Bath application of the biogenic amine dopamine (DA) can change the pyloric motor pattern (Fig. 1) (Anderson and Barker 1981
; Eisen and Marder 1984
; Flamm and Harris-Warrick 1986a
) by affecting both the synaptic strength (Ayali et al. 1998
; Johnson and Harris-Warrick 1990
; Johnson et al. 1993a
,b
, 1995
) and the intrinsic properties of pyloric network neurons (Flamm and Harris-Warrick 1986b
; Harris-Warrick and Flamm 1986
; Harris-Warrick et al. 1995a
, b). The lateral pyloric neuron (LP) and many of the pyloric constrictor neurons (PY) are excited and phase advanced within the pyloric cycle by DA (Harris-Warrick et al. 1995a
,b
). In both cell types this is, at least in part, caused by a dopaminergic increase of the intrinsic rate of recovery after inhibition. DA decreases a transient K+ current (IA) in both LP and PY neurons, and in LP it additionally enhances a hyperpolarization-activated inward current (Ih).

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| FIG. 1.
A and B: effect of dopamine (DA) on the pyloric rhythm (modified from Harris-Warrick et al. 1995b ). A: simultaneous intracellular recordings of 4 cell types are shown under control conditions and during DA (10 4 M) application. B: phase diagrams for activity of pyloric neurons under control conditions and in the presence of DA (10 4 M). C: effect of DA on the resting potential of a pyloric dilator (PD) neuron that was isolated from pyloric chemical synaptic input with PTX (5 × 10 6 M). A 1-s DA pulse (10 3 M) pressure ejected across the stomatogastric ganglion (STG) hyperpolarized the cell by >5 mV and suppressed the firing of action potenials for ~1 min. AB, anterior burster neuron; PD, pyloric dilator neuron; LP, lateral pyloric neuron; PY, pyloric constrictor neuron. Vertical markers: 10 mV.
|
|
The two pyloric dilators (PDs) are members [along with the anterior burster (AB) neuron] of the pacemaker group that drives the pyloric rhythm. DA hyperpolarizes and reduces the number of action potentials per cycle in the PD neurons, leading to a phase delay of PD relative to the AB neuron and a reduction in the overall pyloric cycle frequency (Flamm and Harris-Warrick 1986a
). We investigated the ionic mechanisms underlying these changes by analyzing DA's effects on three outward currents: a transient voltage-activated K+ current (IA), a sustained voltage-activated K+ current (IK(V)), and a voltage- and calcium-dependent K+ current (IO(Ca)). Some of these results were presented in abstract form (Kloppenburg et al. 1997
; Levini et al. 1996
).
 |
METHODS |
Materials
California spiny lobsters, Panulirus interruptus, were obtained from Don Tomlinson and maintained
4 wk in artificial seawater at 16°C until use. All chemicals were obtained from Sigma Chemical (St. Louis, MO).
Cell identification
Animals were anesthetized by cooling in ice for
30 min before dissection. The stomatogastric ganglion (STG), along with the motor nerves and the associated commissural and oesophageal ganglia, were dissected as described by Selverston et al. (1976)
and pinned in a Sylgard-coated dish. The preparations were superfused continuously at 3 ml/min with saline (16°C) of the following composition in mM: 479 NaCl, 12.8 KCl, 13.7 CaCl2, 3.9 Na2SO4, 10.0 MgSO4, 2 glucose, and 11.1 Tris base, pH 7.35 (Mulloney and Selverston 1974
). Extracellular recordings were made from identified motor nerves with bipolar pin electrodes insulated by vaseline. After desheathing the STG, individual somata were impaled with glass microelectrodes (10-25 M
; 2.5 M KCl) and identified with three criteria: 1) 1:1 correspondence of action potentials recorded intracellularly in the soma and extracellularly from an identified motor nerve, 2) characteristic phasing and synaptic input during the pyloric motor pattern, and 3) characteristic shape of the membrane potential oscillations and action potentials in the pyloric rhythm.
DA application
If not indicated differently, DA was bath applied. The volume of the bath was ~3 ml, and the perfusion rate was 3 ml/min. The physiological response was measured after 10 min of bath application of DA (10
4 M), when a steady state of the DA response was well established. Effects of DA bath application on the isolated PD neuron reversed after 30 min washout with normal saline. We also applied DA by pressure ejection across the entire STG (1 s, 10
3 M inside the puffer pipette) with delivery pipettes with a tip diameter of ~50 µm and a suitable ejection pressure of 2 × 104 Pa. To monitor the drug application, Fast Green (1 mg ml
1) was added to the pipette solution.
Physiological recording
The response of the intact pyloric motor pattern to 10
4 M DA was recorded as described previously, except that two or three neurons were impaled for intracellular recordings. Both intracellular and extracellular recordings were digitized and stored on videotape. The threshold for detectable inhibition of PD by DA is 10
5 M, and a maximal effect is observed at 10
4 M (Flamm and Harris-Warrick 1986b
).
Synaptic isolation of the PD neuron
The PD neuron was isolated from all detectable synaptic input with three steps (Flamm and Harris-Warrick 1986b
). First, modulatory inputs from other ganglia were eliminated with a 10
4 M tetrodotoxin (TTX) block placed in a small vaseline well on the stomatogastric nerve, the sole input nerve to the STG (Russel 1979
). Second, the AB and the other PD neuron (and in most cases also the LP and VD neurons) were photoinactivated by intracellular injection of 5,6-carboxyfluorescein and illumination with UV light (Flamm and Harris-Warrick 1986b
; Miller and Selverston 1979
). Third, the remaining glutamatergic synapses were blocked with 5 × 10
6 M picrotoxin (PTX) (Bidaut 1980
). The PD neuron was allowed to recover for
1 h before measurements were made. There might be additional sources of synaptic input to the PD neuron after this treatment (Nusbaum et al. 1992
), but we saw no evidence for these in our experiments.
Current-clamp recordings of synaptically isolated neurons
After isolation, a PD neuron was impaled with two microelectrodes (9-11 M
, 2.5 M KCl or 2.5 M K-acetate with 2 × 10
2 M KCl) for voltage recording and current injection with an Axoclamp-2A amplifier. The cell was held by DC current injection at a constant membrane potential of
50 mV during the entire experiment. Current protocols were generated with the aid of pCLAMP and a TL1 interface (Axon Instruments) running on a Gateway 2000 microcomputer. Typically a series of 200-ms prepulses with incrementally increasing currents was given to hyperpolarize the neuron to between
50 and
100 mV, followed immediately by a 700-ms depolarizing current pulse to
45 mV, just above threshold for action potential generation. Because DA reduces the input resistance of the PD neuron, the current pulse amplitudes were adjusted throughout the experiment to give relatively constant voltage steps, allowing us to compare the spike frequency in the presence and absence of DA at the same membrane potential.
Voltage clamp of synaptically isolated PD neurons
Synaptically isolated PD neurons were impaled with two electrodes for voltage recording and current recording/injection (9-11 M
; 2.5 M KCl or 2.5 M K-acetate with 2 × 10
2 M KCl). The cell was voltage clamped with an Axoclamp-2A amplifier driven by pClamp. Linear leakage and capacitative currents were digitally subtracted with a P/6 protocol (see Armstrong and Bezanilla 1974
).
Current isolation
Currents were isolated with a combination of pharmacological block, voltage inactivation, and digital current subtraction protocols. Sodium currents were blocked by TTX (10
7 M). Calcium currents were blocked by CdCl2 (2-6 × 10
4 M). A hyperpolarization-activated inward current was blocked by CsCl (5 × 10
3 M). Currents from glutamatergic synapses were blocked by 5 × 10
6 M PTX (Bidaut 1980
). Tetraethylammonium (TEA, 2 × 10
2 M) was used to block IK(V) and IO(Ca) simultaneously. IO(Ca) was also indirectly eliminated when the Ca2+ currents were blocked by CdCl2. Although 4-aminopyridine (4AP, 4 × 10
3 M) has been shown to be a selective blocker of IA in other STG neurons (Graubard and Hartline 1991
; Tierney and Harris-Warrick 1992
), it induced a large and reversible leak current in PD (compare currents evoked by small voltage steps in Fig. 3, A and B), and thus was not used. IA was instead eliminated by holding the PD neuron at
40 mV, where IA is almost completely inactivated (Baro et al. 1997
).

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| FIG. 3.
DA evoked outward current and increase in membrane conductance in a synaptically isolated PD neuron. The cell was voltage clamped at 50 mV. To monitor the conductance, brief (100 ms, 1 Hz) 10-mV hyperpolarizing voltage pulses were applied. For clarity, the peak currents evoked by each voltage pulse are marked by an asterisk. A: DA pulse (1 s, 10 3 M) evoked a prolonged outward current, accompanied by an increase in membrane conductance. B: DA-evoked outward current and conductance increase were occluded by the combined presence of 4AP (5 × 10 3 M) and tetraethylammonium (TEA; 2 × 10 2 M). Note that 4AP evokes a large increase in leak conductance in the PD neuron. This rapidly reversed on removal of 4AP.
|
|
Transient K+ current IA
For measurement of IA, the STG was bathed in saline containing 10
7 M TTX, 2 × 10
2 M TEA, 2-6 × 10
4 M CdCl2, 5 × 10
3 M CsCl, and 5 × 10
6 M PTX to greatly reduce non-IA currents. The cell was held at
50 mV. Two series of 10-mV voltage steps between
50 mV and +50 mV were delivered. The first series had no prestep, whereas the second series had a 200 ms prestep to
100 mV to maximally deinactivate IA. The first series, which evoked the residual non-IA currents, was digitally subtracted from the second series, which additionally possessed active IA. The resulting outward current could be completely abolished by 4 × 10
3 M 4AP. Although this digital subtraction procedure gives a relatively pure IA, it also removes the contribution of active IA at or below
50 mV. However, this was typically
5% of the maximal conductance.
Sustained K+ current (IK(V))
To measure IK(V), the STG was bathed in 10
7 M TTX, 2-6 × 10
4 M CdCl2, 5 × 10
3 M CsCl, and 5 × 10
6 M PTX to block most of the non-IK(V) currents, and the cell was held at
40 mV, where IA is almost completely inactivated. Voltage steps in 10-mV increments between
40 mV and +50 mV were delivered to activate IK(V). This current was blocked by 2 × 10
2 M TEA.
Calcium-dependent K+ current (IO(Ca))
For measurement of IO(Ca), the STG was bathed in 10
7 M TTX, 5 × 10
3 M CsCl, and 5 × 10
6 M PTX to greatly reduce non-IO(Ca) currents, and IA was removed by setting the holding potential to
40 mV. The remaining current was then the sum of IO(Ca) and IK(V). After IO(Ca) was blocked (indirectly) by Cd2+, the remaining component (IK(V)) was digitally subtracted from the summed current of IO(Ca) and IK(V) to yield IO(Ca).
The voltage dependence of activation of IA and IK(V) was determined by converting the peak current to a peak conductance, g (assuming EK =
86 mV) (Hartline and Graubard 1992
). The resulting g/V curve was fitted to a third-order (n = 3) and first-order (n = 1) Boltzmann equation of the form
|
(1)
|
where gmax is the maximal conductance and s is a slope factor. For the third order Boltzmann fit, Vact is the voltage at which half-maximal activation of the individual gating steps occurs, assuming a third-order activation relation (Hodgkin and Huxley 1952
). For the first-order Boltzmann fit Vact (= V0.5) is the voltage of half-maximal activation of the peak current. For IO(Ca) the voltage dependence was analyzed by current/voltage plots.
Steady-state inactivation of IA was measured from a holding potential of
50 mV. Two second voltage presteps were delivered at 10-mV increments from
120 to
50 mV, followed by a step to +20 mV, and the peak current was measured. The data, scaled as a fraction of the calculated maximal conductance, were fit to a first-order Boltzmann equation (Eq. 1 with n = 1), based on the model of Hodgkin and Huxley (1952)
.
Statistical analysis
For single pairwise comparisons, Student's t-tests were used to assess statistical significance. Significances were accepted at P = 0.025. Throughout this paper, all calculated ranges are reported as means ± SE.
 |
RESULTS |
DA effect on PD neurons in intact pyloric network
The firing pattern of four of the major cell types in the pyloric network is shown in Fig. 1. The AB and the two PD neurons form the pacemaker group in this circuit. They are electrically coupled and fire synchronously, inhibiting all other neurons in the network. The follower cells recover from this inhibition with different rates and fire with different phases until they are inhibited by the next burst of the pacemaker group (reviewed by Johnson and Hooper 1992
; Miller 1987
).
As we have previously shown, bath application of 10
4 M DA modified the pyloric motor pattern (Ayali et al. 1998
; Flamm and Harris-Warrick 1986a
; Harris-Warrick et al. 1995b
) (Fig. 1, A and B) by changing the strength of synaptic connections between the pyloric neurons (Johnson and Harris-Warrick 1990
; Johnson et al. 1993a
,b
, 1995
) and changing their intrinsic properties (Harris-Warrick et al. 1995a
,b
). The AB, LP, and PY cells were excited and increased their firing rate in response to DA. However, the PD cells were inhibited by DA, which evoked a hyperpolarization and a reduction in the firing rate; in some preparations, the PD neurons fell silent. When it was active, the PD no longer fired synchronously with the AB. It fired a few spikes in the middle of the AB burst and thus had a phase delay compared with the first AB spike of each cycle (Fig. 1B).
In Fig. 1C, a DA pulse (1 s, 10
3 M) was applied to a PD neuron that was isolated from pyloric chemical synaptic input by PTX (5 × 10
6 M). The isolated PD cell hyperpolarized by >5 mV and stopped firing for ~1 min. Similar results were obtained in four experiments.
Effects of DA on synaptically isolated PD neurons
We investigated the effects of DA on the rate of recovery after inhibition in PD cells that were isolated from all detectable synaptic input (n = 5). Figure 2 shows such an experiment. Throughout the experiment, the cell was held at
50 mV by tonic current injection. At these holding potential, the isolated PD neuron did not have spontaneous spike activity. To mimic synaptic inhibition, the cell was hyperpolarized with 200-ms current pulses of varying amplitude. This prepulse was followed by a small depolarizing step to
45 mV, which is just above threshold for the initiation of action potentials. To quantify the rate of recovery after inhibition, we measured the latency from the end of the prepulse to the first action potential. We also monitored the spike frequency by measuring the interspike interval (ISI) between the first and second action potential of the subsequent spike train (Hartline 1979
; Tierney and Harris-Warrick 1992
) and the total number of spikes during the depolarizing step.

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| FIG. 2.
Effect of DA on the rate of recovery after hyperpolarizing presteps in a synaptically isolated PD neuron. A: voltage traces of a PD neuron under control conditions, during DA application, and after wash. The neuron was held at 50 mV by current injection throughout the experiment and did not have spontaneous activity at this potential. The voltage traces are offset to enhance readability. Presteps (200 ms) were applied to hyperpolarize the neuron between 50 mV and 80 mV, followed by a 700-ms depolarizing step to 45 mV. The amplitudes of the injected currents were adjusted during the experiment to maintain these voltage steps. B-D: parameters of the spike train during recovery from 200-ms hyperpolarizing presteps from the neuron shown in A under control conditions ( ) and in 10 4 M DA ( ). B: latency to first spike. The time from the end of the hyperpolarizing prepulse to the first action potential during the subsequent 700-ms depolarizing step to 45 mV is shown as a function of the membrane potential at the end of the hyperpolarizing prestep. C: first interspike interval (ISI). The time between the first and second action potentials during the depolarizing step is shown as a function of the membrane potential at the end of the hyperpolarizing prestep. D: spikes per step. The total number of action potentials elicited during the 700-ms depolarizing step is shown as a function of the membrane potential at the end of the hyperpolarizing prestep.
|
|
At the end of the hyperpolarizing prepulse, the cell began to rebound rapidly, but this slowed down to a prolonged slow ramp depolarization delaying the onset of a spike train. Under control conditions, the latency to the first spike increased as the hyperpolarizing step increased up to approximately
80 mV because of a prolongation of the slow ramp depolarization. Below this membrane potential, the latency remained relatively constant or increased much more slowly because the prolonged slow ramp depolarization became relatively constant. During bath application of DA (10
4 M), synaptically isolated PD cells hyperpolarized and usually became quiescent and showed reduced input resistance. To compare the intrinsic recovery properties of PD under control and DA conditions, we varied the tonic current injection to maintain the resting membrane potential of the PD at the control level and varied the amplitude of the current steps in the prestep protocol such that the voltage steps were near the control values. During bath application of DA (10
4 M), the PD cells showed reduced intrinsic recovery properties compared with control (Fig. 2). DA increased the first spike latency at all hyperpolarizing prestep levels (Fig. 2, A and B). For example, the latency after a hyperpolarizing prestep to
80 mV was prolonged by 30.9 ± 16.3% (P < 0.015; n = 5). DA also reduced the spike frequency during the subsequent spike train (Fig. 2, A and C); after a prestep to
80 mV, the first ISI increased by 21.5 ± 10.1% (P < 0.025; n = 5). This combination of increased latency and reduced spike frequency led to a decrease in the total number of spikes during the depolarizing pulse after the hyperpolarizing presteps (Fig. 2, A and D). After the prestep to
80 mV, the number of spikes decreased by 48 ± 8.3% (P < 0.001; n = 5).
DA modulation of ionic conductances
As a first step to explore the ionic mechanisms of DA inhibition of the PD cells, a brief DA pulse (1 s, 10
3 M) was applied to a synaptically isolated and voltage clamped PD neuron. DA evoked a small voltage-dependent outward current, accompanied by a 37.8 ± 10.5% (n = 5) increase in membrane conductance (Fig. 3A). As seen in Fig. 3B, both of these effects were eliminated by the combined presence of two potassium channel blockers 4AP (5 × 10
3 M), which selectively blocks IA in STG neurons (Tierney and Harris-Warrick 1992
), and TEA (2 × 10
2 M), which blocks IO(Ca) and IK(V) in these neurons. TEA alone reduced the DA evoked outward current by 27.9 ± 6.8% (n = 4) and reduced the DA evoked increase in conductance by 29.4 ± 9.0% (n = 4), suggesting that 4AP blocks the majority of the DA effect. Unfortunately, 4AP activates a large leak current in addition to blocking IA, as seen by the increase in conductance in Fig. 3B, so it was not possible to accurately ascertain the effects of 4AP alone. This 4AP sensitive leak current was only seen in PD neurons and was not seen in our earlier studies of the LP and PY cells (Harris-Warrick et al. 1995a
,b
).
These results suggest that modulation of voltage-activated K+ currents might contribute to the DA-induced changes in intrinsic rebound properties of PD. To test if K+ currents are indeed targets of DA modulation, we performed voltage-clamp studies on synaptically isolated PD cells and determined the effect of DA on a transient voltage-activated K+ current (IA), a sustained voltage-activated K+ current (IK(V)), and a calcium-dependent K+ current (IO(Ca)).
Transient K+ current
Under control conditions, IA activated with voltage steps above
50 mV (Fig. 4, A and B). This current was transient and decayed because of inactivation during a maintained depolarizing voltage step. The conductance/voltage relation (Fig. 4B) was determined from the peak currents evoked by each voltage step. This curve showed a typical voltage dependence for activation for IA and was fitted to a third-order Boltzmann equation. This fit showed half-maximal activation for each of the individual gating steps at
41.9 mV, leading to half-maximal activation of the peak current (V0.5) at
21.5 mV. Once inactivated, inactivation had to be removed by hyperpolarization. The voltage dependence of steady state inactivation (Fig. 4 B) was well fitted by a first-order Boltzmann equation, with a voltage for half-maximal inactivation of
69.1 mV. These parameters for IA in PD were in good agreement with those described by Baro et al. (1997)
.

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| FIG. 4.
Voltage-clamp analysis of the effect of DA on the transient K+ current, IA, in a PD neuron. The cell was held at 50 mV. A: current traces of IA under control conditions, during DA application, and after a wash. Each series represents current responses to increasing voltage steps between 40 and +20 mV in 10-mV increments. IA was isolated by pharmacological blockade and digital subtraction (see METHODS). B: conductance/voltage curves for activation (circles) and inactivation (squares) of IA under control (solid symbols) conditions and in 10 4 M DA (open symbols). Values are means ± SE (n = 7 for activation; n = 6 for inactivation), calculated as a fraction of the calculated maximal conductance under control conditions in each experiment. The activation curves were generated from peak currents after a maximally deinactivating prestep to 100 mV. The curves are fits to a third-order Boltzmann relation (Eq. 1, METHODS) with the following parameters. Control: gmax = 3.32 ± 0.2 µS; Vact = 41.9 mV; s = 15.5 mV. DA: gmax = 3.65 ± 0.2 µS; Vact = 49.5 mV; s = 15.4 mV. Steady-state inactivation is shown in curves with square symbols. The neurons were held at the indicated potential for 2 s before being stepped to +20 mV. The curves are fits to a 1st order Boltzmann relation (Eq. 1, METHODS), with the following parameters. Control: Vinact = 69.0 mV; s = 6.5. DA: Vinact = 69.8 mV; s = 6.4. C: to demonstrate the increase in tonically active IA near the resting potential, the product of the activation and inactivation curve (from B) is plotted as g/gmax = (1/1 + e (V Vact)/sact)3 × (1/1 + e (V Vinact)/sinact)1 for control condition and for DA application. The integral between curve and baseline is increased by 174% during DA application.
|
|
DA increased IA by two mechanisms (Fig. 4). First, it significantly increased the maximal conductance by 10.2 ± 2.8% (P < 0.025; n = 7). Second, it shifted the voltage for half-maximal activation to more negative potentials (Fig. 4B). Under control conditions, the V0.5 for half-maximal activation of the current (from a first-order Boltzmann fit to peak currents) was
21.5 ± 1.3 mV, which shifted significantly to
28.9 ± 1.0 mV (P < 0.02; n = 7) during DA application. However, there was no significant shift in V0.5 for inactivation. The effects of DA on IA were reversible after washing with normal saline (Fig. 4A).
The shift in Vact together with the increase in gmax led to an increase in the tonically active "window current" between the steady-state activation and inactivation curves. This is demonstrated in Fig. 4C by plotting the product of the steady state activation (m3) and inactivation (h) curves. These curves showed the fraction of tonically active IA as a function of membrane potential. During control oscillations (Fig. 1A), the PD neuron hyperpolarized to a mean trough value of 61.6 ± 3.9 mV (mean ± SD; n = 5) (Harris-Warrick, unpublished data), and remained below or close to
60 mV for several hundred milliseconds, sufficient to remove a portion of inactivation at these voltages. Around the normal PD membrane potential, tonic IA was dramatically increased by DA. For example at
50 and
60 mV, DA caused a 242 and 310% increase in resting IA, respectively.
Calcium-activated outward current
IO(Ca) consisted of an inactivating and a noninactivating component (Fig. 5A). Under control conditions, IO(Ca) activated with voltage steps above
30 mV (Fig. 5, A and B). Measurements of the reversal potential determined from tail current measurements with different external K+ concentrations indicated that this current is primarily, but not exclusively, carried by K+ ions (Graubard and Hartline 1991
; Hartline et al. 1985
; Kloppenburg and Harris-Warrick, unpublished data). IO(Ca) could be eliminated by removal of extracellular Ca2+ or addition of >2 × 10
4 M CdCl2 or TEA (2 × 10
2 M). IO(Ca) showed rundown during prolonged voltage clamp recordings, with a decrease in amplitude of >30% during the first 20 min of recording. This rundown appeared to be caused by depolarizing voltage steps and seemed to be cumulative with regard to the number, length and especially the amplitude of the pulses. To minimize rundown during our DA study, we avoided making measurements within the first few minutes of the recording when rundown was maximal, and limited long-lasting, repetitive high-amplitude depolarizing pulses.

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| FIG. 5.
Voltage-clamp analysis of the effect of DA on the calcium dependent outward current, IO(Ca), in a PD neuron. The cell was held at 40 mV. A: current traces of IO(Ca) under control conditions, during DA application, and after a wash. Each series represents current responses to increasing voltage steps between 40 mV and +30 mV in 10-mV increments. IO(Ca) was isolated as described in METHODS. B: current/voltage curves for activation of IO(Ca) under control conditions ( ) and in 10 4 M DA ( ). Values are means ± SE from 5 experiments, calculated as a fraction of the maximal current measured under control conditions at +30 mV in each experiment.
|
|
In our first series of experiments, we gave a single 200-ms depolarizing pulse to +20 mV every minute before, during, and after DA application. This protocol did not cause marked rundown. DA caused a reversible increase in the magnitude of IO(Ca). This effect was then confirmed in seven experiments where a series of depolarizing pulses from
40 mV to +30 mV in 10-mV increments was applied before, during, and after DA application (Fig. 5). From these experiments, it is clear that DA increased the magnitude of IO(Ca) in PD at depolarized voltages; for example, the amplitude of IO(Ca) at +30 mV was reversibly increased by 24 ± 0.5% (P < 0.01; n = 5). IO(Ca) probably depends on both intracellular [Ca2+] and voltage, and the voltage activation curve combines these two independent factors. As a consequence we did not attempt to dissect further the targets of modulation of this current by DA (see DISCUSSION).
Sustained K+ current
When IA and IO(Ca) were eliminated, a small noninactivating potassium current, IK(V), was unmasked. Under control conditions, IK(V) activated with voltage steps above
30 mV (Fig. 6). The current was sustained and did not decay during a maintained depolarizing voltage step. The voltage activation relation (Fig. 6), constructed from the maximal currents evoked by each voltage step, showed a typical voltage dependence for activation of IK(V). When fitted to a third-order Boltzmann equation, the current showed half-maximal activation for each of the individual gating steps at
23.7 mV, leading to half-maximal activation of the peak current at +1.9 mV. IK(V) showed little or no inactivation even with depolarizations lasting
1 s, and there was no detectable voltage dependence of steady-state inactivation (data not shown).

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| FIG. 6.
Voltage-clamp analysis of the effect of DA on the sustained K+ current, IK(V), in a PD neuron. The cell was held at 40 mV. A: currents traces of IK(V) under control conditions, during DA application, and after a wash. Each series represents current responses to increasing voltage steps between 40 mV and +40 mV in 10-mV increments. IK(V) was isolated as described in METHODS. B: conductance/voltage curves for activation of IK(V) under control conditions ( ) and in 10 4 M DA ( ). Conductance was calculated assuming EK = 86 mV (Hartline and Graubard 1992 ). Values are means ± SE from 7 experiments, calculated as a fraction of the calculated maximal conductance under control conditions in each experiment. The curves are fits to a 3rd-order Boltzmann relation (Eq. 1, as described in METHODS), with the following parameters. Control: gmax = 0.48 ± 0.04 µS; Vact = 23.7 mV; s = 20.2 mV. DA: gmax = 0.48 ± 0.04 µS; Vact = 22.2 mV; s = 20.4 mV.
|
|
Bath application of DA (10
4 M) had no detectable effect on IK(V) (Fig. 6). It did not change the maximal conductance, and the small shift in V0.5 from + 1.9 ± 3.7 mV to +3.3 ± 3.8 mV was not significant (P > 0.05; n = 7).
 |
DISCUSSION |
The starting point of this investigation was the finding that exogenously applied DA alters the rhythmic activity of the pyloric network (Anderson and Barker 1981
; Eisen and Marder 1984
; Flamm and Harris-Warrick 1986a
) (see Fig. 1, A and B). In the two PD neurons, DA evokes a hyperpolarization and a reduction in the number of action potentials per cycle. In part, this inhibition is caused by DA alteration of synaptic inputs to the PD neurons; DA enhances LP inhibition of PD (Johnson et al. 1995
). In addition, this inhibition results from direct effects of DA on the PD neuron's own intrinsic properties (Flamm and Harris-Warrick 1986b
). DA's inhibition of the PD neurons has three obvious consequences for the pyloric motor pattern (Fig. 1). First, it causes a decrease in cycle frequency (see Abbott et al. 1991
). This occurs despite DA's direct excitation of the AB neuron and the acceleration of its cycle frequency (Flamm and Harris-Warrick 1986b
) because, when AB is electrically coupled to the hyperpolarized, "leaky" PD neuron, the net effect on the pacemaker group is to reduce cycle frequency (see Abbott et al. 1991
). Second, the PD is phase delayed in its onset of spiking relative to the onset of the AB burst (0 in control conditions to around 0.1 in DA). In some preparations, the PD cells fall silent altogether and thus stop signaling to their muscles (Flamm and Harris-Warrick 1986a
). Third, in the presence of DA, the PD neurons' graded inhibition of the follower neurons, including LP and PY, is markedly reduced or completely abolished (Johnson and Harris-Warrick 1990
). This appears to be due to a reduction of acetylcholine (ACh) release from the PD terminals because the follower cells remain sensitive to ACh. As a partial consequence of this loss of PD inhibition, the LP and PY cells show a phase advance in their burst onset during DA (Eisen and Marder 1984
; Flamm and Harris-Warrick 1986a
).
In voltage clamp, brief pulses of DA evoke an outward current accompanied by an increase in membrane conductance. These effects are largely blocked by the combination of 4AP and TEA, suggesting K+ channel modulation by DA. Direct analyses of three K+ currents revealed that the 4AP-sensitive IA and and the TEA-sensitive IO(Ca) are indeed enhanced by DA. A detailed analysis of IA revealed that DA increases its maximum conductance and shifts its voltage activation curve to more negative potentials. This change in conductance goes in the right direction to explain the hyperpolarization and decreased PIR evoked by DA. In particular, the window region of overlap between steady-state activation and inactivation curves is markedly enlarged during DA (Fig. 4C), and tonic IA is increased two- to threefold in the normal voltage range of the PD neurons. It is unusual to think of the "transient K+ current" (IA) as contributing to the tonic currents that set the resting potential. However, blockade of IA with 4AP causes synaptically isolated pyloric neurons to depolarize by 10-20 mV (Tierney and Harris-Warrick 1992
), suggesting that tonic IA plays a greater role in setting the resting potential than previously thought. Thus DA enhancement of tonic IA may explain, at least in part, DA's tonic hyperpolarization of PD. DA also delays the onset of PD firing relative to the AB burst. This phase delay is due in part to the tonic hyperpolarization of PD described previously. However, DA also delays the rate of PD recovery after inhibition (Figs. 1 and 2), and this could result not only from increase in tonic IA but also an increase in phasic IA activated in the critical subthreshold voltage range for rebound. Thus DA, acting on the same channels, enhances both tonic potassium currents contributing to the PD resting potential and transient potassium currents determining the rate of PIR and phasing of PD activity in the pyloric motor pattern.
In addition to modulating IA, DA increases the magnitude of IO(Ca). Because IO(Ca) is not only voltage but also Ca2+ dependent, it has an important function during prolonged depolarizations that underlie bursts of action potentials (see Hille 1992
). Because of Ca2+ accumulation during a burst of action potentials, IO(Ca) will be increasingly activated during the burst. This will influence the interspike intervals and thus spike frequency, the number of spikes, and the overall duration of the burst. DA increases IO(Ca), which should prolong the interspike interval, decrease the number of spikes per burst, and terminate the burst prematurely. This conclusion fits well with our findings from the rebound experiments in synaptically isolated neurons (Fig. 2) and DA's reduction of PD spike frequency and burst duration in the intact pyloric network (Fig. 1). The I/V curve in Fig. 5B suggests that IO(Ca) is activated only above
30 mV and thus should not contribute to the resting potential. However, bathing the neuron with low extracellular Ca2+ or with Ca2+ channel blockers (Cd2+ or Co2+) causes the PD and other pyloric neurons to depolarize by 10-20 mV (Harris-Warrick, unpublished data). This cannot be a direct effect of reducing ICa, which would hyperpolarize the neurons. Instead, these experiments suggest that IO(Ca) is partially activated at, and contributes to, the resting potential in PD neurons. This hypothesis is further supported by the finding that the DA-induced hyperpolarization from the resting potential of a synaptically isolated neuron can be partially blocked by the IO(Ca) blocker TEA.
We did not perform a detailed analysis of which parameters of IO(Ca) are modulated by DA. The amplitude and time course of IO(Ca) are functions of several parameters, including membrane voltage, Ca2+ current kinetics, intracellular Ca2+ concentration and sequestration, and intrinsic channel properties. Conclusions about which specific parameters of IO(Ca) are modulated by DA cannot be made from whole cell currents induced by simple depolarizing steps. Single channel experiments will be needed to determine if DA acts directly on the channels generating IO(Ca), those generating ICa, or even on the kinetics of intracellular calcium metabolism. However, our results show clearly that DA increases the magnitude of IA and of IO(Ca), suggesting a synergistic effect of both currents on the rebound properties.
Modeling studies (Golowasch et al. 1992
; Harris-Warrick et al. 1995a
) demonstrate that modulation of IK(V) could be an effective mechanism to change neuronal resting potential and firing frequency in STG neurons. However, DA's effects on the intrinsic properties of PD somata appear not to be mediated by this mechanism because IK(V) was unaltered in the presence of DA.
Although our results demonstrate that DA's enhancement of IA and IO(Ca) contribute to its inhibition of the PD neuron, they do not prove that these are the only ionic currents modulated by DA in this neuron. Our voltage-clamp studies measure primarily currents from the cell body, whereas many of the integrative actions of the neuron arise in the neuropil, beyond the space-clamped region of the cell. We would have liked to use TEA and 4AP to try to occlude DA's effects on the firing properties of the unclamped PD neuron to address this question. Unfortunately, 4AP activates a large leak current that significantly reduces the input resistance, and it cannot be used as a simple IA blocker.
From this and other studies two conclusions can be drawn. First, IA is a common target of DA modulation in the pyloric network (Harris-Warrick et al. 1995a
,b
). Although IA is present in all pyloric neurons, its magnitude varies significantly between neuron types (Baro et al. 1997
). Thus, DA could act on IA in different cells but have quantitatively different effects on their firing properties depending on differences in IA density in the cells. In addition, DA can have opposite effects on IA in different neurons. DA inhibits the PD neurons in part by enhancing IA (this paper) and excites the PY and LP neurons in large part by reducing IA (Harris-Warrick et al. 1995a
, b). Second, the actions of DA are not limited to the modulation of a single current. DA excites the LP neuron not only by modulation of IA but also by enhancing Ih (Harris-Warrick et al. 1995b
). In the PD neurons, both IA and IO(Ca) are enhanced by DA, and these currents contribute to DA's actions to inhibit the PD neuron, reduce its rebound properties, and reduce its spike frequency. In addition, DA greatly reduces or abolishes synaptic transmission from PD synapses (Johnson and Harris-Warrick 1990
). Although the modulation of K+ currents (described in this paper) may contribute to reducing release, other ionic currents (such as Ca2+ currents) could be selectively modulated at nerve terminals in a way that is not detectable by voltage clamp of the soma. Experiments are in progress to study these additional effects of DA in distal regions of the neuron.