doi:10.1152/jn.00456.2011 106:1288-1298, 2011. First published 15 June 2011;J NeurophysiolLolahon R. Kadiri, Alex C. Kwan, Watt W. Webb and Ronald M. Harris-Warrickintracellular storespacemaker neuron rely on release of calcium from Dopamine-induced oscillations of the pyloric

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Dopamine-induced oscillations of the pyloric pacemaker neuron relyon release of calcium from intracellular stores

Lolahon R. Kadiri,1 Alex C. Kwan,2 Watt W. Webb,2 and Ronald M. Harris-Warrick1

1Department of Neurobiology and Behavior and 2School of Applied and Engineering Physics, Cornell University,Ithaca, New York

Submitted 19 May 2011; accepted in final form 13 June 2011

Kadiri LR, Kwan AC, Webb WW, Harris-Warrick RM. Dopamine-induced oscillations of the pyloric pacemaker neuron rely on release ofcalcium from intracellular stores. J Neurophysiol 106: 1288–1298, 2011.First published June 15, 2011; doi:10.1152/jn.00456.2011.—Endogenouslybursting neurons play central roles in many aspects of nervous systemfunction, ranging from motor control to perception. The propertiesand bursting patterns generated by these neurons are subject toneuromodulation, which can alter cycle frequency and amplitude bymodifying the properties of the neuron’s ionic currents. In the stoma-togastric ganglion (STG) of the spiny lobster, Panulirus interruptus,the anterior burster (AB) neuron is a conditional oscillator in thepresence of dopamine (DA) and other neuromodulators and serves asthe pacemaker to drive rhythmic output from the pyloric network. Weanalyzed the mechanisms by which DA evokes bursting in the ABneuron. Previous work showed that DA-evoked bursting is criticallydependent on external calcium (Harris-Warrick RM, Flamm RE. JNeurosci 7: 2113–2128, 1987). Using two-photon microscopy andcalcium imaging, we show that DA evokes the release of calciumfrom intracellular stores well before the emergence of voltage oscil-lations. When this release from intracellular stores is blocked byantagonists of ryanodine or inositol trisphosphate (IP3) receptor chan-nels, DA fails to evoke AB bursting. We further demonstrate that DAenhances the calcium-activated inward current, ICAN, despite the factthat it significantly reduces voltage-activated calcium currents. Thissuggests that DA-induced release of calcium from intracellular storesactivates ICAN, which provides a depolarizing ramp current thatunderlies endogenous bursting in the AB neuron.

calcium-activated inward current; ryanodine; voltage clamp; centralpattern generator

RHYTHMIC ACTIVITY plays central roles in many aspects ofnervous system function, ranging from motor control to per-ception. The neural mechanisms underlying rhythmogenesisare not well understood but include both network-driven os-cillators and neuronal oscillators (Harris-Warrick 2010). Neu-ronal oscillators are single neurons that generate an endoge-nous oscillatory voltage pattern based on the ionic currentsthey express. The bursting properties of these neurons aresubject to neuromodulation, which can alter cycle frequencyand amplitude by modifying the properties of the neuron’sionic currents (Harris-Warrick and Johnson 2010; Lotshaw etal. 1986; Marder and Bucher 2001; Marder et al. 2005; Par-tridge et al. 1990).

The pyloric network in the crustacean stomatogastric gan-glion (STG) is an important system for studying the mecha-nisms of rhythmogenesis (Harris-Warrick 1993; Marder and

Bucher 2001; Selverston and Ayers 2006). The pyloric circuitconsists of 1 interneuron (the anterior burster, or AB) and 13motoneurons. The AB neuron is a conditional oscillator: underthe appropriate modulatory conditions, it generates intrinsicrhythmic oscillations and serves as the primary pacemaker todrive the network with a cycle frequency of 1–2 Hz. Whenisolated from all synaptic and modulatory input, the AB neuronloses its oscillatory capabilities. Addition of neuromodulatorssuch as the monoamines dopamine (DA) and serotonin (5-HT)can restore rhythmic bursting in the isolated AB neuron.However, DA and 5-HT appear to use different sets of currentsto drive the oscillations (Harris-Warrick and Flamm 1987).DA-induced bursting of the AB neuron is critically dependenton external calcium, as low external calcium or broad-spec-trum calcium channel blockers halt it, while these blockersonly slow the frequency of 5-HT-evoked bursting. In contrast,low external sodium concentration or application of tetrodo-toxin (TTX) does not affect DA-induced oscillations but abol-ishes 5-HT-induced bursting (Harris-Warrick and Flamm1987).

DA shapes the electrical properties of the AB neuron byaffecting multiple currents in a cell-specific manner. DA re-duces the tonic leak current, the transient potassium current(Peck et al. 2001), and the voltage-gated calcium current[ICa(V)] (Johnson et al. 2003). Additionally, DA enhances thehyperpolarization-activated inward current, Ih (Peck et al.2006), which contributes to (but alone is not sufficient totrigger) the depolarizing ramp driving regenerative voltageoscillations. On the other hand, DA also enhances a slowlyactivating and deactivating inward-rectifying voltage-gated po-tassium current [IK(V)] (Gruhn et al. 2005), which may act torestrict the maximal spike frequency of the oscillating ABneuron (Harris-Warrick and Johnson 2010).

Here we have further studied the cellular mechanisms ofDA-induced pacemaking in the AB neuron. The calcium de-pendence of DA-induced oscillations suggests an involvementof calcium currents and/or calcium-activated inward currents.However, DA reduces ICa(V) by up to 75% (Johnson et al.2003), significantly reducing the influx of calcium ions into thecell. Since this does not appear to be the major source ofcalcium for DA-evoked bursting, we hypothesized that en-hanced release of calcium from intracellular stores might becentral to DA’s actions.

In this paper, we show that DA does indeed evoke anaccumulation of intracellular calcium by inositol trisphosphate(IP3) receptor (IP3R)- and ryanodine (Ry) receptor (RyR)-mediated release from intracellular stores. This increase incalcium concentration begins well before the emergence of

Address for reprint requests and other correspondence: R. M. Harris-Warrick, Dept. of Neurobiology and Behavior, Cornell Univ., W 159 Seeley G.Mudd Hall, Ithaca, NY 14853 (e-mail: rmh4@cornell.edu).

J Neurophysiol 106: 1288–1298, 2011.First published June 15, 2011; doi:10.1152/jn.00456.2011.

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voltage oscillations, and in turn appears to activate the calcium-activated nonselective current ICAN, which provides a depolariz-ing ramp current that contributes to the pacemaker oscillations inthe AB neuron.

MATERIALS AND METHODS

Animals and preparation. Adult California spiny lobsters (Panuli-rus interruptus) were supplied by Don Tomlinson Commercial Fish-ing (San Diego, CA) and kept in tanks with artificial seawater. Animaluse followed a protocol approved by the Institutional Animal Care andUse Committee at Cornell University. Lobsters were anesthetized inice; the stomatogastric nervous system (STNS) was dissected out,pinned on a Sylgard-coated petri dish, and superfused with oxygen-ated lobster saline at 16–17°C unless otherwise specified. The STGwas desheathed, and cells were identified as previously described(Selverston et al. 1976). Experiments were primarily performed on theAB neuron, but some used the pyloric dilator (PD) and lateral pyloric(LP) neurons.

Solutions. Panulirus physiological saline had the following com-position (in mM): 479 NaCl, 12.8 KCl, 13.7 CaCl2, 3.9 Na2SO4, 10.0MgSO4, 2 glucose, and 11.1 Tris base, pH 7.4 (Mulloney andSelverston 1972). To pharmacologically isolate the ionic currents ofinterest, we used 50 mM tetraethylammonium chloride (TEA) and 4mM 4-aminopyridine (4-AP) to block potassium currents, 5 mM CsClto block the hyperpolarization-activated inward current Ih, 0.1 �MTTX to block the sodium current, and 5 �M picrotoxin (PTX) to blockglutamatergic synapses within the STG; the NaCl concentration wasadjusted to compensate for high TEA. In addition to the bath-appliedblockers, we loaded the cell body with intracellular K� channelblockers by iontophoresis, injecting small negative current stepsthrough electrodes loaded with 2 M TEA and 2 M CsCl for 1–1.5 h;the cell was allowed to recover for 1–1.5 h before the experimentstarted. Although these treatments reduced K� currents by �99%, adetectable outward current still contaminated our voltage-clamp stud-ies of the very small calcium-dependent currents. We used flufenamicacid (FFA, 0.1–30 �M) to block ICAN, 30 �M BAPTA-AM to chelateintracellular calcium ions, 10–100 nM Ry to block intracellular RyRchannels, 1–10 �M xestospongin C or D to block intracellular IP3Rchannels, and 10 �M thapsigargin (Tg) to block the sarco/endoplas-mic reticulum calcium-ATPase (SERCA) pump. FFA, Ry, xesto-spongin, and Tg were first dissolved in DMSO to make 100� stocksolutions. Immediately before the experiment, the stock solution wasdiluted in saline to bring the concentration of the blockers to thedesired level; the concentration of DMSO did not exceed 1% in thefinal solution. DMSO alone at this concentration did not have anysignificant effect on the physiology of the neuron. Chemicals werepurchased from Sigma (BAPTA-AM, FFA, Ry), Tocris (Ry), orCayman Chemical (xestospongin).

Two-photon calcium imaging. Calcium indicator dyes were in-jected into the AB neuron by iontophoresis (Kloppenburg et al. 2000).Only AB neurons that maintained rhythmic activity and showedlabeling in fine neurites 30 min after iontophoresis were retained foranalysis. We labeled the AB neuron with potassium salts of CalciumGreen-1 or Indo-1 (both from Invitrogen). Our two-photon micro-scope is based on an upright Olympus BX50WI unit (Díaz-Ríos et al.2007; Kwan et al. 2009). The excitation, generated by a Ti:sapphirelaser (Tsunami, Spectra-Physics), was scanned by a modified Bio-RadRadiance scan head and focused onto the sample by a water immer-sion �40/NA 0.80 objective (Olympus). The Tsunami was driven bya 10-W diode laser (Millenia Pro, SpectraPhysics), whose power was�1.7 W directly out of the laser cavity. The laser intensity wascontrolled by a Pockels cell (350-80LA, Conoptics). The typicalafter-objective laser power used in our experiments was 5–10 mW asmeasured with a standard thermopile sensor. For Calcium Green-1,the specimen was excited at 800 nm and the fluorescence wascollected by a GaAsP photomultiplier tube (H7422, Hamamatsu) after

a band-pass filter between 500 and 650 nm (Chroma). For Indo-1, thespecimen was excited at 720 nm and fluorescence was separated by aDCLP440 dichroic mirror into two detection channels: 360–430 nmwith a bialkali photomultiplier tube (HC125-02, Hamamatsu) and485–505 nm with a GaAsP photomultiplier tube. The line scans weresynchronized to the electrophysiological recordings by connecting thevoltage output of the amplifier to the image acquisition system andrecording membrane potential (Vm) on a third detection channel(Kloppenburg et al. 2000).

For the ratiometric calcium indicator Indo-1, we estimated theabsolute free calcium concentration, [Ca2�]in, by calculating the ratiobetween fluorescence detected at the two emission channels, R �F395/F495, and using the equation [Ca2�] � Keff � (R � R0)/(R1 �R), where Keff is the in situ dissociation constant of Indo-1 and R0 andR1 are the fluorescence ratios at zero and saturating calcium concen-trations. Background fluorescence was estimated from a region with-out neurites and subtracted from the actual fluorescence signals.Because the calcium dissociation constant within fine neurites of STGneurons is not known, we assumed that it is similar to the valuemeasured in salt solution, so Keff � Kd � 250 nM (Grynkiewiecz etal. 1985). To measure R1, we topically applied a calcium ionophore,4-bromo-A-23187 (Invitrogen) to equilibrate the intra- and extracel-lular calcium concentrations. We tried to measure R0 by washing insaline with zero Ca2� and 100 �M BAPTA-AM, but the fluorescencesignals at both detection channels decreased rapidly and precludedmeaningful measurement of R0. Instead of measuring R0, we assumedthat [Ca2�]in is 97 nM as previously measured in STG neurons(Kloppenburg et al., 2000) and used Keff, R1, and Rsaline to calculateR0. We then used these calibration parameters to calculate [Ca2�]in

under other conditions.Electrophysiology. Current clamp (CC) and two-electrode voltage

clamp (TEVC) and were performed with an Axoclamp-2B amplifier,a Digidata 1440 data acquisition board, and pCLAMP10 software (allfrom Molecular Devices, Sunnyvale, CA). For the CC experiments,the AB neuron was synaptically isolated by 5,6-carboxyfluoresceinphotoablation of the three pyloric motoneurons that are electricallycoupled to it—the ventricular dilator (VD) and two PD cells—asdescribed by Miller and Selverston (1979). In some experiments, weblocked synaptic and neuromodulatory input to AB neuron by addingTTX (0.1 �M) and PTX (5 �M) to the saline instead of photoablation.This did not make any difference in DA-induced bursting besidesremoving the very small action potentials (see also Harris-Warrickand Flamm 1987); therefore, these results were pooled for dataanalysis.

ICAN measurements. To measure ICAN activated by influx of extra-cellular calcium in voltage clamp, we delivered depolarizing prestepsfrom Vh � �50 mV to 0 mV to activate ICa(V). This current in turnactivates ICAN, which is detected as a slowly decaying inward tailcurrent upon repolarization. All measured currents were leak sub-tracted before data analysis. For the determination of the ICAN reversalpotential, an activating prestep was followed by a series of hyperpo-larizing steps from �80 mV to �40 mV in 5-mV increments to evokea tail current. The peak slow tail current amplitude was measured andplotted against the value of the voltage step and extrapolated todetermine the reversal potential (Erev). The deactivating portion of thecurrent trace was fitted with an exponential function using Clampex;the time constants were then plotted against voltage steps.

Data analysis. Electrophysiological data analysis was performedwith Clampfit10 (Molecular Devices). The calcium imaging data wereanalyzed with ImageJ (National Institutes of Health). Synchronizationof imaging and physiology was done with custom-written MATLABsoftware. Statistical analyses were performed with JMP 7 (SASSystems). Graphs and figures were made in Excel, Origin 6 (Origin-Lab, Northampton, MA), and Adobe Illustrator CS3 (Adobe Sys-tems). All values are presented as means � SE; the effect wasconsidered statistically significant at P � 0.05.

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RESULTS

Release of calcium from intracellular stores is essential forDA-induced bursting in the AB neuron. In light of our previousresults that DA induces calcium-dependent pacemaker oscilla-tions (Harris-Warrick and Flamm 1987) while greatly inhibit-ing calcium influx via ICa(V) (Johnson et al. 2003), we hypoth-esized that these oscillations rely on intracellular sources ofcalcium ions. DA could enhance Ca2� release from intracel-lular stores, inhibit Ca2� sequestration and buffering in intra-cellular compartments, or both.

We initially assessed the involvement of intracellular cal-cium stores in induction of pacemaker oscillations by chelatingthe intracellular calcium with lipid-permeable BAPTA-AM(Fig. 1A). This compound diffuses into the neuron, where it isdeesterified to yield BAPTA. With time of diffusion the ABoscillations became smaller, and eventually the neuron ceasedto oscillate and remained at a depolarized voltage. Similarresults were seen by blocking the intracellular calcium-releasechannels located on the endoplasmic reticulum (ER) membranesuch as Ry- and IP3-sensitive intracellular calcium receptor

channels, as well as SERCA, a calcium reuptake pump. Ry, aspecific blocker of the intracellular calcium channels respon-sible for calcium-activated calcium release, blocked DA-in-duced AB neuron oscillations at concentrations of 10 nM orhigher within 40 min (n � 7; Fig. 1B). Xestospongin C (Xe) isa specific blocker of the IP3R channel (Gafni et al. 1997); 10�M Xe effectively halted the DA-evoked AB rhythm (n � 3;Fig. 1C). Finally, application of 1 �M Tg, a specific blocker ofthe SERCA pump, stopped an ongoing DA oscillation within40 min (n � 3; Fig. 1D). These data suggest that release ofstored intracellular calcium via RyRs and IP3Rs is essential forDA-induced oscillations in the AB neuron. Notably, as theDA-evoked bursting ceased with all of these blockers, thetrough potential depolarized until the cell fell silent at a highlydepolarized voltage (typically �40 to �42 mV). This was alsoobserved when DA-evoked AB bursting was abolished by lowextracellular calcium or calcium channel blockers (Harris-Warrick and Flamm 1987). This depolarization may be causedby an indirect block of calcium-sensitive potassium current[IK(Ca)] and subsequent depolarization of the Vm, as well asreflecting the effects of DA on other ionic currents such asinhibition of the leak conductance or activation of calcium-dependent inward currents (see below).

Calcium imaging reveals DA-evoked increase of intracellu-lar [Ca2�] in AB neurites. To directly test the possibility thatDA induces an increase in intracellular Ca2� levels, we carriedout two-photon laser microscopic imaging of intracellular cal-cium activity in the AB neuron on two different timescales,using two different calcium-sensitive indicator dyes (CalciumGreen-1 and Indo-1). Briefly, we loaded the AB neuron somawith the calcium indicator dye via the intracellular electrode,let the dye diffuse throughout the dendritic arbor for up to 1 h,and performed synchronized two-photon calcium imaging andelectrophysiological recordings.

Distribution and strength of calcium signal in AB neuron.The dye-filled AB soma and neurites showed clear calciumfluorescence in both intact and DA-stimulated oscillating ABneurons. The signal from the cell body and the primary neuritewas relatively constant, and we were unable to detect anyoscillation-related fluorescence changes there. This is consis-tent with previous reports suggesting that calcium oscillationsoriginate outside the soma in the fine neuropil (Kloppenburg etal. 2000, 2007; Ross and Graubard 1989). As a consequence,we made our measurements in small neurites with diameter �5�m, at distances from the soma of �500 �m.

We performed timed (6–10 images/s) ratiometric measure-ments of Indo-1 fluorescence levels in fine neurites combinedwith simultaneous voltage recordings from the AB soma first inthe absence and then in the presence of DA. Figure 2 shows anexample of the AB soma and its extensive neuritic tree filledwith Indo-1 (Fig. 2A, fluorescence from the 395-nm channel),where the yellow arrowheads indicate typical regions of neu-ropil used for calcium measurements and the dashed squaredemarcates the region enlarged in Fig. 2B. Within this region,the ratios of the fluorescence signals from the 395- and 495-nmchannels were calculated and shown in Fig. 2B in pseudocolor.Additionally, the time course of the calcium signal from 19regions of interest (ROIs) in neuropil was measured over theperiod of the whole experiment.

Fig. 1. Release of calcium from intracellular stores is essential for dopamine(DA)-induced anterior burster (AB) oscillations. A: 30 �M BAPTA-AM(BAPTA) blocked 10�4 M DA-induced bursting in a synaptically isolated AB.In this experiment, the AB neuron was isolated from all synaptic and descend-ing neuromodulatory inputs by photoablation of the pyloric dilator (PD) andventricular dilator (VD) neurons and local application of tetrodotoxin (TTX) toa Vaseline pool around the stomatogastric nerve. B–D: 10 nM ryanodine (Ry),10 �M xestospongin C (Xe), and 1 �M thapsigargin (Tg) blocked DA-inducedoscillations in the AB. In these experiments, the AB neuron was isolated byapplication of TTX and picrotoxin (PTX) directly to the stomatogastricganglion (STG).

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Basal level of intracellular calcium rises during DAapplication. Upon DA application, all visualized fine neuritesdisplayed significant increases in calcium fluorescence of vary-ing amplitude relative to their pre-DA levels (Fig. 2B andFig. 3A). The calcium responses of 27 individual neurites from3 different AB neurons were monitored as DA was applied tothe bath. For individual ROIs in the fine neuropil, whenaveraged calcium measurements were taken at slow speed(6–10 images/s) the DA-induced increase in [Ca2�]in rangedfrom 136% to 555% of the pre-DA level with an average of302 � 153% increase. Thus different neurites of a singleneuron showed marked variability in calcium handling bothunder control conditions and in the presence of DA.

Onset of basal [Ca2�]in rise precedes onset of voltageoscillations. The changes in [Ca2�]in were monitored in par-allel with voltage recordings; for the large majority of neurites(25 of 27), the rise in [Ca2�]in started well before the emer-gence of sustained voltage oscillations (Fig. 3A). The intracel-lular calcium levels began rising �3 min after DA reached theganglion. The interval between the start of the calcium increaseand the onset of voltage oscillations is highlighted with avertical light gray bar in Fig. 3, A and B1. On average, voltageoscillations started 108 � 12 s after the onset of calcium risein fine neurites (n � 27 neurites, 3 AB neurons). This earlieronset of calcium rise in fine neurites, �1.5 min before theemergence of bursting, suggests that the rise in intracellularcalcium might bring about the subsequent depolarization andrhythmic Vm oscillations. DA’s effect was completely revers-ible: washing DA out of the bath solution abolished the Vm

oscillations and brought the levels of [Ca2�]in back to pre-DAlevels (Fig. 3B). Figure 3B2 presents a summary of the meanintracellular [Ca2�] under different conditions: in physiologi-cal saline, [Ca2�] in a spontaneously oscillating AB neuronwas 140 � 8 nM. When AB neuron was synaptically isolated(either by photoablation of the electrically coupled PD and VDneurons or pharmacologically by bath application of TTX andPTX), the mean [Ca2�] decreased to 67 � 3 nM. Uponsubsequent DA application the levels of calcium started risingwithin minutes; the voltage oscillatory activity usually startedwhen the mean [Ca2�]in reached 135 � 14 nM (not shown),while during stable DA oscillations time-averaged [Ca2�]reached 193 � 17 nM (for all conditions, n � 27). Calciumlevels during DA application were significantly different fromcontrol measurements before DA in TTX or after washout(ANOVA, P � 0.01, n � 27).

Effect of preincubation with ryanodine and xestospongin onDA-evoked calcium rise. Our previous data on AB burstingpredicted that application of the intracellular calcium channelblockers Ry and Xe would prevent or block the DA-inducedincrease in calcium levels and voltage oscillations. In threecells that were pretreated for 30–60 min with a cocktail of 10�M Ry and 10 �M Xe, DA either elicited only a small calciumincrease and a short-lived train of slow voltage oscillations ordid not induce any significant increase in intracellular calciumand did not evoke voltage oscillations (Fig. 3C1). The mean[Ca2�] was 82 � 11 nM in Ry � Xe and 87 � 17 nM in thepresence of DA together with Ry � Xe, which were notstatistically different (Fig. 3C2) (P � 0.05, n � 10 ROIs).

Fig. 2. Calcium imaging in the AB neuron filled with thecalcium indicator Indo-1. A: Z stack of the AB neuronlabeled with Indo-1, showing fluorescence from the395-nm channel. Cell body with a bright nucleus is atbottom left. The primary and secondary neurites, alongwith an extensive dendritic tree, are clearly labeled.Yellow arrowheads mark the approximate regions offine neuropil where calcium measurements were typi-cally taken. Size bar, 100 �m. Region in yellow squareis shown enlarged in B. B: the same region of fineneuropil, showing the ratios of the calcium emissionsignal detected at 395 nm and 495 nm. Size bar, 25 �m.Measurements are repeated after blockade of synapticinput with TTX, and again after addition of 10�4 M DA.

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Although DA failed to evoke sustained voltage oscillations inthese neurons, it did slightly depolarize the Vm, probablybecause of its effect on other ionic currents in the AB neuron(Gruhn et al. 2005; Johnson et al. 2003; Peck et al. 2001,2006).

High-temporal-resolution calcium imaging with simultane-ous recording of membrane potential. We used CalciumGreen-1 to track calcium changes on a faster timescale duringspontaneous and DA-evoked AB oscillations. As noted above,

we did not detect any calcium oscillations in the cell soma orprimary neurites, but the fluorescence signals in fine neuritesrevealed clear oscillatory behavior at the same frequency as theVm oscillations.

To monitor calcium changes on a cycle-by-cycle basis, weused fast line scan (frequency 164 Hz) to monitor calciumactivity in single fine neurites along with synchronized elec-trophysiological recordings of Vm in the soma. In the sponta-neously bursting AB (with intact modulatory input to the STG

Fig. 3. Onset of the DA-induced calcium riseprecedes the onset of membrane potential (Vm)oscillations. A: time course of the voltage (top)and free Ca2� concentration ([Ca2�]in; bottom)change during DA application; 10�4 M DA hasreached the bath solution at the 50 s time point.Each dendrite [region of interest (ROI)] is indi-cated by different color; the data points from oneof the neurites are fit with a sigmoid function(plotted as a red solid line). Note that in the timeperiod highlighted with the vertical light gray bar,[Ca2�] started rising before emergence of burst-ing. Voltage oscillations initially are slower andaccelerate as the DA concentration reachessteady state. B1: complete time course of ABoscillations and intracellular calcium measure-ments as DA is added and washed out. As DAwas washed out of the bath solution, the voltageoscillations and calcium signal returned back totheir pre-DA levels within 10–15 min. B2: sum-mary of the changes in the average calcium levelsunder different conditions: physiological saline(140 � 8 nM), TTX (67 � 3 nM), 10�4 M DA(193 � 17 nM), and washout of DA. The neuronwas oscillating in saline and in the presence ofDA and quiescent in TTX and after DA (wash).C: preincubation with intracellular calcium chan-nel blockers (Ry�Xe) abolishes the DA-evokedrise in [Ca2�]in and oscillations in the AB neuron.C1: the AB neuron was preincubated withRy�Xe for 30–60 min before DA application.Intracellular [Ca2�] did not change upon DAapplication; the neuron depolarized, because ofDA’s effects on other currents, but did not oscil-late. C2: there was no significant difference inmean [Ca2�] under different conditions: 82 � 11nM before DA (in Ry�Xe) and 87 � 17 nM inthe presence of DA and Ry�Xe (P � 0.05, n �10 ROIs).

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from the higher-order ganglia), [Ca2�], monitored as thechange in fluorescence level (dF/F), oscillated at the samefrequency as the voltage (Fig. 4A). Application of TTX andPTX to block the descending modulatory inputs effectivelyblocked both voltage and calcium oscillations within severalminutes (Fig. 4B). Addition of DA to the bath saline restoredrhythmic bursting of voltage along with calcium oscillations(Fig. 4C). Finally, Ry treatment of the bursting pacemaker inthe continued presence of DA halted both calcium and voltageoscillations (Fig. 4D). The amplitude of the calcium oscilla-tions was �20% of the basal level of calcium in the presenceof DA. Notably, the calcium oscillations were slightly delayedrelative to the voltage oscillations in both the spontaneouslybursting AB neuron before isolation and the synaptically iso-lated AB treated with DA (Fig. 4E; n � 3). The maxima of thecalcium oscillations were delayed by �150 ms relative to thepeaks of the Vm oscillations. This delay seems to be too long tobe explained by the slow kinetics of calcium activation ofCalcium Green-1 (Parker and Yao 1996).

Dopamine-induced oscillations are blocked by FFA, ablocker of the ICAN. Our imaging studies show that DA evokesincreases in intracellular calcium that could lead to depolariza-tions preceding the onset of voltage oscillations. One possibletarget of the rise in [Ca2�]in is the calcium-activated nonspe-cific current ICAN. We tested the importance of ICAN in DA-

evoked bursting by application of a blocker of this current,FFA. Three micromolar FFA reliably blocked the ongoing DAoscillations in a synaptically isolated AB neuron (n � 5),regardless of the method used to synaptically isolate the AB(Fig. 5A). Notably, as the rhythm ceased, the trough potentialdepolarized until the cell fell silent at �40 to �42 mV. Thiswas also observed when we blocked the rise in intracellularcalcium (Fig. 1) and when DA-evoked AB bursting wasabolished by low extracellular calcium or calcium channelblockers (Harris-Warrick and Flamm 1987). To rule out thepossibility that this depolarization was responsible for thecessation of bursting, we injected a negative bias current(usually 1–5 nA) to bring the Vm down to its preblocker troughvalues of �55 to �60 mV. Although early during FFA appli-cation AB bursting could be rescued by hyperpolarizing theVm, perhaps after enough time to allow equilibration of theFFA concentration, the cell became completely unable to burstor oscillate, even when hyperpolarized. This lack of burstrescue by hyperpolarization suggests that it is the block of theFFA-sensitive inward current and not the possible secondaryeffect of depolarization that causes disruption of the DArhythm. Therefore, we next focused on the characterization ofthe properties and DA modulation of ICAN in the pacemakerneuron.

Fig. 4. The AB neuron calcium signal oscil-lates in phase with voltage oscillations but isdelayed in onset and peak levels. Data werecollected at 164 Hz by a fast line scan over asingle dendrite. Simultaneous recordings ofcalcium signal and membrane: Vm (top) andrelative change in fluorescence (dF/F; bottom).Time markers, in seconds, are below the volt-age trace. A: spontaneously bursting AB neu-ron in physiological saline. B: 10�7 M TTX�5 � 10�5 M PTX blocked both voltage andcalcium oscillations in �10 min. C: 10�4 MDA induced Vm and calcium signal oscilla-tions. D: application of 10 �M Ry stoppedboth voltage and calcium oscillations. E: over-lay of voltage and calcium traces. Calciumoscillations had similar delayed time courseand amplitude both in a spontaneously burst-ing AB neuron with no blockers (red traces)and in a synaptically isolated AB (treated withTTX and PTX) in the presence of DA (blacktraces). Top: voltage trace. Bottom: calciumsignal change (dF/F).

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Properties of ICAN in the anterior burster neuron. We havecarried out a physiological and pharmacological analysis ofICAN by measuring its Erev, deactivation time constant, andsensitivity to FFA. Because this current lacks intrinsic voltagesensitivity, and because of problems of space clamp in thesehighly branched neurons (Kloppenburg et al. 2000), we wereunable to perform a full biophysical analysis. In the presence ofblockers of ionic currents and synaptic input, we measuredICAN as a slowly deactivating tail current following a depolar-izing prestep to 0 mV to activate ICa(V). The tail current wasrecorded over �80 mV to �40 mV in 5-mV increments.Although we blocked �99% of the total K� currents, there wasstill some leak contamination of the very small calcium cur-rents measured in the AB neuron (Fig. 5B). Figure 5B showssample current traces without leak current subtraction. Thepeak amplitude of this tail current was measured at the begin-ning of the hyperpolarizing step, taking advantage of the muchmore rapid deactivation kinetics of the calcium current relativeto that of ICAN in stomatogastric neurons (Johnson et al. 2003).At �65 mV, the mean amplitude of the peak leak-subtractedICAN in the AB was �2.7 � 0.7 nA (Fig. 5E, open squares; n� 8). The leak-subtracted tail current was plotted as a functionof the hyperpolarizing voltage step, fitted with a linear func-tion, and Erev was extrapolated for each cell. The average Erevwas �29 � 0.3 mV. The current decayed during the hyperpo-larizing step, reflecting the time constant of channel deactiva-tion and/or removal of intracellular calcium. The decay of the

ICAN tail current was fitted with an exponential function, andresulting time constants � were plotted against tail currentvoltage. We tested whether there was any voltage dependenceof the deactivation rate. The current tended to decay somewhatmore rapidly at voltages more depolarized than �60 mV, butthere was no statistically significant difference between �values at different voltages, with an average � of 74 � 14 ms.

As expected for ICAN, the tail current was sensitive toapplication of FFA or BAPTA or block of calcium entry byCdCl2. In the high-osmolarity lobster saline, FFA visiblyprecipitated out of solution at 16°C; thus we raised the exper-imental temperature to 21°C to maintain the drug in solutionfor full dose-response experiments. FFA blocked ICAN with anIC50 of 24.7 � 5 �M, (n � 4; Fig. 5C). Preventing a rise inintracellular calcium with 50–70 �M BAPTA completelyblocked ICAN. Finally, blockade of voltage-dependent calciumcurrents with 0.6 mM CdCl2 eliminated the step-activatedICa(V) and thus the resulting ICAN (not shown).

We next measured DA’s effect on ICAN in the AB neuron.The mean peak amplitude of ICAN was increased in the pres-ence of DA (Fig. 5D, thick black trace, arrowhead). For alleight neurons tested, this effect of DA was statistically signif-icant at voltages more hyperpolarized than �45 mV (P � 0.05;Fig. 5E). This ICAN enhancement occurred despite DA’smarked reduction of the amplitude of ICa(V) (by 68 � 7%; Fig.5D, thick black trace, arrowhead; see also Johnson et al. 2003).The maximal CAN conductance in the AB neuron significantly

Fig. 5. Calcium-activated inward current ICAN is a likely targetof DA modulation. A: flufenamic acid (FFA) abolishes DAoscillations in a synaptically isolated AB neuron. Top: the ABneuron was isolated by photoablation, and bursting wasevoked with 10�4 M DA. Bottom: the AB neuron was isolatedfrom modulatory and synaptic input by application of 10�7 MTTX � 5 � 10�5 M PTX, and oscillations were evoked with10�4 M DA. In both cases, 3 �M FFA stopped oscillationswithin 30 min. B: properties of ICAN in the AB neuron: thecurrent is measured during voltage-clamp recordings as aslowly deactivating tail current evoked by an activatingprestep to 0 mV and a series of hyperpolarizing steps (inset atright, voltage protocol). Arrowheads on the current tracesmark the peak voltage-gated calcium current [ICa(V)] activatedby the depolarizing prestep and the peak ICAN tail currentsmeasured after repolarization of the cell. C: dose-responsecurve for FFA block of ICAN (IC50 � 24.7 � 5 �M; n � 4).The amplitude of the blocked current was normalized by thecorresponding control current amplitude. D: DA enhances theamplitude of ICAN while inhibiting ICa(V). Voltage-clamp cur-rent traces under control conditions (thin black trace), duringapplication of 10�4 M DA (thick black trace), and after 30min of washout (thin gray trace). DA inhibited ICa(V) whileenhancing ICAN amplitude (arrowheads). Current traces arenot leak subtracted. The voltage protocol used to mea-sure ICAN is shown in the small inset. E: ICAN current-voltagerelationship under control conditions (C, open squares), in thepresence of DA (filled circles), and after washout (W, graytriangles). All data points are means � SE. Asterisks mark thevoltage steps where the difference between control and DAwas statistically significant (n � 8, P � 0.05, Student’s t-test).

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increased from 58 � 9 nS under control conditions to 145 � 12nS in the presence of DA (248 � 15% of control; n � 8, P �0.05). We also tested DA’s effects on ICAN in two other pyloricnetwork neurons, the PD, which is electrically coupled to theAB neuron, and the LP neuron. DA did not modify ICANamplitude in the PD (105.8 � 25% of control; n � 3) or the LP(103.1 � 29% of control; n � 3) neurons.

The time course of decay of ICAN most likely reflects thereduction in availability of free calcium ions in the intracellularspace near channels mediating ICAN. Consistent with this,within each treatment (control, DA, and washout) there was novoltage dependence of the time constants of decay (ANOVA,P � 0.05, n � 7 for each treatment; not shown). However,when we compared the mean time constants between treat-ments (control and DA), the time constant in DA (150 � 21ms) was significantly slower compared with control (74 � 14ms) at �65 mV and more depolarized potentials (P � 0.05,n � 6). Thus, despite the fact that DA inhibits most of theICa(V), the ICAN in DA is larger and deactivates more slowlythan before DA application.

DA effects on other ionic currents. As reported previously(Peck et al. 2001), DA also reduced the leak current, measuredwith 5-mV hyperpolarizing steps from �60 mV or �55 mV.Under our experimental conditions, the mean conductance ofthe leak current at �6 5 mV was 45 � 17 pS in control, 11 �8 pS during DA application, and 41 � 23 pS after DA washout.This �70% reduction in leak current in the presence of DA islarger than the 10% reduction we previously reported in the AB(Peck et al. 2001, 2006).

DISCUSSION

Our results suggest that DA-induced AB bursting dependson the release of calcium from intracellular stores, which inturn enhances ICAN to provide the additional ramp depolariza-tion leading to the onset of each oscillation. Blockade of eitherof these steps eliminates or prevents pacemaker oscillations.

Dopamine-induced rise in intracellular calcium is centralfor pacemaker oscillations. Many bursting neurons rely onintracellular calcium stores as a source of calcium in bothinvertebrates (Levy 1992; Partridge and Swandulla 1987; Par-tridge and Valenzuela 1999; Sawada et al. 1990; Swandullaand Lux 1985; Yazenian and Byerly 1989) and vertebrates (DelNegro et al. 2008; Pace et al. 2007a, 2007b; Peña et al. 2004;Rubin et al. 2009). We show that in the pyloric pacemaker ABneuron chelation of calcium ions or block of calcium releasefrom the intracellular stores prevents or eliminates ongoingDA-evoked pacemaker oscillations. Calcium imaging of theAB neuron with a ratiometric dye shows that DA elevates basalcalcium levels in the neurites by more than twofold over thequiescent state (67 � 3 nM pre-DA, 193 � 17 nM in DA). Weare able to detect this DA-induced elevation of [Ca2�]in be-ginning more than a minute and a half before the emergence ofsustained voltage oscillations (Fig. 3A) and, in some cells, evenbefore the neuron starts to depolarize.

During AB oscillations, fast line scanning of neurites filledwith Calcium Green-1 shows that the intracellular calciumlevel oscillates with amplitude of �20% of the basal level ofcalcium in the presence of DA and with the same frequency asthe voltage oscillations. However, the calcium oscillations aredelayed from the voltage oscillations by �150 ms (0.08 of the

period). One interpretation of this result is that a tonic modu-lator-evoked release of calcium from intracellular stores acti-vates ICAN, which acts as a major contributor to the tonic rampcurrent, depolarizing the membrane voltage; above a voltagethreshold, this depolarization activates the small DA-insensi-tive portion of ICa(V), which then participates (possibly alongwith ICAN) in the rapid rise of the oscillation to its peak. Theessential role of calcium release from intracellular stores ismaintained even during stable oscillations, since Ry blockadeof calcium-induced calcium release abolishes these ongoingcalcium oscillations along with voltage oscillations (Fig. 4D).

Cross talk between IP3 and Ry receptors and their respectivepathways. The ER calcium stores appear to play a critical rolein DA-induced AB bursting, since blockade of Ry-sensi-tive and IP3 receptors or depletion of stores by blockade of theSERCA inhibits this bursting. The mechanisms by which DAenhances calcium release from these stores are not yet known.Clark and colleagues cloned three types of DA receptors fromthe lobster STG: D1�Pan coupled with Gs and Gq, D1�Pancoupled with Gs, and D2�Pan coupled with Gi proteins (Clarkand Baro 2006, 2007; Clark et al. 2008). All three receptortypes were reported to localize to the synaptic neuropil (Clarket al. 2008), where we made our calcium measurements. Invertebrates, DA can trigger mobilization of intracellular cal-cium through concurrent activation of D1 and D2 receptors ora D1–D2 heteromer, via activation of Gq, phospholipase C(PLC), and the IP3 cascade (Hasbi et al. 2010; Undie et al.1994), which in turn provides calcium to activate furthercalcium release via the RyRs. In Drosophila photoreceptors,this signaling cascade leads to calcium release from IP3Rs andcalcium influx across the cell membrane via two types of TRPchannels (Hardie and Minke 1993). Thus, in the AB neuron,DA most likely acts on the D1�Pan receptor or its heteromer toactivate the Gq-PLC-IP3 pathway.

In addition, DA could also activate D1�Pan or D1�Pan, whichare coupled to the Gs-cAMP-PKA pathway (Clark et al. 2008),leading to increased phosphorylation of RyR protein and cal-cium release from the ER, as demonstrated in heart sinoatrialnode pacemaker cells (Vinogradova et al. 2006). Since bothtypes of ER receptor channels are sensitive to calcium, they aresubject to positive autofeedback, leading to regenerative open-ing of RyRs and/or IP3Rs beyond a certain threshold of[Ca2�]in and amplification of the initial calcium signal(Verkhratski 2005). Replenishment of the intracellular calciumstores is critically important to prolonged DA bursting; thismay occur via ICAN, ICa(V), and/or store-operated calcium entry(SOCE). Although we did not test this directly in the ABneuron, in rat hepatocytes SOCE appears to be a more effectiveway of refilling the stores than ICAN (Gregory et al. 2003).

The FFA-sensitive ICAN is enhanced by DA and contributesto sustained pacemaker oscillations. We showed that ICANplays an important role in DA-induced AB bursting. First,FFA terminated ongoing dopamine-evoked oscillations andblocked ICAN with an IC50 of 24 �M (Fig. 5A). FFA isroutinely used at 100 –500 �M to reduce ICAN in bothinvertebrates and vertebrates (Derjean et al. 2005; Ghamari-Langroudi and Bourque 2002; Green and Cottrell 1997; Leeand Tepper 2007; Morisset and Nagy 1999; Pace et al.2007a; Partridge and Valenzuela 2000). At these high con-centrations, above 100 �M, FFA has multiple side effects,including partial inhibition of calcium channels and NMDA

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receptors (Wang et al. 2006) and induction of calciumrelease from ER (Gardam et al. 2008; Lee et al. 1996; Shawet al. 1995) and mitochondria (Tu et al. 2009). We avoidedthese side effects by using much lower concentrations (be-low 10 �M). In our system, FFA block causes the membranepotential to stabilize around �40 to �42 mV (Fig. 5A), nearthe upper voltage limit of the slow oscillatory wave. Thisend-point depolarization was also seen in the experiments inwhich we blocked AB oscillations by disruption of intracel-lular calcium dynamics (Fig. 1). While we do not understandthe basis for this depolarization, it may be the result ofcalcium compartmentalization into separate domains andlimiting the calcium supply to calcium-activated potassium[K(Ca)] channels that normally stabilize the potential near itsresting state. This was not the main cause of the cessation ofbursting, as the AB neuron also failed to burst upon artificialhyperpolarization to its initial resting potential.

A second argument for the role of ICAN in DA-evoked ABbursting is that DA significantly enhances this current’s max-imal conductance and slows its deactivation rate. The ICANpeak amplitude almost doubled during DA application (Fig. 5,D and E), despite DA inhibition of ICa(V) (Johnson et al. 2003),which would normally act to reduce ICAN. Monoamine-inducedmodulation of ICAN was previously described in the AplysiaR15 neuron (Lotshaw et al. 1986) and Helix bursting neurons(Partridge et al. 1990), as well as in the DG neuron of theCancer STNS (Zhang et al. 1995). In the AB neuron, DA couldmodulate ICAN either directly, via second messenger-mediatedmodulation of these channels, or indirectly by elevating intra-cellular calcium levels, which would interact cooperativelywith calcium entering from the extracellular space; our datasuggest that at least this second mechanism occurs. Alterna-tively, DA could slow the rate of reuptake of calcium intointracellular stores after entering via voltage-activated chan-nels, thus increasing the maximal ICAN and slowing its deac-tivation rate.

Model of DA-induced bursting of the lobster pyloric pace-maker neuron. Based on previous work and our data, wesuggest the following model of DA-induced bursting in ABneurons (Fig. 6).

1) DA binds to D1�Pan and/or D1�Pan receptors and in-creases the intracellular [Ca2�] in the fine neuropil. The ERcalcium channels and the SERCA play a major part in thisprocess. IP3Rs are likely activated via the Gq-PLC-phospha-tidylinositol 4,5-bisphosphate (PIP2) pathway with a subse-quent activation of additional IP3Rs and RyRs as calcium isreleased from the ER.

2) This rise in intracellular calcium activates ICAN. DAincreases the peak current amplitude and slows the rate ofdeactivation of the current, probably due to elevated levels ofcytoplasmic calcium, although a direct modulatory action onthe channels is also possible.

3) ICAN activation acts as a ramp current in addition to Ih todepolarize the cell after the termination of the previous burst.This ramp brings the cell to voltage threshold to activate otherinward currents possibly including the DA-insensitive portionof ICa(V) to initiate additional calcium entry and the full burst.This additional influx of Ca2� could also recruit more ICANduring the burst phase to contribute to the burst itself.

4) Depolarization and high intracellular calcium activatecalcium-dependent outward currents, such as IK(Ca), which

must have either slower activation kinetics or a higher calciumconcentration dependence compared with ICAN. Other currents,such as sodium-sensitive potassium currents [IK(Na)] or pumpcurrents, may also contribute to this accumulation of outwardcurrents. The outward currents eventually prevail over theinward currents driving the depolarization; this repolarizes thecell, ending the cycle.

5) Extracellular calcium is essential for replenishment of theintracellular calcium stores and is carried inside via ICAN,ICa(V), and/or SOCE.

6) In addition to these effects, previous work in our labora-tory has shown that DA also reduces the tonic leak current,which will make the neuron electrically tighter and morecompact, enhancing the effects of the rather small ICAN andother subthreshold currents (Fig. 6B). DA reduces the transientpotassium current (Peck et al. 2001), which would normallycounteract the ramp ICAN current. DA also enhances the hy-perpolarization-activated inward current Ih (Peck et al. 2006),which would also contribute to the depolarizing ramp drivingregenerative oscillations in AB. Paradoxically, DA also en-hances a slowly activating and deactivating inward-rectifyingIK(V) (Gruhn et al. 2005), which may act to restrict the maximalspike frequency of the oscillating AB neuron (Harris-Warrickand Johnson 2010; Fig. 6).

Multiple pacemaking mechanisms. There are multiple pos-sible ionic mechanisms to generate bursting in neurons, and therelative contribution of each may vary under different condi-tions (Harris-Warrick and Flamm 1987; Harris-Warrick andJohnson 2010; Peña et al. 2004). In the vertebrate respiratorycentral pattern generator (CPG), there may exist differentgroups of pacemaker neurons, each with a distinct rhythmo-

Fig. 6. Multiple effects of DA on the ionic currents in AB neuron.A: schematics of the changes in the voltage and [Ca2�] during DA application,based on our results with indo-1 and Calcium Green-1 imaging. B: summaryof DA effects on AB neuron. DA inhibits transient potassium current IA, leakcurrent (Ileak), and ICa(V), enhances IK(V) and hyperpolarization-activated cur-rent Ih, and increases intracellular [Ca2�]. C: mechanism of the DA-inducedoscillations in the AB neuron (see DISCUSSION for the description of themechanism of action of DA). IP3R, inositol trisphosphate receptor; RyR, Ryreceptor.

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genic mechanism—either sodium dependent or calcium de-pendent (Del Negro et al. 2002; Pace et al. 2007a, 2007b; Peñaet al. 2004). Our work shows how DA evokes AB bursting bya calcium-dependent mechanism (Harris-Warrick and Flamm1987). However, the same AB neuron uses a completelydifferent mechanism to induce bursting in the presence of5-HT, which is driven by a sodium-dependent mechanism(Harris-Warrick and Flamm 1987; Kadiri and Harris-Warrick,unpublished observations). Thus if in the respiratory CPG theremay be different groups of pacemaker neurons in the sameCPG, in the pyloric CPG a single pacemaker neuron canemploy two quite distinct ionic mechanisms to drive rhythmicbehaviors.

ACKNOWLEDGMENTS

We thank B. R. Johnson and W. R. Zipfel for their feedback on theexperiments.

Present addresses: L. R. Kadiri, Cold Spring Harbor Laboratory, OneBungtown Rd., Cold Spring Harbor, NY 11724; A. C. Kwan, Division ofNeurobiology, Department of Molecular and Cell Biology, Helen Wills Neu-roscience Institute, University of California, Berkeley, CA 94120.

GRANTS

This work was supported by National Institutes of Health (NIH) predoctoralgrant T32 GM-007469 to L. R. Kadiri, NIH Grant 9-P41-EB-001976 to W. W.Webb, and NIH Grant NS-17323 to R. Harris-Warrick.

DISCLOSURES

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

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