155

Distributed Effects of DopamineModulation in the Crustacean Pyloric Networka

RONALD M. HARRIS-WARRICK,b BRUCE R. JOHNSON,

JACK H. PECK, PETER KLOPPENBURG, AMIR AYALI, AND

JACK SKARBINSKI

Section of Neurobiology and Behavior, Seeley G. Mudd Hall, CornellUniversity, Ithaca, New York 14853, USA

ABSTRACT: It is now clear that neuromodulators can reconfigure a single motor net-work to allow the generation of a family of related movements. Using dopamine mod-ulation of the 14-neuron pyloric network from the crustacean stomatogastricganglion as an example, we describe two major mechanisms by which network out-put is modulated. First, the baseline electrophysiological properties of the networkneurons can be altered. Dopamine can affect the activity of each neuron indepen-dently. For example, DA modulates IA in nearly every neuron in the pyloric network,but in opposite directions in different cells. Furthermore, DA usually modulates com-binations of ionic currents. In some cases, currents with opposing actions on cellexcitability are simultaneously affected, and the net response reflects the sum of theseopposing effects. Second, neuromodulators can alter the strength of synaptic interac-tions within the network, quantitatively “rewiring” the network. Every synapse in thenetwork is affected by DA, with some increased and others decreased in strength. DAacts both pre- and postsynaptically to affect transmission: these actions are fre-quently opposing in sign, and the net response arises as the sum of these opposingactions. Finally, spike-evoked and graded transmission at the same synapse can beoppositely affected by DA. These results emphasize the distributed nature of modu-lation in motor networks.

To survive, an animal must be able to alter its behavior to choose the appropriate vari-ant of a basic movement. Simple rhythmic behaviors, such as locomotion, are orga-

nized by central pattern generator (CPG) networks, which coordinate the signals for thetiming, phasing, and intensity of motoneuron activity to drive the behavior.1–3

Neuromodulatory inputs from outside the CPG, using monoamines, peptides and othercompounds, can reconfigure a CPG to allow the same anatomically defined circuit to gen-erate a family of related behaviors.1,4 – 6 In addition, neuromodulators can modify thestrength of interactions between separate CPGs to build up more complex conjoint net-works driving multicomponent behaviors.6 –9

The cellular mechanisms underlying circuit reconfiguration are poorly understood andare a major field of research in neuroethology.3,10 –12 In this paper, we describe our generalconclusions from a series of experiments on how dopamine modulates the motor patterngenerated by the pyloric network in the crustacean stomatogastric ganglion (STG). Theseexperiments show that nearly all the cellular and synaptic components that form this

aThis work was supported by NIH grants NS17323 and NS35631 and USDA Hatch grant 191310.bCorresponding author; e-mail: rmh4@cornell.edu

network are modified by dopamine. Often, dopamine has opposing effects on differentcomponents of the network. The final motor pattern is thus a net population response ofthe network to these many individual modulatory actions.

THE PYLORIC NETWORK IN THE SPINY LOBSTER STOMATOGASTRIC GANGLION

The STG is a small ganglion of 30 neurons that controls rhythmic movements of theforegut in crustaceans.13,14 It contains two entire CPGs, the slow gastric mill network andthe pyloric network that controls rhythmic pumping and filtering movements of the poste-rior region of the foregut. The pyloric network contains only 14 neurons, all of which canbe unambiguously assigned to one of six major classes.15,16 With intact modulatory inputsfrom higher ganglia, the pyloric network generates a rhythmic motor pattern with a cyclefrequency between 0.5 and 2 Hz; each neuron fires bursts of action potentials at a charac-teristic phase and intensity in the cycle (FIG. 1). The neural network generating this motorpattern is fully mapped, thanks primarily to work from the laboratories of Allen Selverstonand Eve Marder.17,18 FIGURE 1A shows a simplified 4-cell version of the neural network(representing 12 of the 14 neurons in the network), along with recordings from the fourmajor neurons (FIG. 1B) and a phase diagram that shows when each neuron is active in acycle (FIG. 1C). The neurons communicate by electrical coupling (both rectifying and non-rectifying) and chemical inhibition, using either acetylcholine or glutamate as their chem-ical transmitters.19

How does the network in FIGURE 1A generate the motor pattern in FIGURE 1B?Research from many labs suggests the following simplified summary.16 The anteriorburster (AB) and two pyloric dilator (PD) neurons are conditional bursters, capable of gen-erating rhythmic oscillations and bursts of action potentials in the presence of appropriateneuromodulators. They are electrically coupled and function as the major pacemakers forthe network because of their rapid oscillatory properties. They inhibit all the follower cells,including the lateral pyloric (LP) and eight pyloric constrictor (PY) neurons. At the end ofthe AB/PD burst, the follower cells recover from inhibition by postinhibitory rebound andstart firing again, but at different times: the LP recovers more rapidly and fires at an ear-lier phase than the PY neurons (FIG. 1C). Eventually the PY neurons start firing and inhibitthe LP cell; the PY neurons continue to fire until inhibited by the AB/PD burst at the begin-ning of the next cycle. Thus, the basic pyloric rhythm is determined by a complex inter-action between the pattern of synaptic connectivity (e.g., the pacemakers inhibit thefollower cells) and the intrinsic firing properties of the component neurons (e.g., the pace-makers have rapid rhythmic bursting properties, and the LP neuron has more rapid postin-hibitory rebound than the PY neurons).

DOPAMINE MODULATION OF THE PYLORIC MOTOR PATTERN

Dopamine (DA) is present in axons entering the STG from anterior ganglia,20,21 and,when bath applied, has dramatic modulatory effects on the pyloric motor pattern22,23 (FIG.1D–F ). Virtually every parameter of the motor pattern is altered: (1) An overall decreasein cycle frequency occurs. (2) There are changes in the intensity of neuronal activity, withPD and VD decreasing their firing frequency (and sometimes falling silent altogether)while the remaining neurons are excited and fire more vigorously. (3) There are changesin the phasing of activity of the different cells, with PD and VD phase delayed while LP,PY, and IC are phase advanced. These changes arise from both alterations in the intrinsic

156 ANNALS NEW YORK ACADEMY OF SCIENCES

firing properties of the network neurons and changes in the strength of synaptic interac-tions that form the network. We will discuss each of these major actions in turn.

HARRIS-WARRICK et al.: DOPAMINE MODULATION 157

FIGURE 1. The pyloric network and its response to dopamine. (A) Simplified 4-cell pyloric net-work. Names of neurons are given in the text. Chemical inhibitory synapses are noted by filled cir-cles, electrical coupling by resistor symbols, and rectifying electrical coupling by diode symbols. (B)Simultaneous recordings from AB, PD, LP, and PY neurons with modulatory inputs via stomatogas-tric nerve intact. (C) Phase diagram of action potential activity of the four neurons, with the firstaction potential in AB defined as phase 0. (D) Altered pyloric network in presence of 10-4 Mdopamine. Strengthened synapses are shown in darker lines, weakened synapses by dashed lines, andsilenced synapses are not shown. (E) Activity of the four neurons in the presence of 10-4 Mdopamine, from the same preparation as B. Voltage and time markers for B and E: 10 mV, 1 s. (F)Phase diagram of action potential activity of the four neurons during dopamine application. (FromHarris-Warrick et al.33)

DOPAMINE MODULATES THE INTRINSIC FIRING PROPERTIES OF EVERYNEURON IN THE NETWORK, BUT IN DIFFERENT WAYS

A distinct advantage of the STG preparation is the ability to isolate a neuron or a synap-tically interacting subset of neurons from the rest of the circuit in situ by a combination oftetrodotoxin (TTX) block of inputs carried by axons from other ganglia, photoinactivation ofpresynaptic neurons, and pharmacological blockade of selected synapses.24,25 Thus, we canstudy each neuron or synapse in isolation and determine its baseline properties and directresponses to neuromodulators. Dopamine directly affects every neuron in the network.26,27

The AB neuron becomes an endogenous burster, whereas the LP, PY, and IC neurons depo-larize and fire tonically. Recent work has shown that the isolated LP neuron and some of thePY neurons exhibit slow oscillations and bursting during application of DA if a hyperpolar-izing bias current is injected (J. Peck, B. Johnson, and A. Ayali, unpublished data). However,because these oscillations are very slow compared to the normal cycle frequency of thepyloric network, the LP and PY cells can be considered as tonically firing neurons whoseactivity is shaped by periodic synaptic inhibition. In contrast to these excitatory actions, DAinhibits the PD and VD neurons, which fire fewer spikes and occasionally fall completelysilent. In summary, DA does not have a single major mechanism of action in the pyloric net-work. Rather, it affects all the neurons in different ways to shape the motor pattern.

DOPAMINE MODULATES THE SAME POTASSIUM CURRENT, IA,IN NEARLY EVERY PYLORIC NEURON, BUT IN DIFFERENT DIRECTIONS

AND BY DIFFERENT MECHANISMS

The transient potassium current, IA, is a low-voltage-activated outward current that islargely inactivated at rest. Inactivation is partially removed by hyperpolarization duringsynaptic inhibition, and the channels then open transiently in the subthreshold voltagerange.28 IA plays an important role in determining the cycle frequency, spike frequency,and phasing of activity of the follower cells in the pyloric network.29,30 DA modulates theproperties of IA in every pyloric neuron we have studied, but in different ways (TABLE 1).The PD and VD neurons are inhibited by DA, which acts in part by enhancing IA. In thePD neuron,31 DA enhances the maximal conductance of IA and shifts its voltage activationcurve in the negative direction with no effect on its voltage inactivation curve (FIG. 2A).As a consequence, the “window” current of tonically active IA is enhanced about two- tothreefold around the resting potential. This contributes a net outward current to the restingcurrents and helps to hyperpolarize the PD neuron during DA application.

In contrast, the LP, PY, and AB neurons are excited by DA, which decreases IA in all threecell types (TABLE 1). IA inactivates in a double exponential manner, with rapidly and slowlyinactivating components. In the PY cell, DA reduces the maximal conductance of IA byselectively reducing the maximal conductance of the slowly inactivating component with noeffect on the rapidly inactivating component. In addition, DA shifts the voltage activation

158 ANNALS NEW YORK ACADEMY OF SCIENCES

TABLE 1. Known Ionic Targets of Dopamine on Pyloric Neurons

Cell Known Ionic Targets

AB ↓ IA

PD ↑ IA, ↑IO(CA)

LP ↓ IA, ↑ Ih

PY ↓ IA

VD ↑ IA, ↑ Ih

HARRIS-WARRICK et al.: DOPAMINE MODULATION 159

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and inactivation curves for IA in the positive direction (FIG. 2B). As a consequence of DA’sactions in the PY cell, there is less total IA to be activated and it requires more depolarizationto activate it; therefore there is less “window” current active at the resting potential, and thecurrent inactivates more rapidly than under control conditions.32 DA also decreases IA in theLP neuron, but both rapidly and slowly inactivating components of the current are reduced.33

Thus, although IA is decreased in both LP and PY neurons, the detailed biophysical mecha-nisms of the decrease are different.

In conclusion, IA is a common target of modulation by DA in nearly all the pyloric neu-rons, but DA alters IA by different mechanisms in different cells (TABLE 1). These distinctresponses could result from different DA receptors, second messenger mechanisms,34 ormolecular forms of IA channels that are modulated by DA in each cell type.35 Indeed, wehave shown that IA has different kinetic and voltage dependence properties in the differentpyloric neurons (Baro et al., this volume).

DOPAMINE MODULATES MULTIPLE IONIC CURRENTS IN MANYPYLORIC NEURONS, AND THESE SOMETIMES HAVE OPPOSING

EFFECTS ON THE NEURON

Although DA modulates IA in virtually every pyloric neuron, it also modulates addi-tional currents in some of the neurons (TABLE 1). This is expected if DA acts via a secondmessenger mechanism which could simultaneously affect multiple targets.34 For example,DA inhibits the PD neuron via at least two separate mechanisms: (1) enhancement of IA, asdescribed above and (2) enhancement of a calcium-dependent outward current, IO(Ca).

31 IO(Ca)

is activated with larger depolarizations than IA and probably plays a role in regulating spikefreqency. Its enhancement by DA may thus contribute to the reduction in spike frequencyof the PD neurons.

In contrast to inhibition of the PD, DA excites the LP neuron via at least two conduc-tance changes: (1) the decrease in IA described above and (2) an enhancement of Ih, a slowhyperpolarization-activated inward current, whose voltage activation curve is shifted in thepositive direction by DA.33 Both of these actions contribute to the depolarization of LP.However, modeling and dynamic clamp experiments show that reduction of IA is the pre-dominant mechanism for DA’s excitation of the LP neuron.

The isolated VD neuron fires tonically, and often gives a biphasic response to a briefpuff of DA, with an initial reduction followed by a prolonged enhancement of spontaneousspike frequency (FIG. 3; J. Skarbinski, unpublished data). The initial inhibition is reducedby bath application of the IA blocker, 4-aminopyridine (4-AP), and is mediated by anenhancement of IA (J. Peck, unpublished data). In contrast, the delayed enhancement isabolished by low concentrations of extracellular Cs+ and results from an enhancement ofIh. DA shifts the Ih voltage activation curve to the right and increases its activation rate.Thus, DA acts on IA and Ih to both inhibit and excite the VD neuron. When DA is bathapplied, the net effect is usually inhibition, with a decrease in spike frequency. However,we have occasionally observed cells with little net response or a biphasic response to DA.These variants could reflect cell-specific differences in the net strength of the excitatoryand inhibitory responses to DA.

As summarized in TABLE 1, these biophysical studies show that dopamine affects aunique palette of ionic currents in each cell. It is almost certain that DA affects additionalcurrents in these cells that we have not yet detected. Some of these currents may be pref-erentially expressed at synaptic sites, and cannot be readily measured from our somaticvoltage clamp studies.

160 ANNALS NEW YORK ACADEMY OF SCIENCES

DOPAMINE MODULATES THE STRENGTH OF EVERY SYNAPSE IN THEPYLORIC NETWORK, BUT IN VARIED AND SYNAPSE-SPECIFIC WAYS

The second major mechanism for neuromodulators to generate flexible outputs from aneuronal circuit is to alter the strengths of synaptic interactions that form the network. Inthe pyloric network, neurons interact by both spike-evoked transmission and graded synap-tic transmission, where transmitter release varies as a continuous function of membranepotential and release can occur even at the resting potential.36,37 We have studied the effectsof DA and two other monoamines, serotonin and octopamine, on graded synaptic trans-mission between isolated cell pairs in the pyloric network.38– 41 Every synapse in the net-work was reproducibly affected by DA, but in different directions and to different extents(FIGS. 4 and 5). Many synapses were strengthened whereas others were weakened (FIG. 4Aand B). A few synapses fell silent and became nonfunctional during DA application. Forexample, the PD neuron output synapses cease to effectively inhibit their follower neuronsduring DA application (FIG. 4C). In contrast, some synapses appear to be functional onlyin the presence of appropriate neuromodulators such as DA. For example, the PY→LPchemical synapse falls silent when the cell pair is isolated from modulatory inputs, but isreactivated when DA is applied (FIG. 4D). Electrical coupling can also be modulated, andis usually decreased by DA (FIGS. 4E and 5). Some neurons interact by a combination ofchemical inhibition and electrical coupling; DA can have opposite modulatory effects oneach synaptic component, effectively reversing the net sign of the synaptic interaction. Forexample, as seen in FIGURE 4F, under control conditions the PY cell is electrically coupledto the LP while a chemical inhibitory synapse from PY→LP is silent; thus when the PY isdepolarized, the LP neuron also depolarizes. Application of DA markedly weakens theelectrical coupling while strengthening the chemical inhibition, so that when the PY isdepolarized, the LP neuron then hyperpolarizes.42

HARRIS-WARRICK et al.: DOPAMINE MODULATION 161

FIGURE 3. Effect of dopamine on isolated VD neuron. The VD neuron was isolated from all synap-tic input, and fired tonically at about 2.5 Hz; instantaneous spike frequency given in Y axis. At thearrow, a 2-s puff of 10-3 M DA was applied. The initial reduction in tonic spike frequency is blockedby 4 mM 4-AP and was shown by voltage clamp studies to result from an increase in IA. The delayedenhancement in tonic spike frequency is blocked by 5 mM CsCl and was shown by voltage clamp toresult from an increase in Ih. (Unpublished data of J. Skarbinski, J. Peck, and R. Harris-Warrick.)

These studies show that DA modifies the properties of all the synapses throughout thenetwork, enhancing some while weakening others (FIG. 5). It is not possible to point to asingle synapse as the most important target of DA action.

DOPAMINE CAN EXERT OPPOSING EFFECTS AT PRE- ANDPOSTSYNAPTIC SITES OF A SINGLE GRADED CHEMICAL SYNAPSE

In order to identify the cellular targets where DA acts to modify graded synapticstrength, we compared DA’s effects on isolated synapses and on the postsynaptic neuron’sresponse to iontophoretic applications of glutamate, the transmitter of four of the six pyloricneuron classes43 (FIGS. 5 and 6 ). In 5 of the 10 graded glutamatergic synapses, the postsy-naptic response to glutamate changed in a way that was compatible with the overall effectof DA on the synapse. For example, the glutamatergic LP→PY synapse was strengthenedby DA; consistent with this, the responsiveness of the PY neuron to glutamate was alsomarkedly strengthened (FIG. 6A and B). In part this enhancement arises from a DA-evokedincrease in the input resistance of the PY neuron (FIG. 5). However, in the other five gluta-matergic synapses, DA’s effects on postsynaptic glutamate responsiveness were not com-patible with DA’s net effects on the graded chemical synapse. For example, the LP→PDgraded synapse is markedly strengthened by DA, but the responsiveness of the PD neuronto glutamate is dramatically weakened (FIGS. 5 and 6A, B). There are at least two mecha-nisms for this postsynaptic reduction in glutamate responsiveness. First, Cleland andSelverston44 demonstrated that DA can directly reduce the glutamate receptor response inunidentified cultured STG neurons. Second, DA acts indirectly by significantly reducingthe PD input resistance,39 so current flow from the synapse to the recording site is reduced

162 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 4. Examples of the effect of 10– 4 M dopamine on synaptic interactions in the pyloric net-work. Each pair of neurons was isolated from all other synaptic input by a combination of TTX block-ade of stomatogastric nerve modulatory inputs, 6-carboxyfluorescein photoinactivation of selectedneurons, and pharmacological blockade of other synapses. Action potentials were eliminated with 10–7

M TTX. Postsynaptic responses to 1-s current injections in the presynaptic neuron are shown. (Datafrom refs. 38–41.)

(FIG. 5). Despite these postsynaptic effects, the LP→PD synapse is strengthened, and weconclude that DA must enhance the presynaptic graded release of transmitter sufficiently tooutweigh the reduced responsiveness of the postsynaptic cell. Similar disparities were seenat several other synapses, where the postsynaptic glutamate responsiveness was of theopposite sign to the net synaptic response. These opposite actions provide further flexibil-ity to the final network response to DA. If another neuromodulator were, for example, tomodify DA’s presynaptic effect on transmitter release without altering its postsynapticeffects, the net effect of added DA on that synapse could be reversed. Certainly many othermodulators are present in descending axons to the STG that could do this.10,45

DOPAMINE CAN HAVE OPPOSING EFFECTS ON GRADED AND SPIKE-EVOKED TRANSMISSION AT THE SAME SYNAPSE

As a partial consequence of these opposing actions of DA at pre- and postsynaptic sitesof a synapse, DA can have opposite effects on the strength of graded and spike-evokedchemical transmission at a single synapse (FIG. 6A and C). We have focused on the outputsynapses from the LP neuron onto the PD, VD, and PY cells.46 DA strengthens graded trans-mission at all three of these synapses (measured with step depolarizations in the presenceof TTX). In contrast, DA markedly reduces spike-evoked transmission from LP to the PDand VD neurons, while enhancing spike-evoked transmission at the LP→PY synapse. Thechanges in spike-evoked transmission are consistent with changes in the responsiveness ofthe postsynaptic neuron to the LP transmitter, glutamate, and the effect of DA on its input

HARRIS-WARRICK et al.: DOPAMINE MODULATION 163

FIGURE 5. Summary of the effects of dopamine on the pyloric network. Chemical inhibitorysynapses are given by filled circles; electrical and rectifying electrical coupling are given by resistorsand diodes, respectively. Neurons that are excited and synapses that are strengthened by 10-4 M DAare shown by bold circles and lines; neurons that are inhibited and synapses that are weakened by DAare shown in dashed circles and lines. Effects of DA on neuronal input resistance are given by circledsymbols within the neurons. Effects of DA on each neuron’s response to glutamate iontophoresis aregiven in the pipette symbols adjacent to each neuron. Effects of DA on release of glutamate, whereknown, are shown in the terminal circle of the synapse. (Adapted from Johnson and Harris-Warrick.43)

164 ANNALS NEW YORK ACADEMY OF SCIENCES

FIG

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resistance, which are summarized schematically in FIGURE 5. As described above, the PYglutamate response and input resistance are both increased by DA, consistent with DA’senhancement of LP→PY graded and spike-evoked transmission. The PD and VD neuronsshow reduced responses to glutamate which are caused in part by a reduced input resistanceduring DA. In the PD neuron, a linear relation exists between DA’s effects on postsynapticinput resistance and on the strength of spike-evoked transmission. Dopamine must enhancethe LP’s presynaptic graded release, but not spike-evoked release, to compensate for thesechanges in postsynaptic responsiveness so that all three graded synapses are functionallystrengthened.46 The molecular mechanisms underlying differential modulation of spike-evoked and graded release are not known.

CONCLUSION

Our experiments show that the effects of a neuromodulator on a CPG network are highlydistributed throughout the network. The final motor pattern is a population response of allof the network neurons and synapses to the actions of the modulator. We were surprised tofind how frequently DA has opposing effects on the activity of a cellular or synaptic com-ponent in the network. The overall frequency of the pyloric network is determined in largepart by the sum of opposing effects of DA on the AB and PD neurons in the pacemakergroup. The final firing pattern of the VD cell is determined by the sum of opposing changesin inward and outward currents. DA’s modulation of the strengths of many synapses in thenetwork reflect opposing actions on the pre- and postsynaptic cells. Dopamine can modu-late spike-evoked and graded synaptic transmission in opposite directions at a singlesynapse. Such opposing and apparently inconsistent actions are not unique to our system.In the leech, Lockery and Kristan47,48 studied changes in synaptic strength during sensitiza-tion of the bending reflex. They also found that nearly every synapse is altered in strength,and some of these changes were of the “wrong sign” for the overall response.

There are several possible reasons why DA has so many apparently conflicting effectson the pyloric network. One explanation is that the overall output from the network is thetarget of evolutionary selection, and some opposing effects are tolerated to allow theproper final outcome. Alternatively, these effects could themselves be altered by additionalmodulators to allow the sign of DA’s effects on a component of the network to bechanged.45 In addition, the opposing effects might have different dose-response relationsto DA, and may be differentially recruited as a function of the intensity of dopaminergicneuron activity.23 Finally, it is possible that DA is released differentially from severaldopaminergic neurons into different parts of the neuropil, and the response could dependon which dopaminergic neurons are active. In any case, it is likely that in other networksa neuromodulator may not act in an entirely consistent manner to evoke its final change innetwork output.

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