Conopressin Affects Excitability, Firing, and Action Potential Shape Through Stimulation of Transient and Persistent Inward Currents in Mulluscan Neurons

Paul F. van Soest and Karel S. Kits

Membrane Physiology Section, Research Institute of Neurosciences, Vrije Universiteit, 1081 HV Amsterdam, The Netherlands

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

van Soest, Paul F. and Karel S. Kits. Conopressin affects excitability, firing, and action potential shape through stimulation of transient and persistent inward currents in mulluscan neurons. J. Neurophysiol. 79: 1619-1632, 1998. The molluscan vasopressin/oxytocin-related neuropeptide conopressin activates two persistent inward currents in neurons from the anterior lobe of the right cerebral ganglion of Lymnaea stagnalis that are involved in the control of male copulatory behavior. The low-voltage-activated (LVA) current is activated at a wide range of membrane potentials, its amplitude being only weakly voltage dependent. The high-voltage-activated (HVA) current is activated at potentials positive to -40 mV only and shows a steep voltage dependence. Occurrence of both currents varies from cell to cell, some expressing both and others only the HVA current. In most neurons that have the LVA current, a conopressin-independent persistent inward current (INSR) is found that resembles the HVA current in its voltage dependence. The functional importance of the LVA and HVA currents was studied under current-clamp conditions in isolated anterior lobe neurons. In cells exhibiting both current types, the effect of activation of the LVA current alone was investigated as follows: previously recorded LVA current profiles were injected into the neurons, and the effects were compared with responses induced by conopressin. Both treatments resulted in a strong depolarization and firing activity. No differences in firing frequency and burst duration were observed, indicating that activation of the LVA current is sufficient to evoke bursts. In cells exhibiting only the HVA current, the effect of conopressin on the response to a depolarizing stimulus was tested. Conopressin reversibly increased the number of action potentials generated by the stimulus, suggesting that the HVA current enhances excitability and counteracts accommodation. Conopressin enhanced action potential broadening during depolarizing stimuli in many neurons. Voltage-clamp experiments performed under ion-selective conditions revealed the presence of transient sodium and calcium currents. Using the action potential clamp technique, it was shown that both currents contribute to the action potential. The calcium current, which is activated mainly during the repolarizing phase of the action potential, is augmented by conopressin. Thus conopressin may directly modulate the shape of the action potential. In summary, conopressin may act simultaneously on multiple inward currents in anterior lobe neurons of Lymnaea to affect firing activity, excitability, and action potential shape.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Lys-conopressin, a nonapeptide related to vasopressin and oxytocin (Cruz et al. 1987), appears to be a common transmitter in various mollusks. Potent physiological effects of vasopressin and oxytocin on neurons from Aplysia and Otala (Barker and Gainer 1974; Barker et al. 1975), as well as the discovery of a vasopressin/oxytocin-related peptide in the central nervous system (CNS) of these animals (Ifshin et al. 1975), already suggested the existence of an endogenous molluscan vasopressin/oxytocin-related transmitter. Since then, conopressin has been shown to modulate the activity of several types of central neurons of Aplysia (Martínez-Padrón and Lukowiak 1993; Martínez-Padrón et al. 1992a,b) and Lymnaea (van Kesteren et al. 1995a,b; van Soest and Kits 1997).

Conopressin is expressed abundantly in most neurons in the anterior lobe of the right cerebral ganglion of Lymnaea stagnalis (van Kesteren et al. 1995a). Two G protein-coupled Lymnaea conopressin receptors have been identified, at least one of which also is expressed in a large portion of the anterior lobe neurons (van Kesteren et al. 1995b, 1996). Most of the anterior lobe neurons send projections into the penis nerve and are involved in the control of male copulatory behavior (De Boer et al. 1996, 1997). Furthermore, several of the peptides that are produced by the anterior lobe neurons, including conopressin, affect parts of the male copulatory apparatus (De Lange et al. 1997; van Golen et al. 1995; van Kesteren et al. 1995a,b). Thus conopressin plays an important role in the control of male copulatory behavior in Lymnaea. The occurrence of coexpression of conopressin with its receptors suggests that at least part of its function involves auto-transmission and hence regulation of the activity of the anterior lobe neurons themselves. In this respect, the conopressin system resembles the oxytocinergic neurons of the mammalian hypothalamus, where autoexcitation was shown to underlie oxytocin release during reproductive activity (see e.g., Lambert et al. 1993).

Previously, we have shown that conopressin excites neurons in the anterior lobe of Lymnaea (van Kesteren et al. 1995b; van Soest and Kits 1997). In the identified anterior lobe neuron RCB1, it activates two persistent inward currents that mainly are carried by sodium. Both currents differ in voltage dependence, agonist sensitivity, and dynamics of desensitization and washout. The first type, called the high-voltage-activated (HVA) current, is activated at potentials positive to -40 mV by relatively low concentrations of conopressin and exhibits little desensitization. This current resembles the pacemaker currents underlying burst firing in several mollusks (reviewed by Adams and Benson 1985; Benson and Adams 1987). The second type, called the low-voltage-activated (LVA) current, is relatively voltage independent (i.e., it can be activated at all potentials between -90 and +10 mV), requires higher doses of conopressin to activate, and desensitizes within minutes. Finally, some of the cells also have a persistent inward current that is independent of conopressin. This current, which was called INSR because of similarity to the pacemaker currents mentioned above, resembles IHVA in its voltage dependence, but is mainly carried by calcium (van Soest and Kits 1997).

In agreement with the nonuniform distribution of conopressin receptors throughout the anterior lobe (van Kesteren et al. 1995b), the responses to conopressin varied from cell to cell. Some cells had both the LVA and HVA currents, whereas others had only the HVA current or showed no response to conopressin at all (van Soest and Kits 1997). The differential occurrence of both currents among cells indicates that they may serve functionally different purposes, especially because there is a large diversity in firing patterns within the anterior lobe (Khennak and McCrohan 1988). The electrical activity in the anterior lobe as a whole is enhanced strongly during penis eversion, a process in which conopressin may play a central role (De Boer et al. 1997).

These data led us to hypothesize that conopressin serves a broad modulatory role in the anterior lobe, aimed at enhancing excitation and neuropeptide release. The similarity of IHVA to pacemaker currents suggests that it may be involved primarily with modulation of ongoing activity, whereas the LVA current is expected to provide a depolarizing input to cells irrespective of their current state. Furthermore, conopressin may modulate the shape of the action potential, either directly through modulation of currents, or indirectly through a frequency-dependent mechanism. All these effects are expected to augment the release of the various peptide transmitters, including conopressin itself. In the present study, we investigated the functional importance of the conopressin-activated persistent currents in anterior lobe neurons. To this end, the effects of activation of the LVA and the HVA current on firing activity and excitability were tested under current-clamp conditions. In addition, we asked whether conopressin could affect directly the transient currents underlying the action potential in these cells to augment spike broadening.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Animals and preparations

All experiments were performed on acutely isolated neurons from adult, laboratory-bred specimens of L. stagnalis (L.). The animals were kept in aerated, circulating water under a 12:12 h light:dark regime and fed lettuce ad libitum. The CNS was dissected from the animal and incubated in a 0.2% solution of trypsin (Sigma, type III) in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered saline (HBS, see further) at 37°C for 35 min. After the incubation, the CNS was rinsed twice, and pinned down in a dish containing HBS supplemented with 5 mM glucose. The sheet of connective tissue covering the anterior lobe of the right cerebral ganglion was removed using fine hooks, after which the lobe was severed from the ganglion. Single anterior lobes were transferred to 35-mm culture dishes (Corning Costar, Cambridge, MA) containing glucose-supplemented HBS and mechanically dissociated. The cells were allowed to sit for >= 1 h before the dishes were placed in the experimental setup. For the experiments, small to medium-sized cells (40-80 µm) were selected.


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FIG. 1. Action potential clamp under nonselective conditions. A, top: action potential of an anterior lobe neuron under current clamp, evoked by a 5-ms, 1-nA depolarizing stimulus. Bottom: same action potential but inverted. A fixed voltage offset was added to set the starting membrane potential to the same value as in top trace. B, top: total current evoked by voltage clamping the neuron with the recorded action potential from A. Apart from a small residual, no current is required to clamp the cell (see METHODS section for a more complete description). Note that the stimulus is accurately reconstructed. Bottom: current evoked by the inverted action potential. No or very little ionic current will be activated by the hyperpolarizing waveform. C: net current obtained by adding the 2 current traces from B, revealing the total passive current required to charge the cell membrane during an (inverted) action potential.

Recordings

All experiments were performed on isolated neurons, in the whole cell configuration. Large-tip patch pipettes were pulled from borosilicate capillaries (Clark Electromedical Instruments, Reading, UK) and typically had resistances of 1.5-2.5 MOmega , resulting in access resistance values that were only slightly larger. Series resistance normally could be compensated for 70-80%. A standard whole cell recording setup was used, including an Axopatch 1C (Axon Instruments, Foster City, CA) amplifier that had been adapted to allow accurate membrane potential measurements in current-clamp mode (see also Magistretti et al. 1996) Data were digitized using a CED-1401 12-bit AD/DA-converter (Cambridge Electronic Design, Cambridge, UK) and stored on an IBM-compatible PC using software written by T. A. de Vlieger (voltage-clamp software) and P. F. van Soest (voltage ramp and action potential clamp software). Various sampling rates were used, and signals were filtered at or below the Nyquist frequency using a Bessel filter built into the amplifier.


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FIG. 2. Injection of prerecorded low-voltage-activated (LVA) current under current clamp mimics the effect of conopressin application. A1: current activated by a 20-s pressure application of 1 µM conopressin (bar) in an anterior lobe neuron clamped at -50 mV. A2: injection of recorded and inverted current from A1 under current clamp results in a depolarization and a burst of action potentials. In this panel and in A, 3 and 4, the membrane potential was set to -50 mV before the start of the experiment using constant current injection. A3: pressure application of 1 µM conopressin results in a burst that is very similar to those induced by current injection (A, 2 and 4). A4: same as A2, after washout of conopressin. B: pseudo steady-state I-V relation of the neuron in A under control conditions and in the presence of 1 µM conopressin, obtained using a voltage ramp protocol. In these experiments, the command potential was swept from -90 to +10 mV at 5 mV/s. Labels LVA, HVA, and NSR indicate the regions of the I-V relation where the LVA and high-voltage-activated (HVA) currents and the conopressin-independent current INSR are most clearly discernible. C: cumulative histogram of the number of action potentials vs. time in the bursts induced by current injection (1× current) and conopressin application (conopressin), showing nearly identical frequency and duration of both types of burst. D: total spike count in bursts induced in 11 anterior lobe neurons by conopressin application (CP) and current injection at the original amplitude (1×, both before conopressin application and after washout), reduced amplitude (0.5×), and increased amplitude (1.5×). Injection of the reduced current results in a lower number of spikes, whereas the other treatments do not show significant differences.

Voltage recordings also were stored on digital audio tape using a DC-enabled Unitrade (Philadelphia, PA) DAT-recorder, and subsequently digitized and analyzed off-line using the CDR software by Dr. J. Dempster (University of Strathclyde, Glasgow, UK). Hardcopy output was generated using a Brush 2200 (Gould, Cleveland, OH) pen recorder.

Peptides and solutions

Lys-Conopressin G (Cys-Phe-Ile-Arg-Asn-Cys-Pro-Lys-Gly-NH2; Saxon Biochemicals GmbH, Hannover, Germany) was applied using a pressure ejection system from glass pipettes (tip diameter ranging from 5 to 10 µm), which were placed 100-200 µm from the cell body. Stock solutions were stored at -20°C and diluted to a final concentration of 1 µM in HBS.

HBS was composed of (in mM) 30 NaCl, 1.7 KCl, 4 CaCl2, 1.5 MgCl2, 10 NaCH3SO4, 5 NaHCO3, and 10 HEPES, pH set at 7.8 with NaOH. For the isolation procedure, normal HBS was supplemented with 5 mM glucose. Sodium-selective HBS (i.e., used for recording sodium currents) consisted of (in mM) 40 NaCl, 7.5 tetraethylammonium-chloride (TEA-Cl), 2 4-amino pyridine (4AP), 5 MgCl2, and 10 HEPES. Calcium-selective HBS (i.e., used for recording calcium currents) consisted of (in mM) 40 TEA-Cl, 4 CaCl2, 1 MgCl2, 2 4AP, and 10 HEPES. In both cases, the pH was set at 7.8 with TEA-OH.

Standard pipette solution contained (in mM) 2 NaCl, 64 KCl, 2.3 CaCl2, 10 HEPES, 11 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 2 MgATP, and 0.1 GTP-tris(hydroxymethyl)aminomethane (Tris). The pH was set at 7.4 using KOH. Pipette solution used for recording sodium and calcium currents consisted of (in mM) 64 CsCl, 2.3 CaCl2, 10 HEPES, 11 EGTA, 2 MgATP, and 0.1 GTP-Tris. The pH was set at 7.4 using CsOH.


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FIG. 3. Action potential broadening during LVA current-induced bursts does not depend on the presence of conopressin. A: increase in action potential duration during bursts induced by injection of prerecorded LVA current (current injection, open circle ) is similar to that during bursts evoked by conopressin application (conopressin, bullet ). Data from 2 current injection-induced bursts, recorded under control conditions and after washout of conopressin, respectively, are shown. Each symbol represents the duration at half-maximal height of 1 action potential. Inset: cumulative spike count during the bursts in this neuron. B1: sample action potentials (1st and 100th from start of burst) recorded under control conditions. B2: corresponding action potentials recorded during the burst evoked by 1 µM conopressin in the same cell. Although small differences in action potential shape were sometimes observed, conopressin had no consistent effect on spike broadening in 10 cells.


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FIG. 4. Increased excitability during conopressin-induced activation of the HVA current. A1: anterior lobe neurons under current clamp were stimulated with 15-s depolarizing pulses. Amplitude was chosen so as to evoke >= 1 action potential under control conditions. Membrane potential was set to -50 mV before the start of each experiment using a small amount of constant current. A2: response to a 100-pA stimulus under control conditions. Cell shows moderate accommodation. (In this and the other 2 panels, some spikes appear truncated due to sampling artifacts.) A3: response to the same stimulus in the presence of 1 µM conopressin. Accommodation has disappeared, and the cell remains active during the full duration of the stimulus. A4: accommodating response is restored after washout of conopressin. B: pseudo steady-state I-V relation of the cell in A under control conditions and in the presence of 1 µM conopressin, obtained using a voltage ramp protocol. Conopressin-induced HVA current that is activated at potentials positive to -40 mV is clearly visible. C: in 14 anterior lobe neurons, depolarizing stimuli evoked a larger number of action potentials in the presence of 1 µM conopressin (cp) than under control conditions (pre). Effect is fully reversible after washout (wash). Bars represent means ± SD. D: average number of action potentials (mean ± SD) induced by stimuli ranging from 25 to 200 pA tested on a single cell under control conditions (open circle ) and in the presence of 1 µM conopressin (bullet ). Conopressin enhances excitability at all stimulus amplitudes. Numbers above error bars indicate number of cells tested.

Data analysis

In the current-clamp experiments, several parameters, including burst duration, burst frequency, and spike width, were analyzed off-line. Depending on whether data were obtained from tape or had been digitized during the experiment, either the CDR software by Dr. J. Dempster (see earlier text) or custom software written by P. F. van Soest was used. In either case, similar results were obtained. Spike width was measured in the conventional manner, as the duration of the action potential at half-maximal amplitude.

All data are presented as means ± SD. Statistical significance is indicated by P values, which were obtained from paired Student's t-tests, unless stated otherwise.

Action potential clamp

Action potential clamp was performed using methods described previously by Barra (1995, 1996) and Gola et al. (1986). Briefly, under current-clamp conditions, an action potential was evoked by a brief depolarizing stimulus. The recorded action potential was subsequently used as the stimulus waveform in a voltage clamp experiment (Fig. 1A, top). The holding potential was set to the original resting membrane potential, and the action potential was played back.

We tested the applicability of the action potential clamp technique to our cells by performing initial experiments under nonselective conditions. Under ideal circumstances, the replayed action potential should activate exactly those currents that charged the cell membrane when the action potential was generated, so no net current flow should be required to clamp the cell. This assumes that cellular properties did not change in the meantime. In practice, small residual currents are usually observed (Fig. 1B, top). To obtain an estimate of the capacitive current, the recorded action potential was inverted and played back (Fig. 1A, bottom). Because of its hyperpolarizing character, the inverted action potential is not expected to activate ion channels so the current required to charge the membrane capacitance has to be supplied by the amplifier (Fig. 1B, bottom). Summation of both traces yields the net passive current induced by the inverted action potential. As the current flows through the pipette instead of across the membrane, the orientation of the current induced by the inverted action potential follows the convention of plotting inward current as negative. Therefore the passive current thus obtained approximates the total net current flow through ion channels during a normal action potential (Fig. 1C) (see also Barra 1996).

To gauge the contribution of sodium and calcium currents to the action potential, prerecorded action potentials (recorded under nonselective conditions, see earlier text) were used as command input in voltage-clamp experiments employing sodium- or calcium-selective saline. Here, a slightly different approach was used to separate the capacitive current from the total current. First, the action potential was played back five times at a reduced amplitude (20% in the case of calcium currents) to avoid activation of sodium and calcium channels, and the resulting current traces were summed to obtain the full-size capacitive current. Second, the action potential was played back at its original amplitude, activating the appropriate ion channels. The capacitive current then was subtracted from the total current to yield the calcium current that was activated by the action potential. Sodium currents were measured in a similar manner, but the action potential was reduced to 10% to avoid contamination of the capacitive current trace by the low voltage-activated transient sodium current. To compensate for offset differences, all traces were zeroed (by subtracting the average of the first 10 samples) before subtraction.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Previously, two distinct conopressin-activated persistent inward currents have been described. To assess the functional importance of each current for the firing pattern of the anterior lobe neurons, we sought ways to discriminate between their effects. As selective blockers for either current are lacking, we used their differential occurrence among unidentified neurons. Most of the anterior lobe neurons have the HVA current, which is activated by conopressin at potentials positive to -40 mV, whereas some anterior lobe neurons also have the LVA current, which is activated by conopressin at all potentials between -90 and +10 mV. Finally, some neurons do not exhibit any conopressin-induced current.

We characterized the conopressin response of individual anterior lobe neurons by testing the effect of the peptide on their pseudo steady-state I-V relation, obtained with the use of voltage ramp protocols (see van Soest and Kits 1997). On the basis of the I-V relation, we selected cells expressing both the LVA and HVA current or only the HVA current. The isolated HVA current manifested itself as an downward deflection of the steady-state I-V relation at potentials positive to -40 mV (Fig. 3B, "conopressin" trace). The combination of the LVA and HVA current caused an inward shift of the entire I-V relation between -90 and +10 mV (Fig. 2B, conopressin trace). Furthermore, the voltage-dependent but conopressin-independent current, INSR, was visible at potentials positive to about -35 mV under control conditions (Fig. 2B, control trace). After establishing which currents were present, the recording was switched to current-clamp mode to test the effects of the conopressin-induced currents on firing rate and excitability. In most cases, a small amount of constant current was required to set the membrane potential to -50 mV.


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FIG. 5. Action potential broadening during step depolarizations is enhanced in the presence of conopressin. A: increase in action potential duration during 15-s step depolarizations (see Fig. 3) is enhanced in the presence of 1 µM conopressin as compared with the control situation. Effect is reversible after washout of the peptide. Each symbol represents the duration at half-maximal height of 1 action potential. Similar effects were observed in 8 of 13 cells tested. B1: sample action potentials (the 1st, 6th, 11th, and 16th from the burst) under control conditions. B2: corresponding action potentials recorded in the presence of 1 µM conopressin.

Excitation induced by the LVA current

The role of the LVA current was investigated in neurons expressing both the LVA and the HVA current because cells expressing the LVA current in isolation have not been found. Most of these neurons also have the conopressin-independent persistent current, INSR (Fig. 2B). The response to activation of the LVA current was studied by injecting prerecorded LVA current into the cell (Fig. 2A1). To obtain the isolated LVA current, the response to a 20-s application of 1 µM conopressin was recorded under voltage-clamp conditions at -50 mV, at which potential the HVA current is not activated and does not contribute to the response (van Soest and Kits 1997). After recovery, the recorded LVA current was inverted and played back to the same cell under current-clamp conditions, causing a depolarization and vigorous spiking activity (Fig. 2A2 and A4). As the LVA current is only weakly voltage dependent, its amplitude during a burst is not expected to deviate much from the amplitude at -50 mV.

Interestingly, the bursts that were induced with this technique were very similar to the bursts that were evoked by direct application of 1 µM conopressin (Fig. 2A3), in which case both the LVA and HVA currents will be active. Figure 2C shows the cumulative spike count during current injection-evoked bursts in the absence of conopressin (i.e., before application and after washout) and during a conopressin-induced burst. No significant differences in spike count were observed (n = 11; Fig. 2D). Total burst duration, measured as the time between the first and the last action potential, did not change significantly either. On average, LVA current-induced bursts lasted 46.7 ± 15.4 (SD) s before conopressin application and 42.8 ± 15.5 s after washout, whereas the conopressin-induced bursts lasted 55.1 ± 21.3 s (n = 11, paired t-test: P > 0.05).

For comparison, the recorded LVA current also was injected at 50% and at 150% of its original amplitude. The number of action potentials induced by the 50% current injection was significantly smaller than that induced by the other treatments (n = 5; P < 0.05). The injection of current at 150%, on the other hand, did not result in a further increase as compared with the 100% current injection and conopressin application (Fig. 1D).

These data suggest that activation of the LVA current is sufficient to account for the responses to conopressin observed under current clamp in these cells. Apparently, activation of the HVA current is not required for a complete response (in terms of firing rate and duration) under the present experimental circumstances.

Action potential broadening during bursts due to the LVA current

Because the bursting behavior of anterior lobe neurons is expected to mediate peptide release, we analyzed whether the LVA current affected the shape of the action potential in these cells. To this end, we compared the duration of action potentials in bursts induced by current injection and conopressin application. In all cells tested, spike duration increased strongly during bursts (Fig. 3A). Under control conditions (i.e., in the bursts induced by current injection), the duration increased from 11.7 ± 1.5 ms for the 1st action potential to 47.3 ± 26.4 ms for the 50th. In the conopressin-induced bursts, spike duration increased from 12.2 ± 2.2 ms to 46.8 ± 21.2 ms (data not shown). Thus the spike widths measured in the absence and presence of conopressin were similar (Fig. 3; n = 10, P = 0.94). These observations imply that strong frequency-dependent action potential broadening occurs during bursts in these cells that could either be induced by current injection or by conopressin application. Furthermore, we can presume that conopressin-induced effects other than the LVA current, such as the HVA current, do not contribute appreciably to this process.


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FIG. 6. Conopressin enhances the fast sodium current of anterior lobe neurons. A: voltage-clamp recording of an anterior lobe neuron in sodium-selective saline. Step depolarizations to -20 mV and higher, from a holding potential of -80 mV, evoke a fast transient sodium current that fully inactivates within 10 to 20 ms. Inset: voltage protocol used to clamp the cells. From a holding potential of -80 mV, the command potential was stepped to test potentials ranging from -60 to +40 mV in 10-mV increments. (For clarity, only a subset of the traces is shown in main figure and inset.) B: averaged I-V relation for the sodium current in 4 cells. Data represent means ± SD. C: effect of conopressin on the sodium current was tested by applying 40-ms depolarizing pulses to +10 mV (or -10 mV if the current at +10 mV exceeded 10 nA) at 30-s intervals. Amplitude of the sodium current is increased in the presence of 1 µM conopressin. D: plot of the peak amplitude (mean ± SD, n = 12) of the sodium current vs. time, showing the increase induced by a 90-s conopressin application (bar). Effect desensitizes already before washout of the peptide.

HVA current enhances excitability

The role of the HVA current was investigated in cells having a prominent HVA current and lacking the LVA current (see Fig. 4B). Most of these cells did not have the conopressin-independent INSR either. As is to be expected, conopressin application does not result in a response in these cells when the resting membrane potential is negative to approximately -40 mV. Accordingly, the effects of peptide application were tested on the response to 15-s depolarizations induced by current injections of 25-200 pA. The amplitude was chosen so as to evoke at least one action potential under control conditions and then was kept constant during the remainder of the experiment. Before each stimulus, the membrane potential was set to -50 mV by constant current injection.

Figure 4 shows a typical example of a neuron that only exhibited the HVA response to conopressin in its steady-state I-V relation. Under current clamp, application of 1 µM conopressin resulted in a clear increase in the number of spikes observed in response to the depolarizing stimulus (Fig. 4A). In 14 cells, the 15-s stimulus evoked 25.2 ± 23.9 action potentials under control conditions, 62.9 ± 30.0 in the presence of conopressin, and 31.8 ± 19.7 after washout of the peptide (P = 0.009; Fig. 4C). To test whether the effect of conopressin on spike count was dependent on the amount of current injected, stimuli ranging from 25 to 200 pA were applied subsequently to the same cell. In five cells, the spike count was higher in the presence of conopressin than under control conditions, at all stimulus amplitudes(P = 0.013, analysis of variance; Fig. 4D). This indicates that the excitatory effect of activation of the HVA current is relatively independent of stimulus amplitude.

Conopressin enhances spike broadening

Next we analyzed the effect of conopressin on the increase in action potential width during the 15-s stimulus in these cells lacking the LVA current. Figure 5A shows a typical example of the increase in spike width versus time under control conditions, in the presence of 1 µM conopressin, and after washout. Some spike broadening occurred during the stimulation under control conditions, but this process was enhanced greatly in the presence of conopressin. The lower panels show the 1st, 6th, 11th, and 16th spike of the control recording (Fig. 5B1) and in the presence of conopressin (Fig. 5B2). In 8 of 13 cells, a substantially larger increase in action potential width was observed in the presence of conopressin as compared with the control situation. None of these cells had the conopressin-independent sustained current INSR. All but one of the remaining five cells, which showed little or no conopressin-induced enhancement of spike broadening, did have a prominent INSR. In these cells, strong spike broadening occurred already under control conditions, and application of conopressin had no additional effect. In this respect, the responses resembled the bursts evoked by injection of LVA current (see earlier text). Taken together, these observations suggest that the persistent HVA inward currents, both the endogenous INSR and the conopressin-induced HVA current, may facilitate spike broadening. Part of the effect might be due to an increase in firing rate and the resulting enhancement of frequency-dependent spike broadening.


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FIG. 7. Conopressin enhances the HVA calcium current of anterior lobe neurons. A: voltage-clamp recording of an anterior lobe neuron in calcium-selective saline. Step depolarizations from a holding potential of -80 mV to test potentials positive to -10 mV evoke a slowly inactivating calcium current. Inset: voltage protocol used to clamp the cells. From a holding potential of -80 mV, the command potential was stepped to test potentials ranging from -50 to +50 mV in 10-mV increments. (For clarity, only a subset of the traces is shown in main figure and inset.) B: averaged I-V relation of the peak (bullet ) and late (open circle ) calcium current in 14 cells (data represent means ± SD). Amplitude of late current was measured at the end of the depolarizing step. C: amplitude of the calcium current is enhanced by application of 1 µM conopressin. Figure shows the last control trace and the 1st trace in the presence of conopressin, 30 s after the start of the application. D: plot of the peak amplitude of the calcium current (mean ± SD, n = 12) vs. time, showing the increase induced by a 90-s conopressin application (bar). Effect builds up gradually, and declines slowly after washout of the peptide.

Effects of conopressin on fast inward currents

The observation that conopressin could affect the shape of the action potential led us to investigate its effect on the fast inward currents that are expected to underlie the action potential in these cells. To this end, the currents were isolated pharmacologically, allowing the contribution of individual currents to the action potential to be established. As reported before, isolation of potassium currents was not feasible because the anterior lobe neurons deteriorated rapidly in the appropriate media (van Soest and Kits 1997).

In sodium-selective saline (i.e., in the absence of extracellular Ca2+ and in the presence of Ca2+- and K+-channel blockers), a fast transient sodium current was observed on depolarizing pulses (Fig. 6A). This current activated around -30 to -20 mV and reached its maximal amplitude around 0 to +10 mV (Fig. 6B). Figure 6C shows the effect of 1 µM conopressin on the sodium current activated by a40-ms test pulse to +10 mV (or -10 mV if the current would have otherwise exceeded 10 nA). Test pulses were applied at 30-s intervals. Immediately after the fifth pulse, conopressin was applied for 1.5 min by means of pressure ejection. In 12 cells, the peak current amplitude increased from 4.5 ± 2.7 nA for the last control pulse to 5.4 ± 3.0 nA for the first pulse in the presence of conopressin (Figs. 6C and 5D; paired t-test: P = 0.001). As can be seen in Fig. 6D, the effect desensitized rapidly and was reversed nearly completely at the end of the 90-s application.

Calcium-selective saline (i.e., in the absence of Na+ and K+ and in the presence of K+-channel blockers) was used to record the isolated calcium current (Fig. 7A). It activated at a slightly higher potential than the sodium current, i.e., around -10 mV, and peaked around +30 mV. The current activated rapidly, but inactivated only partially during 300-ms test pulses. The peak current and the current measured at the end of the depolarizing step (late current) showed an identical voltage dependence (scaling of the late current to the peak current resulted in exactly overlapping I-V curves; not shown). This suggests that the calcium current in anterior lobe neurons is homogeneous (Fig. 7B). Application of 1 µM conopressin augmented the calcium current activated by a test pulse from -80 to +10 mV (Fig. 7C). In 12 cells, the average peak current amplitude increased from 4.2 ± 1.1 to 5.5 ± 2.4 nA within 90 s in the presence of conopressin (mean ± SD, paired t-test: P = 0.029, Fig. 7D). In eight other cells, the effect of a shorter 30-s application of conopressin was tested. Here, the peak current amplitude increased similarly from 3.6 ± 2.0 to 4.6 ± 2.7 nA (P = 0.011; not shown). In both cases, the effect was largely reversible within a few minutes of washing (see Fig. 7D).

Action potential clamp

Knowing that transient sodium and calcium currents are modulated by conopressin in most anterior lobe neurons, we assessed the contribution of these currents to the action potential to determine whether conopressin is capable of affecting the action potential directly. To this end, voltage-clamp experiments were performed using prerecorded action potentials as the stimulus waveform. Because of the use of sodium- and calcium-selective salines in these experiments, it was not possible to determine whether the cells had the LVA and/or HVA current. Accordingly, the experiments were performed on randomly selected cells.

To gauge the activation of the individual inward currents during an action potential, the experiments were performed under sodium- and calcium-selective conditions. Because it was not possible to record action potentials from the cell under study, a typical action potential recorded under standard conditions was used as the stimulus waveform (Fig. 8A). To separate the active current from the passive currents, the action potential was first played back five times at 20% of its original amplitude, inducing only passive current. Subsequently, the action potential was played back at its full amplitude, activating the ionic current under study in addition to the passive current. In the case of sodium currents, which are activated at lower membrane potentials, the amplitude was reduced to 10%.


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FIG. 8. Transient sodium current is activated during the upshoot of the action potential. A: action potential recorded from an anterior lobe neuron, which was used to stimulate neurons under voltage clamp. Inset: full trace, including the stimulus artifact. B: currents induced by the action potential under conditions selective for recording sodium currents. Passive current was obtained by rescaling current induced by the action potential at 10% of its original amplitude. Total current was induced by the action potential at full amplitude. C: isolated sodium current activated by the action potential obtained by subtracting the passive current from the total current in B. Sodium current is active mainly during the depolarizing phase of the action potential and rapidly approaches zero at the peak voltage.

Figure 8B shows the rescaled passive current activated by the reduced action potential and the current activated by the full action potential. Subtraction yields the isolated sodium current (Fig. 8C), which is activated predominantly during the upshoot of the action potential (n = 16). To test the effect of conopressin, action potential clamp experiments were carried out under control conditions and in the presence of the peptide, and the resulting sodium currents were compared. However, the effects of conopressin on the amplitude of the sodium current were small and variable. Only 4 of 10 cells tested displayed a robust increase in current amplitude. The sodium current of the remaining cells did not appear to respond to conopressin. On average, the amplitude of the sodium current did not change (n = 10, P = 0.299). In a similar manner, cells were clamped using an action potential under calcium-selective conditions (Fig. 9, A and B). The calcium current, obtained by subtraction (Fig. 9C), is activated predominantly during the repolarizing phase of the action potential (n = 11). In contrast to the sodium current, the calcium current was consistently augmented by conopressin, increasing from 5.2 ± 1.0 to 6.3 ± 1.2 nA (n = 4, P = 0.033).


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FIG. 9. HVA calcium current is activated during the repolarising phase of the action potential, and is enhanced by conopressin. A: action potential recorded from an anterior lobe neuron, which subsequently was used to stimulate neurons under voltage clamp. Inset: full trace, including the stimulus artifact. B: rescaled currents induced by the action potential, when played back at a reduced amplitude (passive current) and at its normal amplitude (total current), both under control conditions (control) and in the presence of 1 µM conopressin (conopressin). As expected, conopressin enhances the total current but not the passive current. C: isolated calcium current activated by the action potential, obtained by subtracting the passive current from the total current in B. Calcium current is active mainly during the repolarizing phase of the action potential, and its amplitude is increased by conopressin.

It should be noted that this technique has some limitations, particularly the fact that recursive and mutual effects of increases in sodium and/or calcium current and changes in action potential shape are not taken into account. Even so, these observations indicate that at least the high voltage-activated calcium current is enhanced by conopressin and, because it is activated during the repolarizing phase, might underlie conopressin-induced spike broadening.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

In the present study, we provide experimental evidence that the neuropeptide conopressin acts on multiple inward currents to enhance the firing activity, excitability and action potential duration of Lymnaea anterior lobe neurons. The persistent inward LVA current, which can be activated at a wide range of potentials, depolarizes the neurons toward the spike threshold and induces a vigorous burst. The persistent inward HVA current, which is activated at potentials positive to -40 mV only, does not affect neurons at their resting membrane potential but increases the number of action potentials generated on stimulation. Cells having the conopressin-independent persistent current INSR are highly excitable even in the absence of conopressin, suggesting thatINSR and IHVA serve a similar purpose. However, IHVA differs in that its activation will be dependent on input. In addition to activating persistent currents, conopressin augments the transient calcium current underlying the action potential in anterior lobe neurons. The latter mechanism may explain the enhancement of action potential broadening observed in the presence of conopressin. All of these effects are likely to enhance the release activity of these peptidergic neurons.

Functional aspects of LVA and HVA current

The present experiments indicated that activation of the LVA current causes a strong depolarization in isolated anterior lobe neurons. Because neurons manifesting only the LVA current in response to conopressin were not found, we injected prerecorded LVA current under current-clamp conditions to simulate its activation. Obviously, the recorded current does not vary with membrane potential, as would the "real" LVA current. However, because the amplitude of ILVA is only weakly voltage dependent (van Soest and Kits 1997), the injected current will approximate ILVA quite closely.

In most cells, injection of prerecorded LVA current resulted in a burst that was virtually indistinguishable from the bursts induced by direct conopressin application. This indicates that, under the present experimental conditions, conopressin-induced activation of the HVA current is not required for generation of the burst. It may be that the conopressin-independent INSR, which is present in most of these cells, already provides ample excitatory drive. After all, the voltage ranges of activation of IHVA and INSR largely overlap, suggesting that both currents serve a similar purpose (see also next section). However, it may be that the HVA current does contribute significantly to bursts in intact neurons in situ that probably have a lower input resistance and are less excitable.

These observations implied that the observed "discharges" represent a standard response to any adequate depolarizing stimulus. The observation that injection of prerecorded LVA current at 1.5 times its original amplitude resulted in nearly identical bursts supports this idea. Apparently, the responses to both conopressin application and current injection have reached a maximum, in terms of firing rate, and potential effects of activation of the HVA current on top of INSR may be masked by limits in the cell's ability to fire. It should be noted that the present study addressed the effects of near-saturating responses to conopressin and that lower doses might induce weaker responses, thus leaving room for additional effects of the HVA current.

Activation of the persistent HVA current coincides with a strong increase in excitability of anterior lobe neurons. In cells expressing only the HVA current and not the LVA current, the number of action potentials generated by a15-s fixed-amplitude stimulus was increased strongly and reversibly in the presence of conopressin. Furthermore, it was shown that this effect was relatively independent of the level of depolarization, indicating that the conopressin-induced HVA current may augment any excitatory input that is sufficiently strong to induce spiking. We presume that the HVA current acts during the interspike interval to increase the rate of depolarization toward spike threshold, thereby increasing the firing rate and counteracting accommodation. During the action potential itself, the effects of conopressin-induced persistent currents probably will be small compared with those of the transient sodium and calcium currents. However, effects of conopressin on the fast inward currents underlying the action potential (see further text) also may contribute to the increased excitability.

Modulation of excitability

Neuronal excitability appears to depend strongly on the balance of inward and outward currents that are active around the cell's resting membrane potential (see e.g., Benson and Adams 1987). One way of enhancing excitability would be to reduce a background potassium current, such as the serotonin-sensitive S current in Aplysia sensory neurons (see e.g., Goldsmith and Abrams 1992; Klein et al. 1986; Mercer et al. 1991). On the other hand, enhancement of persistent inward currents is expected to be equally effective in enhancing neuronal excitability. For example, a persistent inward current somewhat similar to IHVA was shown to underlie the afterdischarge of Aplysia bag cells, a phenomenon that is fully dependent on long-term changes in excitability (Wilson et al. 1996). Endogenous bursting properties of molluscan neurons have been attributed to the presence of a region of NSR in the steady-state I-V relation (Smith et al. 1975; Wilson and Wachtel 1974), and, in line with this notion, inhibition of a sustained inward current underlying the NSR abolished bursting pacemaker properties (Wilson and Wachtel 1978). Since then several reports of agonist-induced bursting, accompanied by appearance of a region of NSR, have been published (see e.g., Funase et al. 1993; Levitan and Levitan 1988; see also next section).

However, it has become clear that the presence of sustained inward current is in itself not sufficient to evoke bursting pacemaker activity. Although the nature of the currents involved appears to differ from preparation to preparation, most reports agree that at least two currents are required to mediate the depolarization-hyperpolarization cycle. Alternating activation of sodium and potassium currents was implicated in bursting behavior of certain Aplysia and Otala neurons (Barker and Gainer 1975). In contrast, a sodium- and calcium-dependent inward current, and an apparent outward current caused by closure of calcium channels, cooperated to generate bursting in Aplysia neuron R15 (Adams 1985; Adams and Benson 1985; Adams and Levitan 1985). Similarly, calcium-dependent activation of a mixed cationic inward current, and calcium-dependent inactivation of a sustained calcium current, were shown to underlie bursting in identified Aplysia L2-L6 neurons (Kramer and Zucker 1985a,b). Accordingly, it seems reasonable to assume that the conopressin-induced HVA current and INSR in Lymnaea anterior lobe neurons serve enhancement of excitability in a general sense, not necessarily leading to classical bursting pacemaker activity.

Similar effects of vasopressin and oxytocin in other mollusks

The potent effects of conopressin on Lymnaea anterior lobe neurons raise the question whether conopressin is a common transmitter in mollusks. In Aplysia, conopressin modulates several types of central neurons involved in the control of the gill withdrawal reflex and respiratory pumping (Martínez-Padrón and Lukowiak 1993; Martínez-Padrón et al. 1992a,b). In addition, it has been known for a long time now that vasopressin/oxytocin-related peptides are capable of modulating the activity of molluscan neurons. Both vasopressin and oxytocin induced bursting pacemaker activity in an identified Otala lactea neuron, accompanied by a reduction of the slope resistance of the I-V relation (Barker and Gainer 1974; Barker et al. 1975). A peptide fraction related to vasopressin and oxytocin, and also capable of inducing bursting behavior, could be extracted from the Otala and Aplysia CNS (Ifshin et al. 1975). Similarly, vasopressin and oxytocin excited and induced bursting behavior in several identified neurons of Achatina fulica (Ku et al. 1986). Studies into the conductance changes underlying these effects have yielded some apparently contradictory results. Oxytocin was reported to modulate chloride currents in Helix neurons (Osipenko and Bogomoletz 1989). In an identified Achatina neuron, however, oxytocin activated a persistent sodium current, causing a region of negative slope resistance to appear in the steady-state I-V relation (Funase 1990). The effects of vasopressin and oxytocin on Lymnaea anterior lobe neurons resemble those of conopressin, also suggesting the involvement of a persistent sodium current. These observations are compatible with the idea that vasopressin and oxytocin exert their effects on molluscan neurons by binding to conopressin receptors, although their affinity appears to be substantially lower (van Soest and Kits 1997). Thus conopressin (and possibly other vasopressin/oxytocin-related neuropeptides) appears to exert consistently excitatory effects in mollusks, most likely through activation of one or more persistent inward currents.

Effects of conopressin on transient inward currents and the action potential

Many anterior lobe neurons displayed enhanced action potential broadening in the presence of conopressin. This observation suggested that conopressin modulated the transient currents underlying the action potential in these cells. In the present study we focused on inward currents, i.e., the sodium and calcium current.

Conventional voltage-clamp recordings made using sodium-selective saline revealed a rapidly inactivating inward current in all anterior lobe neurons tested. Subsequent action potential-clamp experiments demonstrated that this current is activated during the action potential. Nearly all of the sodium influx occurs during the depolarizing phase, suggesting that the transient sodium current underlies the initial depolarization. Conopressin slightly enhanced (~20%) the amplitude of the transient sodium current activated by a step depolarization but did not have a substantial or consistent effect on the sodium current activated by an action potential. At present, it is unclear whether this should be attributed to normal experimental variation or differences between the two techniques. It may be that the amplitude of the sodium current activated during the action potential is limited by the decreasing driving force, as the current rapidly approaches zero at the peak of the action potential (see Fig. 7C). In either case, considering both magnitude and variability of the effect, it does not seem likely that the subtle modulation of the sodium current has a prominent function.

All cells tested expressed a rapidly activating, and slowly inactivating, calcium current. This current strongly resembles the slowly inactivating, HVA calcium current described in other peptidergic Lymnaea neurons (Dreijer and Kits 1995). The action potential-clamp experiments demonstrate that the calcium current is activated during the action potential and that it reaches its maximal amplitude during the repolarizing phase. Probably the balance between calcium and potassium currents will determine the rate of repolarization and therefore the shape (especially the duration) of the action potential. The calcium current in anterior lobe neurons is augmented by conopressin, both when it is evoked by step depolarizations and under action potential clamp. Taken together, these results suggest that conopressin can have profound effects on action potential shape in these neurons and thus may produce, or at least facilitate, action potential broadening. Conopressin may augment this process both directly, through its effect on the calcium current, and indirectly, through the increased firing rate (enhancing frequency-dependent broadening, see further text). Action potential broadening is, in turn, expected to have a substantial effect on the amount of transmitter released, as was shown for other molluscan neurons (see e.g., Coates and Bulloch 1985; Hochner et al. 1986; Sugita et al. 1992). It, however, should be noted that the effect of conopressin on the transient currents may have been underestimated, as the normal, recursive interaction between current and action potential shape was abolished by the experimental procedure (i.e., the use of fixed action potentials recorded under control conditions).

The method of voltage clamping cells with recorded action potentials has been applied to molluscan neurons before. In Helix neurons, inward sodium and calcium currents and subsequent outward voltage- and calcium-dependent potassium currents were shown to be important for de- and repolarization, respectively (Crest and Gola 1993; Gola et al. 1986). More recently, a slightly modified technique was used to study the currents underlying the action potential in other Helix neurons. Blocking experiments revealed a fast sodium current that was active mainly during the depolarizing phase and a somewhat slower calcium current during the repolarizing phase (Barra 1995, 1996). The results from these studies were similar to ours, implicating a similar involvement of sodium and calcium currents even though the methods used to isolate the currents differed considerably.

Although the present study did not address potassium currents, their modulation also may mediate action potential broadening. Serotonin-induced action potential broadening in Aplysia sensory neurons has been attributed mainly to depression of potassium currents, although a contribution of increased calcium currents could not be ruled out (Baxter and Byrne 1990; Goldsmith and Abrams 1992; Hochner and Kandel 1992; Sugita et al. 1992, 1994). Likewise, dynamic-clamp experiments revealed that several potassium and calcium currents change during frequency-dependent spike broadening in R20 neurons of Aplysia. In the latter case, however, cumulative voltage-dependent inactivation of the transient outward A current (IA) appears to be the major factor underlying broadening (Ma and Koester 1995, 1996). Thus inactivation of potassium currents also may be involved in frequency-dependent action potential broadening in the anterior lobe neurons of Lymnaea. Here, conopressin could enhance broadening indirectly, through its effects on the firing behavior. Finally, possible effects of conopressin on potassium currents might add to this.

Functional role of conopressin

Although several aspects of conopressinergic signaling remain to be clarified, it is tempting to consider what will happen once conopressin is released onto the anterior lobe neurons. The conopressin-induced depolarization, in addition to the increased excitability, most likely will result in a widespread increase in activity. Conopressin-induced action potential broadening in turn may facilitate further release of transmitters, including conopressin, by the anterior lobe neurons. Thus a cascade of positive feedback might generate a "discharge"-like period of activity, similar to the oxytocin-induced activity occurring in oxytocinergic neurons in the hypothalamus of mammals (see e.g., Lambert et al. 1993). Interestingly, discharge-like phenomena coinciding with the occurrence of male copulatory behavior have been observed during in vivo recordings of anterior lobe neurons of Lymnaea (De Boer et al. 1997), providing indirect evidence for the occurrence of such transient changes in excitability. Thus the present results shed new light on the mechanisms through which conopressin is involved in the central control of copulatory behavior in Lymnaea.

In summary, conopressin exerts several effects on anterior lobe neurons, all of which seem to act in concert to enhance the activity of these cells. First, the LVA current provides a powerful depolarizing drive that drives the membrane potential toward spike threshold. Second, the persistent HVA current greatly enhances the cells excitability, causing an increase in the number of action potentials induced by any given input. Third, the high voltage-activated calcium current underlying the action potential is enlarged, thus probably affecting the shape (especially the duration) of the action potential. These observations show that a single peptide transmitter may act on several physiological parameters simultaneously to enhance the electrical activity of peptidergic neurons.

    ACKNOWLEDGEMENTS

  The authors thank Prof. T. A. de Vlieger, Dr. A. B. Brussaard, H. D. Mansvelder, and H. van Tol-Steye for comments on the manuscript.

    FOOTNOTES

  Address for reprint requests: K. S. Kits, Membrane Physiology Section, Research Institute of Neurosciences, Vrije Universiteit, de Boelelaan 1087, 1081 HV Amsterdam.

  Received 10 October 1997; accepted in final form 16 December 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society