a Cs⁺-Asp-based intracellular saline (inset). Stepping the neurons from a holding potential (HP) of −60 mV to 40 mV in 200 ms, 10 mV intervals, evokes a voltage-dependent Ca²⁺ current that is modestly inactivating during the test pulse. Compared with a neuron given DMSO (top), the current from a cell exposed to PMA (bottom) is three-fold larger. Scale bar applies to both traces. B, Plotting peak Ca²⁺ current against step voltage reveals a nonlinear, voltage-dependent relationship. The current is inward and peaks between 0 and 10 mV for both DMSO (clear circles) and PMA (black circles). Yet, the peak in PMA is significantly greater than DMSO at potentials more positive than −30 mV (−30 mV, t₍₅₎ = 3.404, *p < 0.02; −20 mV, t₍₅₎ = 3.106, *p < 0.03; −10 mV, t₍₆₎ = 3.568, *p < 0.02; 0 mV, t₍₅₎ = 4.369, *p < 0.008; 10 mV, t₍₅₎ = 3.872, *p < 0.02; 20 mV, t₍₅₎ = 4.282, *p < 0.08; 30 mV, t₍₅₎ = 4.115, *p < 0.01; 40 mV, t₍₅₎ = 3.744, *p < 0.02; all unpaired Student's t test). N values indicate number of individual neurons recorded following DMSO or PMA. C, A fast-phase ETP-like waveform (top) is given to single bag cell neurons voltage-clamped at −40 mV while recording Ca²⁺ current. The waveform elicits an ∼200 pA current in a neuron treated with DMSO beforehand (gray trace; bottom). By comparison, a cell in PMA displays a two-fold larger Ca²⁺ current (black trace; bottom). Abscissa also applies to the top panel. D, The peak amplitude of the ETP-like waveform-induced Ca²⁺ current is significantly greater in PMA versus DMSO (t₍₁₁₎ = 3.353, *p < 0.007; unpaired Student's t test). N values indicate number of individual neurons recorded following DMSO or PMA.