Neuromodulator-evoked synaptic metaplasticity within a...

Neuromodulator-evoked synaptic metaplasticity within a central patterngenerator network

Mark D. Kvarta, Ronald M. Harris-Warrick, and Bruce R. JohnsonDepartment of Neurobiology and Behavior, S. G. Mudd Hall, Cornell University, Ithaca, New York

Submitted 9 July 2012; accepted in final form 27 August 2012

Kvarta MD, Harris-Warrick RM, Johnson BR. Neuromodula-tor-evoked synaptic metaplasticity within a central pattern generatornetwork. J Neurophysiol 108: 2846–2856, 2012. First publishedAugust 29, 2012; doi:10.1152/jn.00586.2012.—Synapses show short-term activity-dependent dynamics that alter the strength of neuronalinteractions. This synaptic plasticity can be tuned by neuromodulationas a form of metaplasticity. We examined neuromodulator-inducedmetaplasticity at a graded chemical synapse in a model central patterngenerator (CPG), the pyloric network of the spiny lobster stomato-gastric ganglion. Dopamine, serotonin, and octopamine each producea unique motor pattern from the pyloric network, partially throughtheir modulation of synaptic strength in the network. We characterizedsynaptic depression and its amine modulation at the graded synapsefrom the pyloric dilator neuron to the lateral pyloric neuron (PD¡LPsynapse), driving the PD neuron with both long square pulses andtrains of realistic waveforms over a range of presynaptic voltages. Wefound that the three amines can differentially affect the amplitude ofgraded synaptic transmission independently of the synaptic dynamics.Low concentrations of dopamine had weak and variable effects on thestrength of the graded inhibitory postsynaptic potentials (gIPSPs) butreliably accelerated the onset of synaptic depression and recoveryfrom depression independently of gIPSP amplitude. Octopamine en-hanced gIPSP amplitude but decreased the amount of synaptic de-pression; it slowed the onset of depression and accelerated its recov-ery during square pulse stimulation. Serotonin reduced gIPSP ampli-tude but increased the amount of synaptic depression and acceleratedthe onset of depression. These results suggest that amine-inducedmetaplasticity at graded chemical synapses can alter the parameters ofsynaptic dynamics in multiple and independent ways.

graded synaptic transmission; synaptic depression; lobster pyloricneurons; dopamine; serotonin; octopamine

SHORT-TERM SYNAPTIC DYNAMICS, such as facilitation and depres-sion, alter synaptic strength in activity-dependent ways (Abbottand Regehr 2004). Synaptic dynamics have been studied mostextensively at synapses using action potential (AP)-evokedtransmitter release (Fioravante and Regehr 2011; Fisher et al.1997; Thomson 2000; Zucker and Regehr 2002). Transient,activity-dependent changes in synaptic strength can alter syn-aptic transmission to increase the response to novel stimuli,enhance directional sensitivity, adapt to repeated sensory stim-uli, and detect bursts of activity in sensory networks (Chacronet al. 2009; Fortune and Rose 2001; Klug 2011). They can alsocontribute to gain control and network stabilization in centralnetworks (Abbott and Regehr 2004) and help regulate motornetwork activity (Gelman et al. 2011; Li et al. 2007; Nadim andManor 2000; Parker and Grillner 2000; Sanchez and Kirk2002).

Chemical synapses using graded transmitter release, whererelease is a continuous function of presynaptic membranepotential, also show short-term dynamics (Goutman and Glo-watzki 2007; Jackman et al. 2009). The roles of graded syn-aptic dynamics have been analyzed in the pyloric network ofthe lobster stomatogastric ganglion (STG). In this 14-neuroncentral pattern generator (CPG) network, graded chemicalsynapses help determine the phasing of pyloric neurons toproduce coordinated motor patterns (Hartline et al. 1988). Thesynaptic strength in this network dynamically tracks activitylevels and thus alters network connectivity as a function ofcycle frequency (Mouser et al. 2008; Nadim and Manor 2000).Synaptic depression during pyloric network activity is thoughtto control the transition to different oscillatory modes of thenetwork (Nadim et al. 1999) and to promote phase mainte-nance over different cycle frequencies (Manor et al. 2003;Nadim et al. 2003).

Short-term synaptic plasticity can be regulated through neu-romodulation. This is a form of metaplasticity (Philpot et al.1999), where neuromodulators alter the characteristics of ac-tivity-dependent changes in synaptic strength (Fischer et al.1997; Kreitzer and Regehr 2000; Parker 2001; Parker andGrillner 1999; Qian and Delaney 1997). In the pyloric network,we have previously demonstrated that monoamines such asdopamine and octopamine can alter the steady-state synapticdepression at pyloric synapses (Johnson et al. 2005, 2011). Inaddition, the peptide proctolin can change the sign of synapticplasticity from depression to facilitation at a pyloric gradedsynapse to help stabilize the cycle period (Zhao et al. 2011).

In this article we examine amine modulation of synapticdynamics from the pyloric dilator (PD) neuron to the lateralpyloric (LP) neuron, the PD¡LP synapse. This synapse pro-vides an important output from the pacemaker kernel, whichsets the cycle frequency of its follower cells and thus helpsregulate network activity. The PD¡LP graded synapse showssynaptic depression when the PD neuron is driven with pre-synaptic square pulses (Graubard et al. 1983; Johnson andHarris-Warrick 1990) and when the PD neuron is artificiallydriven with realistic voltage waveforms that resemble its nor-mal activity (Rabbah and Nadim 2007). Graded synapticstrength at nondepressed PD¡LP synapses is reduced bydopamine and serotonin and enhanced by octopamine (Johnsonand Harris-Warrick 1990), but the effects of amines onPD¡LP synaptic strength under conditions of realistic rhyth-mic activity are unknown. Here we show that the three aminescan have independent effects on synaptic strength, the magni-tude and temporal dynamics of synaptic depression, and therecovery from depression.

Address for reprint requests and other correspondence: B. R. Johnson, Dept.of Neurobiology and Behavior, S. G. Mudd Hall, Cornell Univ., Ithaca, NY14853 (e-mail: [email protected]).

J Neurophysiol 108: 2846–2856, 2012.First published August 29, 2012; doi:10.1152/jn.00586.2012.

2846 0022-3077/12 Copyright © 2012 the American Physiological Society www.jn.org

MATERIALS AND METHODS

General Procedures

California spiny lobsters (Panulirus interruptus) were supplied byDon Tomlinson Commercial Fishing (San Diego, CA) and maintainedin marine aquaria at 16°C. After lobsters were anesthetized in ice, thestomatogastric nervous system (STNS) was removed and pinned in aSylgard-coated petri dish in chilled Panulirus saline of the followingcomposition (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 andSelverston 1974). The STG was desheathed, enclosed in a 1-ml poolwalled with Vaseline, and superfused at 5 ml/min with oxygenatedPanulirus saline. Experiments were performed at 19°C to enhance thestrength of graded synaptic transmission (Johnson et al. 1991). Do-pamine (DA; 10�5 M), octopamine (Oct; 10�5 M), and serotonin(5-HT; 10�5 M) were dissolved in saline just before application(amine conditions). If the amine effects did not reverse after a 15- to60-min wash, the experiment’s results were discarded. Amines werepurchased from Sigma Chemical.

Electrophysiological Recording and Cell Identification

Pyloric neuron activity was monitored by using pin electrodes torecord extracellular APs from appropriate motor roots (differentialAC amplifier, model 1700; A-M Systems) and intracellular electrodes(3 M KCl, 10–15 M�) to record from cell bodies in the STG. Weidentified pyloric neuron somata during ongoing rhythmic activityby matching the extracellularly recorded APs with intracellularlyrecorded APs, by the characteristic shape and amplitude of mem-brane potential oscillations and APs of pyloric neurons, and by theknown synaptic connections between pyloric neurons (Johnsonet al. 2011).

Isolation of Graded PD¡LP Synapse

Inhibitory glutamatergic synapses in the pyloric network wereblocked by 5 � 10�6M picrotoxin (PTX; Sigma Chemical) topharmacologically isolate the PD¡LP synapse (Bidaut 1980). Theanterior burster (AB) neuron, which is electrically coupled to thePD neurons, was photoinactivated by intracellular iontophoresis of5,6-carboxyflourescein and illumination with bright blue light(Miller and Selverston 1979). This eliminated any amine-inducedAB activity spreading to the PD neuron through the AB¡PDelectrical synapse. We waited at least 1 h after AB photoinactiva-tion to start our experiments, to allow the preparation to recoverfrom the acute effects of the light exposure (Flamm and Harris-Warrick 1986).

Dynamics of PD¡LP Graded Synaptic Transmission

LP graded inhibitory postsynaptic potentials (gIPSPs) were re-corded after 10�7 M tetrodotoxin (TTX; Tocris) was added to Panu-lirus saline to block APs. We studied the PD¡LP graded chemicalsynapse using two different presynaptic stimulation protocols. Thefirst used single, 3-s square pulses as voltage-clamp commands todrive the PD neuron, whereas the second used a train of 12 linkedrealistic PD waveforms as voltage-clamp commands to drive PDoscillations (see Johnson et al. 2005, 2011 for realistic waveformconstruction). Averaged and filtered PD waveforms from pyloricrecordings had a mean amplitude of 15.5 � 3.5 mV and a mean cycleperiod of 688 � 199 ms (n � 10); we used this shape and cycle periodas presynaptic stimuli. Although realistic presynaptic waveforms aremore physiologically relevant than square pulse waveforms, we usedboth stimulation regimes for two reasons. First, some basic charac-teristics of synaptic depression and its modulation at the PD¡LPsynapse can only be measured with square pulse stimulation, for

example, accurate measurements of the onset of synaptic depression(see below). Second, previous studies of graded synaptic transmissionbetween pyloric neurons have often used square pulse stimulation (forexample, Graubard et al. 1983; Johnson et al. 1995; Manor et al. 1997;Zhao et al. 2011), and we wanted to compare our results with theseearlier studies and determine whether postsynaptic responses differedbetween the two stimulus forms. Both stimulation protocols usedtwo-electrode voltage clamp to drive the PD from a holding potential(Vhold) of �55 mV in increasing amplitudes of 5-mV increments up to�25 mV. Stepping the PD up to �25 mV evokes near-maximumgIPSPs in the LP neuron (Johnson and Harris-Warrick 1990; Rabbahand Nadim 2007). We separated stimulation runs by a minimum of 30 s,which was sufficient to eliminate all effects of the previous stimula-tion. We used two-electrode current clamp to hold the postsynaptic LPmembrane potential at �50 mV; postsynaptic electrodes were filledwith 0.6 M K2SO4 � 0.02 M KCl and presynaptic electrodes with 3MKCl. In these experiments we used Axoclamp-2A and 2B amplifiers(Molecular Devices).

Under control and amine conditions (at least 5 min of aminesuperfusion), we measured the initial peak and steady-state amplitudesof the gIPSPs to calculate input-output curves and dynamics ofsynaptic depression (see Fig. 1, A and B). The steady-state amplitudewas measured as the gIPSP plateau level at the end of the square pulsestimulation or as the mean amplitude of the last five gIPSPs inresponse to the realistic PD waveform train. A synaptic depressionindex (DI) was calculated as the fractional gIPSP decrease from theinitial peak response at steady state: DI � (initial peak amplitude �steady-state amplitude)/initial peak amplitude. Thus a larger DI valuecorresponds to greater synaptic depression. The time constant (�Dep)of the gIPSP depression was measured during presynaptic squarepulse stimulation by fitting the voltage decay from the initial peak tothe steady-state amplitude with a single-exponential function (see Fig.1A). The rate of synaptic depression was rapid enough that it could notbe measured accurately from the separated peaks of the realisticwaveform series.

In a separate measurement, we quantified the time constant ofrecovery from synaptic depression (�Rec) for both realistic and squarewaveform stimulation under control and amine conditions. We evokedsteady-state depression by stimulating the PD neuron (Vhold �55 mV)as described above, followed by a single square pulse or realisticoscillation test stimulation at increasing times after the end of the lastconditioning stimulus. The percent recovery of the test pulse back tothe initial peak value over time was fitted with a single exponential ineach experiment to calculate �Rec (see Fig. 2, B and C).

Data Acquisition and Analysis

Electrophysiological recordings were digitized at 4 kHz using aPCI-6070-E board (National Instruments) and stored on a personalcomputer using custom-made recording software (Scope) written inLabWindows/CVI (National Instruments). This software also con-trolled the injection of oscillatory and square pulse waveforms asvoltage-clamp commands into the PD neuron (Johnson et al. 2005,2011). All data were analyzed with the related custom-made softwareprogram ReadScope, also written in LabWindows/CVI; these pro-grams were kindly provided by Dr. F. Nadim (http://www.stg.rutgers.edu/software/index.html). Data were exported to Clampfit software(Molecular Devices) to calculate �Dep and �Rec. For statistical com-parisons, we used JMP software (SAS Institute) to run multivariateanalysis, followed by post hoc tests to determine the statisticalsignificance of differences between individual data groups. Statisticaldifferences between mean values were accepted with P � 0.05(2-tailed probability) for F and t values. Mean measured values andpercentages are means � SD.

2847METAPLASTICITY AT A GRADED CHEMICAL SYNAPSE

J Neurophysiol • doi:10.1152/jn.00586.2012 • www.jn.org

http://www.stg.rutgers.edu/software/index.html

http://www.stg.rutgers.edu/software/index.html

RESULTS

Synaptic Dynamics of the PD¡LP Graded Synapse UnderControl Conditions

We first characterized the baseline synaptic dynamics of thePD¡LP graded synapse under control conditions, using single3-s square pulses (n � 10) and repeated presynaptic activationwith trains of 12 realistic oscillations (n � 7). As describedpreviously for square pulse stimulation of the PD neuron(Graubard et al. 1983; Johnson and Harris-Warrick 1990;Rabbah and Nadim 2007), above the threshold for graded PDtransmitter release (approximately �50 mV), the LP responseis a rapid initial peak hyperpolarization that rapidly depressesto a significantly smaller steady-state hyperpolarization (P �0.001 across PD depolarizations; Fig. 1, A and C). The LPresponds to a train of repeated PD oscillations with an initialpeak gIPSP followed by subsequent gIPSPs whose amplitudesdepress with repetition to a significantly smaller steady-statevalue over several oscillations (P � 0.001 across PD depolar-

izations; Fig. 1, B and D; see also Rabbah and Nadim 2007).Peak and steady-state gIPSP amplitudes increased with in-creasing PD voltage commands from �40 to �25 mV underboth stimulation protocols (Fig. 1, C and D). The amplitude ofthe initial peak LP gIPSP tended to be larger when elicited withPD square pulse stimulation than with realistic waveformstimulation (compare Fig. 1, C and D, open and filled circles),and this difference was significant during PD depolariza-tions to �30 and �35 mV (P � 0.03 and 0.05, respectively).However, the mean steady-state gIPSP amplitudes weresignificantly greater when elicited with oscillations com-pared with square steps at strong PD depolarizations (to�30 and �25 mV; compare Fig. 1, C and D, open and filledsquares; P � 0.05 and 0.03, respectively). Since squarepulse stimulation evoked larger peak initial gIPSPs andsmaller steady-state amplitudes than oscillation stimulation,it also evoked greater synaptic depression at all PD stimu-lation voltages (Fig. 1E; P � 0.001 at all PD voltages). Forexample, the mean DI was twice as large at PD depolariza-

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Fig. 1. Synaptic dynamics of graded synaptic transmissionat the PD¡LP chemical synapse. A: lateral pyloric (LP)graded inhibitory postsynaptic potentials (gIPSPs) in re-sponse to 3-s pyloric dilator (PD) neuron square pulsedepolarizations. Initial peak and steady-state IPSP ampli-tudes and the time constant for synaptic depression (�Dep)were measured for all PD depolarizations from a holdingpotential (Vhold) of �55 mV. B: LP gIPSPs during a trainof 12 realistic PD oscillations of increasing amplitudefrom Vhold � �55 mV. The first gIPSP amplitude (initialpeak) and the mean of the last 5 gIPSPs amplitudes (steadystate) were measured for all PD depolarizations. C: meaninput/output relationship for the amplitude of the initialpeak (open circles) and steady-state (open squares) gIPSPsduring square pulse PD stimulation (n � 11). D: meaninput/output relationship for initial (filled circles) andsteady-state (filled squares) LP gIPSP amplitudes duringPD oscillation stimulation (n � 7). E: mean depressionindex (DI) for square pulse (open bars) and oscillation(filled bars) stimulation across PD stimulation voltages.*Significantly greater DI for PD square pulse stimulationthan for PD oscillation stimulation (P � 0.001 for all PDvoltages). The DI did not show voltage dependence witheither stimulation protocol (P � 0.20 for both).

2848 METAPLASTICITY AT A GRADED CHEMICAL SYNAPSE


tion to �25 mV with the use of square waves compared withoscillations (Fig. 1E). This probably results from the greaterdepression during continued presynaptic stimulation thanduring trains of oscillations, where the synapse could par-tially recover between oscillations (see DISCUSSION). Therewas no significant effect of PD voltage on the magnitude ofthe depression index over the voltage range of either PDstimulation protocol (Fig. 1E; P � 0.23 and 0.21 for squarepulse and oscillations, respectively).

We also characterized the time constants of synaptic depres-sion and recovery from depression at the PD¡LP gradedsynapse using the square pulse series. The decay of the LPresponse to PD stimulation (Fig. 1A) was well fit by a singleexponential (fit correlation r � 0.99 � 0.001, n � 11). Themean time constant of synaptic depression, �Dep, was 400 �200 ms during PD square pulse steps to �25 mV. There was no

voltage dependence of �Dep during square step stimulation(P � 0.95; Fig. 2A). Because of the comparatively slow cyclefrequency of the oscillation stimulation (cycle period 688 ms), wewere unable to calculate the faster �Dep from the oscillation data.

In a separate measurement, we characterized the timeconstant of recovery from depression, �Rec, by using bothstimulation protocols (PD steps to �25 mV only) and fittingthe gIPSPs at increasing poststimulation intervals with asingle exponential (Fig. 2, B and C; square pulse: exponen-tial fit correlation r � 0.95 � 0.03; oscillation: fit correla-tion r � 0.98 � 0.01). Recovery from synaptic depressionwas over twice as fast with square pulse PD stimulation(n � 13) as with oscillation stimulation (n � 12; Fig. 2D;P � 0.003). This may result from increased mobilization ofthe recovery process during maintained presynaptic depo-larization (see DISCUSSION).

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Fig. 3. Dopamine (DA; 10�5 M) modulation of synapticstrength at the PD¡LP synapse. A–C: examples showingvariable effects of DA to reversibly enhance (A), reduce(B), or have no effect (C) on the initial LP gIPSP ampli-tude with PD square pulse stimulation. D: mean effects ofDA on amplitudes of initial peak and steady-state LPgIPSPs elicited by PD square pulse (open bars, initialpeak; light gray bars, steady state) and oscillation stimu-lation (solid bars, initial peak; dark gray bars, steady state)at �25-mV PD depolarization. E: example showing DAenhancement of the initial peak gIPSP during PD oscilla-tion stimulation. Ctl, control.

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Fig. 2. Synaptic depression and recovery at the PD¡LPgraded synapse. A: mean �Dep across PD square pulsestimulation voltages (n � 11). �Dep did not show anyvoltage dependence (P � 0.95). B: measurement of thetime constant of LP gIPSP recovery from depression(�Rec) in response to PD square pulse depolarization to�25 mV. C: measurement of �Rec in response to PDoscillation depolarization to �25 mV. D: mean �Rec forLP gIPSPs elicited by PD square pulse (n � 13) andoscillation stimulation (n � 10). *Significantly longer�Rec with PD oscillation stimulation (P � 0.003).



Amines Change the Synaptic Dynamics of the PD¡LPGraded Synapse

DA, 5-HT, and Oct affect synapses in the pyloric network bya complex set of pre- and postsynaptic actions (Harris-Warrickand Johnson 2010; Johnson and Harris-Warrick 1997). Inaddition, they can alter synaptic strength indirectly by modi-fying the waveforms of the pre- or postsynaptic neuronaloscillations (Johnson et al. 2005). To focus on synaptic meta-plasticity, we attempted to limit the other consequences ofamine modulation. For square pulse stimulations, we held thepresynaptic neuron at �50 mV by voltage clamp both beforeand during amine application. For oscillation stimulation, weused the realistic waveforms obtained from PD oscillationsduring the normal pyloric rhythm for both control and amineconditions. Thus the effects of amine-evoked changes in PDoscillations were eliminated. The postsynaptic LP neuron washeld at �55 mV under all conditions to prevent indirect effectsof neuronal membrane potential changes evoked by the amines(Flamm and Harris-Warrick 1986).

Dopamine. The physiological DA concentrations normallyachieved in vivo will depend on the spike frequency of thedopaminergic neurons and are not known. We demonstratedpreviously that 10�4 M DA greatly reduces or abolishes the LPgIPSP in response to square pulse PD stimulation (Johnson andHarris-Warrick 1990). This also occurs using realistic wave-form PD stimulation (data not shown), so we decided instead touse a lower concentration of 10�5 M DA, which does noteliminate PD¡LP transmission (Fig. 3), to examine DA’seffects on PD¡LP graded synaptic dynamics. At this lowerconcentration, DA had weak and highly variable effects on theLP gIPSPs in different preparations. With the use of squarepulse PD stimulation, DA reversibly increased initial gIPSPamplitudes in about one-half of the preparations (Fig. 3A) andreduced or had little effect on gIPSP amplitudes in the otherhalf (Fig. 3, B and C); all of these effects reversed uponwashout of DA. We suggest that this variability is caused bythe known opposing effects of DA to decrease presynaptic PDtransmitter release and increase postsynaptic LP input resis-tance (Harris-Warrick and Johnson 2010), but to differingamounts in different preparations. On average, 10�5 M DA didnot significantly change the mean amplitude of the peak andsteady-state components of the LP gIPSP during PD squarepulse stimulation (Fig. 3D, n � 5, P � 0.3 for both). Incontrast, during oscillation stimulation, DA significantly in-creased the mean first gIPSP amplitude when compared acrossall PD depolarizations (example shown in Fig. 3E; P � 0.02,n � 16). However, this effect was weak and only produced atrend to increase initial peak gIPSP amplitude at single PDvoltage step amplitudes (Fig. 3D; P � 0.08, n � 4 each). DAdid not significantly change the steady-state gIPSP amplitudeacross PD oscillation steps (Fig. 3D; P � 0.26).

With regard to synaptic depression, there was no significanteffect of DA on the amplitude of synaptic depression, asmeasured by the DI, for either square pulses (control DI: 0.68 �0.07, DA DI: 0.70 � 0.10 at �25 mV) or oscillations (controlDI: 0.32 � 0.06, DA DI: 0.37 � 0.17 at �25 mV) at any PDvoltage (P � 0.10 for both).

Despite these weak and variable effects on the amplitudes ofLP gIPSPs, DA consistently accelerated both the onset of, andthe recovery from, synaptic depression. Figure 3C shows a

typical square wave measurement, where �Dep was acceleratedby DA compared with the control gIPSP response, with littlechange in gIPSP amplitude. The mean �Dep was significantlydecreased over the entire PD voltage range (Fig. 4A; n � 5,P � 0.005), but the DA effect showed no significant voltagedependence (P � 0.78). Recovery from depression was alsofaster with both square pulse and realistic waveform stimula-tion (Fig. 4A). This can be seen in the example using oscillationstimulation in Fig. 4B, which shows more complete recovery of

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Fig. 4. Dopamine acceleration of synaptic depression and recovery at thePD¡LP synapse. A: mean effects of DA on �Dep (left, open bars; n � 5) and�Rec (middle, open bars; n � 5) during PD square pulse stimulation and on �Rec

during PD oscillation stimulation (right, filled bars; n � 4). *Significantlyshorter � in DA compared with control conditions (P � 0.005 for all).B: example showing more rapid recovery of gIPSP amplitude during DA aftera 1-s interval following PD oscillation stimuli and no effect of DA on the initialpeak gIPSP. Gray horizontal lines mark the amplitude of the initial peakgIPSP. C: lack of correlation between DA effects on initial peak LP gIPSP andits effects on depression and recovery in individual experiments. �RecSq, �Rec

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the depressed gIPSP 1 s after the stimulus train during DA(89% recovery) than under control conditions (72% recovery).The mean �Rec at PD depolarization to �25 mV was signifi-cantly faster in DA for both square pulse PD stimulation (Fig.4A, 39 � 23% �Rec decrease; P � 0.001, n � 5) and realisticwaveform PD stimulation (Fig. 4A, 43 � 19% �Rec decrease;P � 0.03, n � 4). This acceleration of onset and recovery fromdepression did not depend on the sign of the rather variable DAeffect on gIPSP amplitudes described above. As summarized inFig. 4C, in all but one experiment the �Dep and �Rec values weresmaller while DA either increased or decreased gIPSP ampli-tude. Thus, although DA (10�5 M) had highly variable effectson gIPSP amplitude, it had reliable, significant, and indepen-dent effects to accelerate the time course of synaptic depressiononset and recovery from synaptic depression.

Octopamine. We previously demonstrated that 10�5 M Octstrengthens the PD¡LP synapse when tested with square wavePD stimulation (Johnson and Harris-Warrick 1990). In ourcurrent experiments, Oct consistently increased the amplitudeof gIPSPs over the full range of presynaptic voltages usingboth square pulse (n � 5) and realistic (n � 4) PD waveforms(Fig. 5, A and B). The peak and steady-state responses tosquare pulse stimulation were significantly larger during Octapplication at all PD depolarizations (P � 0.0001 and P �0.004, respectively): during a PD step to �25 mV, the meanpeak gIPSP increased by 55 � 29% whereas the steady-stategIPSP increased by 157 � 32% (Fig. 5C). As shown in Fig.5C, Oct increased the steady-state gIPSP amplitude signifi-cantly more than the initial peak amplitude across the voltagesteps (P � 0.04). With the oscillation protocol, Oct alsoincreased the first peak and steady-state gIPSP amplitudesacross all PD stimulation voltages (P � 0.0001 for both); forexample, at �25 mV, the peak amplitudes increased by 57 �33% whereas the steady-state amplitudes increased by 48 �37% (Fig. 5C). Unlike the results using square pulse stimula-tion, during oscillation stimulation there was no significant

difference between Oct’s enhancement of the peak and thesteady-state gIPSP amplitudes at any PD voltage (P � 0.15;Fig. 5C). There was no voltage dependence of the Oct effect ongIPSP amplitude.

During square pulse stimulation, the greater Oct enhance-ment of the steady state over the peak gIPSP amplitude resultedin a significant decrease in the DI across PD depolarizations(16 � 6% decrease at �25 mV, P � 0.0001; Fig. 5D).However, there was no equivalent DI decrease during oscilla-tion stimulation, because Oct did not differentially increase thefirst peak and steady-state amplitudes of the gIPSP (Fig. 5, Cand D; P � 0.15).

Octopamine significantly altered the onset and recovery ofsynaptic depression in response to square pulse PD stimulationin a way that is quite different from DA. Octopamine signifi-cantly slowed �Dep across PD steps (increase by 80 � 41% at�25 mV; n � 5, P � 0.0001; Fig. 6A). This slowing ofsynaptic depression was not caused simply by the largergIPSPs evoked during Oct. When gIPSP amplitudes werematched in the presence and absence of Oct with the use ofdifferent amplitude presynaptic PD steps in the same prepara-tion, �Dep remained slower during Oct (Fig. 6B). In contrast,Oct significantly accelerated the recovery from depressionduring square pulse PD stimulation (Fig. 6C) by an average of47 � 24% using �25-mV steps (n � 4, P � 0.026; Fig. 6A).However, when measured with oscillation stimulation, Oct hadhighly variable effects on �Rec that could either accelerate orslow down and were not significant overall (control: 1,120 �610 ms, Oct: 1,510 � 1,510 ms; P � 0.63). Thus the maineffect of Oct was to increase PD¡LP synaptic strength duringboth PD stimulation protocols. With the use of square pulse PDstimulation, Oct reduced the depression amplitude and slowedthe onset of synaptic depression while speeding up the rate ofrecovery from depression.

Serotonin. We previously showed that 10�5 M 5-HT weak-ens the PD¡LP synapse when tested with square wave PD

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Fig. 5. Octopamine (10�5 M) enhances synapticstrength and reduces synaptic depression at thePD¡LP synapse. A and B: examples showing Octenhancement of initial peak and steady-state gIPSPamplitudes during PD square pulse (A) and oscillationstimulation (B). Black traces, control conditions; graytraces, Oct condition. C: Oct enhances mean absoluteamplitudes of initial peak and steady-state LP gIPSPselicited by PD square pulse (open bars, initial peak;light gray bars, steady state; n � 5) and oscillationstimulation (filled bars, initial peak; dark gray bars,steady state; n � 4) at �25-mV PD depolarization.*Significant increase (P � 0.004 for all). #Signifi-cantly less Oct enhancement of the initial peak gIPSPamplitude than the steady-state gIPSP amplitude dur-ing PD square pulse stimulation (P � 0.04). D: Octsignificantly decreases synaptic depression with PDsquare pulse but not with oscillation stimulation at�25-mV PD depolarization. *Significant reduction(P � 0.0001).



stimulation (Johnson and Harris-Warrick 1990). In our recentexperiments, we reproduced these results using both squarepulse (n � 4) and oscillation (n � 6) PD waveforms (Fig. 7, Aand B). With the use of square pulse stimulation, the meaninitial peak and steady-state responses were consistently andsignificantly smaller during 5-HT application at all PD depo-larizations (P � 0.0001 for initial peak and P � 0.004 forsteady state). This effect did not show any voltage dependence:with �25-mV steps, the peak gIPSPs fell by 28 � 17% and thesteady-state gIPSPs fell by 40 � 12% (Fig. 7C). Similar resultswere seen during oscillation PD stimulation at all voltages(P � 0.0035 and P � 0.0001 for initial peak and steady-stateresponses, respectively): at �25-mV PD depolarization, thepeak gIPSPs fell by 20 � 12% and the steady-state gIPSPs fellby 27 � 10% (Fig. 7C). During 5-HT application, synapticdepression was significantly greater across all PD depolariza-tions during square pulse (12 � 8% greater at �25 mV; P �0.0015; Fig. 7D) and oscillation stimulation (32 � 38% greaterat �25 mV; P � 0.004; Fig. 7D).

Serotonin significantly accelerated the time course of syn-aptic depression measured with square pulse PD stimulationand tended to accelerate the time course of recovery fromdepression (Fig. 8A), although this effect did not reach statis-tical significance. The acceleration in �Dep was not caused by asmaller gIPSP during 5-HT. For example, the trace in Fig. 8Bshows that when sized matched (using different presynapticvoltage steps), the gIPSP recorded during 5-HT depressedmore quickly than in control conditions. Serotonin significantlyaccelerated �Dep with square pulses across PD voltages (P �0.02); at �25-mV PD depolarization, �Dep was reduced by 25 � 34%(Fig. 8A; n � 4). In 3 of 3 experiments, 5-HT decreased �Recby 28 � 14%, but this did not reach statistical significance withthe small sample size and variability (P � 0.10; Fig. 8A).Serotonin also decreased �Rec in 5 of 5 experiments by 26 �27%, measured by oscillation stimulation to �25 mV (seeexample in Fig. 8C), but this also did not reach our criterion forstatistical significance (P � 0.07; Fig. 8A). Thus 5-HT reducesPD¡LP synaptic strength and increases the rate of onset ofsynaptic depression and the amount of synaptic depressionwhile tending to weakly accelerate the recovery fromdepression.

DISCUSSION

Amine-Induced Metaplasticity at the PD¡LP GradedSynapse

Graded chemical synapses of the pyloric and gastric CPGnetworks of the lobster STG show marked synaptic depressionwith prolonged voltage steps or repeated trains of presynapticactivation; all pyloric synapses are tonically depressed at nor-mal pyloric cycle frequencies (reviewed by Hartline andGraubard 1992; see also Johnson et al. 2005, 2011; Mamiyaand Nadim 2004; Manor et al. 1997). Synaptic depression iscommonly seen at graded chemical synapses, such as thosebetween retinal neurons and from hair cells to afferent fibers(Edmonds et al. 2004; Wan and Heidelberger 2011), althoughnot all graded chemical synapses show short-term activity-dependent changes in synaptic strength (Narayan et al. 2011;Simmons 1981). Aside from studies with pyloric networksynapses, little previous work has examined neuromodulationof synaptic dynamics at graded chemical synapses.

We have previously described amine effects on the strengthof the PD¡LP graded synapse (Johnson and Harris-Warrick1990), and here we demonstrate that the magnitude and kinet-ics of short-term synaptic dynamics are also modulated byamines at this synapse. The most surprising conclusion fromour results is that the synaptic strength and the magnitude ofsynaptic depression, as well as the kinetics of its onset andrecovery, can be independently modulated by each amine. Wesuggest that this may occur through multiple pre- and postsyn-aptic mechanisms of neuromodulatory actions that shape syn-aptic strength in a functioning network.

Dopamine. We have previously demonstrated that DA (10�4

M) increases graded synaptic strength at the LP¡PY andLP¡PD synapses, but only changes (increases) the amount ofsynaptic depression at the LP¡PY synapse (Johnson et al.2005, 2011). Thus DA can have differential effects on synapticdepression and synaptic strength depending on the postsynaptictarget. We used 10�5M DA in the present study because

CPD

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Fig. 6. Octopamine modulation of synaptic depression and recovery at thePD¡LP synapse. A: Oct increases �Dep (left, n � 5) and decreases �Rec (right,n � 4) at �25-mV PD square pulse depolarization. *Significant effects (P �0.03 for both). B: example using PD square pulse depolarizations of differingamplitudes to adjust the LP gIPSP to the same amplitude under control and Octconditions, showing the Oct-induced increase in �Dep in size-matched LPgIPSPs (black traces, control; gray trace, Oct). C: example of normalized initialpeak gIPSPs in control (black traces) and Oct (gray traces), with more rapidgIPSP recovery in Oct during PD square pulse depolarizations to �25 mV.



10�4M DA abolishes functional synaptic transmission at thePD¡LP graded synapse (Johnson and Harris-Warrick 1990).At the lower concentration, DA consistently accelerated �Depand �Rec despite small and variable effects on PD¡LP synapticstrength and on the magnitude of synaptic depression. Thisdemonstrates that the kinetics of synaptic depression can bemodulated independently of synaptic strength and the magni-tude of synaptic depression. At present, we cannot correlate10�5 M DA’s variable actions on synaptic strength with itseffects on other cellular properties. None of the known effectsof DA on pre- or postsynaptic ionic currents described previ-ously (Harris-Warrick and Johnson 2010) can account for theDA-induced changes in depression kinetics. DA may be actingpostsynaptically to accelerate the kinetics of transmitter recep-tor desensitization and recovery from desensitization (Papkeet al. 2011). Motor patterns produced by the lobster pyloricnetwork are qualitatively different when treated with 10�4 and10�5 M DA (Flamm and Harris-Warrick 1986). Concentration-dependent DA effects on the onset and recovery of synapticdepression may contribute to shaping distinct DA-inducednetwork activity.

Octopamine. Octopamine’s enhancement of synaptic strengthwas accompanied by reduced synaptic depression, slowed �Dep,and accelerated �Rec for PD square pulse stimulation but not forPD oscillation stimulation. Part of the synaptic strength in-crease by Oct may be due to its increase in postsynaptic LPinput resistance (Johnson et al. 1993), but the change insynaptic depression suggests more dynamic effects on synaptictransmission. Action potential-evoked synaptic transmission atcrustacean and vertebrate central synapses depresses more atsynapses with high transmitter output than with low transmitteroutput and depresses less with low transmitter output (Millarand Atwood 2004; Thomson 2000). Reduced synaptic depres-sion coupled with increased synaptic strength during Oct ap-plication does not fit this pattern, which would directly linktransmitter release probability with short-term synaptic dynam-

ics. Instead, at the PD¡LP graded synapse, enhancement ofpresynaptic calcium currents by Oct could lead to both greatertransmitter release and more rapid mobilization of vesicles intothe readily releasable pool, thus leading to reduced depressionand accelerated recovery from depression (Babai et al. 2010;Gomis et al. 1999; see below). However, the effects of Oct onPD ionic currents are not known.

Serotonin. Serotonin reduced PD¡LP graded synapticstrength but increased synaptic depression; again, this does notfit a pattern directly linking synaptic dynamics with the amountof transmitter release. Serotonin’s effects could reflect presyn-aptic mechanisms such as a reduction in voltage-dependentcalcium currents, which would reduce release and slow the rateof vesicle mobilization. Serotonin may have additional post-synaptic effects besides decreasing LP input resistance (John-son et al. 1993), as described above for DA. Serotonin showeda trend to accelerate both the onset and recovery from depres-sion, which would allow greater flexibility of the synapse asthe cycle frequency changes; the accelerated recovery maylimit the reduction in synaptic strength during the normaloscillating activity of the neurons in the pyloric rhythm. Morework is needed to clarify the mechanisms for the metaplasticeffects of amines, but it is clear that amines have complex andsometimes opposing effects on short-term dynamics of gradedsynaptic transmission in the pyloric network.

Characteristics of Synaptic Dynamics at the PD¡LPGraded Synapse

The use of both square pulse and oscillation stimulationprotocols allowed us to probe characteristics of synaptic de-pression and its modulation at the PD¡LP graded synapse ofthe pyloric that might not be evident with either stimulationprotocol alone. First, we found that the magnitude of thesynaptic depression was greater with square pulse stimulationof PD than with trains of PD oscillations, emphasizing that

BA

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Fig. 7. Serotonin (5-HT; 10�5 M) decreases synapticstrength and enhances synaptic depression at thePD¡LP synapse. A and B: examples showing 5-HTreduction of initial peak and steady-state gIPSP am-plitudes during PD square pulse (A) and oscillationstimulation (B). Black traces, control conditions; graytraces, 5-HT. C: 5-HT reduces the amplitudes ofinitial peak and steady-state LP gIPSPs elicited byPD square pulse (open bars, initial peak; light graybars, steady state; n � 4) and oscillation stimulation(filled bars, initial peak; dark gray bars, steady state;n � 6) at �25-mV PD depolarization. *Significant atP � 0.005 for both stimulation protocols. D: 5-HTincreases synaptic depression. *Significant at P �0.004 for both stimulation protocols.



realistic oscillation stimulation allows a more physiologicalcharacterization of pyloric synapses (Manor et al. 1997).Weaker synaptic depression with realistic oscillation stimula-tion may be explained simply by the partial recovery fromdepression that occurs between the PD oscillations. Second, wecould more accurately determine the time course of onset ofdepression with long square pulse PD stimulation. The onset ofdepression, measured with square pulse PD stimulation, had atime constant of 400 ms and could not be accurately mea-sured with oscillations occurring every 688 ms. Values for �Depvary from milliseconds to tens of seconds at other AP-evokedand graded chemical synapses (Cho et al. 2011; Wang and

Manis 2008; Zucker and Regehr 2002). The recovery fromdepression was over twice as fast with square pulse PDstimulation (�Rec 600 ms) as with PD oscillations (�Rec

1,600 ms). Our �Rec times are longer than those previouslyreported by Rabbah and Nadim (2007) at the PD¡LP gradedsynapse, but the stimulation protocols differed between the twostudies. Time constants for recovery from depression rangefrom tens of milliseconds at hair cell synapses to seconds atother graded synapses, again depending on experimental con-ditions (Rabl et al. 2006; Wan and Heidelberger 2011). Finally,amines sometimes affected synaptic depression and its onsetand recovery kinetics differentially (see DISCUSSION above),suggesting more complex actions on synaptic dynamics thanwould be seen with PD oscillation stimulation alone.

The most common mechanism of synaptic depression atboth AP-evoked and graded chemical synapses is transmitterdepletion during prolonged or repeated synaptic activity (vonGersdorff and Matthews 1997; Zucker and Regher 2002); otherreported presynaptic mechanisms include calcium channel in-activation (Xu et al. 2007), autoreceptor activation (Davieset al. 1990), and accumulating refractoriness of the transmitterrelease mechanism (Waldeck et al. 2000). Our experimentswere not designed to determine the site of depression, but theysuggest a mixed mechanism for depression at the PD¡LPgraded synapse. Changing the length of the presynaptic squarepulse or the duration of the PD oscillations altered the level ofdepression (Rabbah and Nadim 2007). In addition, the faster�Rec using square pulse PD stimulation, which would accumu-late more intracellular calcium, is consistent with a calcium-dependent replenishment of the readily releasable transmitterpool, seen at AP-evoked (Neher and Sakaba 2008) and gradedsynapses (Babai et al. 2010; Gomis et al. 1999). We also foundthat the magnitude of PD¡LP depression and its onset kineticswere not sensitive to presynaptic voltage stimulation, suggest-ing that depression was independent of the amount of trans-mitter release. The presynaptic voltage independence of boththe depression magnitude and �Dep is inconsistent with a simplepresynaptic transmitter depletion model of synaptic depression.The lack of correlation between the amount of synaptic de-pression and gIPSP amplitude at the PD¡LP graded synapseis also seen at AP-evoked synapses from other preparationswhere synaptic depression is clearly due to a presynapticmechanism but its magnitude is independent of PSP amplitude(Hefft et al. 2002; Wu et al. 2005). There may also be apostsynaptic contribution at our synapse, such as receptordesensitization (Jonas and Westbrook 1995; Papke et al. 2011;Trussell et al. 1993).

Possible Functional Importance of Metaplasticity in thePyloric Network

Short-term synaptic depression plays an important role inorganizing a flexible pyloric motor pattern. The magnitude ofdepression and its onset and recovery kinetics dynamicallydetermine the synaptic strength at different pyloric cycle peri-ods and when pyloric rhythm periods are perturbed by inter-actions with linked networks such as those from the cardiac sacand gastric mill motor networks (Ayali and Harris-Warrick1998; Russell and Hartline 1981; Thurma and Hooper 2002,2003). Synaptic depression promotes phase maintenance as thepyloric network changes its cycle frequency (Greenburg and

1 s

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Fig. 8. Serotonin modulation of kinetics of synaptic depression and recovery atthe PD¡LP synapse. A: 5-HT reduces �Dep (left, open bars; n � 4) and hasonly weak effects on �Rec (middle, open bars; n � 3) during �25-mV PDsquare pulse depolarization and on �Rec (right, closed bars; n � 5) during�25-mV PD oscillation depolarization. *Significant effect (P � 0.002).B: example with PD square pulse depolarizations adjusted in control and 5-HTconditions to show the 5-HT-induced acceleration of �Dep in size-matched LPgIPSPs (black traces, control; gray traces, 5-HT). C: example showing nor-malized initial peak gIPSPs in control (black traces) and 5-HT (gray traces)and more rapid gIPSP recovery in 5-HT 1 s after the end of PD oscillations.Gray horizontal lines mark the amplitudes of initial peak gIPSPs.



Manor 2005; Manor et al. 2003; Nadim et al. 2003) and contributesto stabilization of the rhythm period (Mamiya and Nadim2004). It can enable a switch between different forms ofnetwork bistability (Manor and Nadim 2001; Nadim et al.1999) and can even determine the sign of a synaptic interaction(Johnson et al. 2005; Mamiya et al. 2003).

Neuromodulator-induced metaplasticity could help maintainor reset synaptic strengths between network neurons to adjustthe postsynaptic neurons’ firing phase. For example, the 10�5

M DA effects to accelerate both �Dep and �Rec with no changein gIPSP amplitude or depression could stabilize PD¡LPsynaptic strength as the cycle frequency changes in DA (John-son et al. 2011). Octopamine enhances LP excitability but doesnot change the LP firing onset phase (Johnson et al. 2011). Thegreater PD¡LP synaptic strength in Oct, with reduced depres-sion and faster onset and recovery kinetics, could balanceenhanced LP excitability to maintain LP phasing at its pre-Octvalues (Johnson et al. 2011). There is also no change in LPonset firing phase during 5-HT despite a reduction in LPexcitability (Johnson et al. 2011). A faster �Dep might contrib-ute to weakening the PD¡LP gIPSP and to enhanced depres-sion, and this may be mitigated by the accelerated �Rec tomaintain the LP firing onset phase. At the crab LP¡PY gradedsynapse, the peptide proctolin changes the sign of synapticplasticity from depression to facilitation at certain presynapticvoltage levels and functions to help stabilize the cycle period(Zhao et al. 2011).

Metaplasticity through the actions of many different neuro-modulators is a common feature of many neural networks(Barriere et al. 2008; Carey et al. 2011; Kreitzer and Regehr2000; Parker 2001; Sakurai and Katz 2009) and should beconsidered one of the building blocks of network operation.Although well studied at AP-evoked synapses, there is rela-tively little known about metamodulation at graded chemicalsynapses. The pyloric network of crustaceans provides anexcellent model system to explore the ranges of expression ofmetaplasticity at graded synapses and its functional role inorganizing network operation and plasticity (Johnson et al.2005, 2011; Zhao et al. 2011).

ACKNOWLEDGMENTS

We thank Françoise Vermeylen from the Cornell Statistical Consulting Unitfor help with the statistical analysis.

Present address of M. D. Kvarta: Department of Physiology, Program inNeuroscience, Medical Scientist Training Program, University of MarylandSchool of Medicine, 655 W. Baltimore Street, Baltimore, MD 21201.

GRANTS

This research was supported by National Institute of Neurological Disordersand Stroke Grant NS17323 (to R. M. Harris-Warrick).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

M.D.K., R.M.H.-W., and B.R.J. conception and design of research; M.D.K.performed experiments; M.D.K. and B.R.J. analyzed data; M.D.K., R.M.H.-W., and B.R.J. interpreted results of experiments; M.D.K. and B.R.J. preparedfigures; M.D.K., R.M.H.-W., and B.R.J. drafted manuscript; M.D.K., R.M.H.-W., and B.R.J. edited and revised manuscript; M.D.K., R.M.H.-W., and B.R.J.approved final version of manuscript.

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