Neuromodulator-Evoked Synaptic Metaplasticity within a Central Pattern Generator Network 1

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Mark D. Kvarta1,2, Ronald M. Harris-Warrick1 and Bruce R. Johnson1 3

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1Department of Neurobiology and Behavior, S.G. Mudd Hall, Cornell University, Ithaca, NY 5

14853 USA 6

2Current Address: Department of Physiology, Program in Neuroscience, Medical Scientist 7

Training Program, University of Maryland School of Medicine, 655 W. Baltimore Street 8

Baltimore MD 21201 9

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Abbreviated Title: Metaplasticity at a graded chemical synapse 11

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Corresponding Author: 13

Bruce R. Johnson 14

Department of Neurobiology and Behavior, S.G. Mudd Hall, Cornell University 15

Ithaca, NY 14853 USA 16

Telephone: 607-254-4323 17

FAX: 607-254-1303 18

E-Mail: brj1@cornell.edu 19 20

Articles in PresS. J Neurophysiol (August 29, 2012). doi:10.1152/jn.00586.2012

Copyright © 2012 by the American Physiological Society.

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21 Abstract 22 23 Synapses show short-term activity-dependent dynamics that alter the strength of neuronal 24

interactions. This synaptic plasticity can be tuned by neuromodulation as a form of 25

metaplasticity. We examined neuromodulator-induced metaplasticity at a graded chemical 26

synapse in a model central pattern generator (CPG), the pyloric network of the spiny lobster 27

stomatogastric ganglion. Dopamine, serotonin and octopamine each produce a unique motor 28

pattern from the pyloric network, partially through their modulation of synaptic strength in the 29

network. We characterized synaptic depression and its amine modulation at the graded PDLP 30

synapse, driving the PD neuron with both long square pulses and trains of realistic waveforms 31

over a range of presynaptic voltages. We show that the three amines can differentially affect the 32

amplitude of graded synaptic transmission independently of the synaptic dynamics. Low 33

concentrations of dopamine had weak and variable effects on the strength of the graded synaptic 34

potentials (gIPSPs), but reliably accelerated the onset of synaptic depression and recovery from 35

depression independently of gIPSP amplitude. Octopamine enhanced gIPSP amplitude but 36

decreased the amount of synaptic depression; it slowed the onset of depression and accelerated 37

its recovery during square pulse stimulation. Serotonin reduced gIPSP amplitude but increased 38

the amount of synaptic depression, and accelerated the onset of depression. These results 39

suggest that amine-induced metaplasticity at graded chemical synapses can alter the parameters 40

of synaptic dynamics in multiple and independent ways. 41

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Key words: graded synaptic transmission; synaptic depression; lobster pyloric neurons; 46

dopamine, serotonin, octopamine 47

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Introduction 49

50 Short-term synaptic dynamics, such as facilitation and depression, alter synaptic strength 51

in activity-dependent ways (Abbott and Regehr 2004). Synaptic dynamics have been studied 52

most extensively at synapses using action potential-evoked transmitter release (Fioravante and 53

Regehr 2011; Fisher et al. 1997; Thomson, 2000; Zucker and Regehr 2002). Transient, activity-54

dependent changes in synaptic strength can alter synaptic transmission to increase the response 55

to novel stimuli, enhance directional sensitivity, adapt to repeated sensory stimuli, and detect 56

bursts of activity in sensory networks (Chacron et al. 2009; Fortune and Rose 2001; Klug 2011). 57

They can also contribute to gain control and network stabilization in central networks (Abbott 58

and Regehr 2004), and help regulate motor network activity (Gelman et al. 2011; Li et al. 2007; 59

Nadim and Manor 2000; Parker and Griller 2000; Sanchez and Kirk 2002). 60

Chemical synapses using graded transmitter release, where release is a continuous 61

function of presynaptic membrane potential, also show short-term dynamics (Goutman and 62

Glowatzki 2007; Jackman et al. 2009). The roles of graded synaptic dynamics have been 63

analyzed in the pyloric network of the lobster stomatogastric ganglion (STG). In this 14 neuron 64

central pattern generator (CPG) network, graded chemical synapses help determine the phasing 65

of pyloric neurons to produce coordinated motor patterns (Hartline et al. 1988). The synaptic 66

strength in this network dynamically tracks activity levels, and thus alters network connectivity 67

as a function of cycle frequency (Nadim and Manor 2000; Mouser et al. 2008). Synaptic 68

depression during pyloric network activity is thought to control the transition to different 69

oscillatory modes of the network (Nadim et al. 1999), and to promote phase maintenance over 70

different cycle frequencies (Nadim et al. 2003; Manor et al. 2003). 71

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Short-term synaptic plasticity can be regulated through neuromodulation. This is a form 72

of metaplasticity (Philpot et al. 1999), where neuromodulators alter the characteristics of 73

activity-dependent changes in synaptic strength (Fischer et al. 1997; Kreitzer and Regehr 2000; 74

Parker 2001; Parker and Grillner 1999; Qian and Delaney 1997). In the pyloric network, we 75

previously demonstrated that monoamines such as dopamine and octopamine can alter the steady 76

state synaptic depression at pyloric synapses (Johnson et al. 2005, 2011). In addition, the peptide 77

proctolin can change the sign of synaptic plasticity from depression to facilitation at a pyloric 78

graded synapse to help stabilize the cycle period (Zhao et al. 2011). 79

In this paper we examine amine modulation of synaptic dynamics at the pyloric PDLP 80

synapse. This synapse provides an important output from the pacemaker kernel, which sets the 81

cycle frequency of its follower cells, and thus helps regulate network activity. The PDLP 82

graded synapse shows synaptic depression when the PD neuron is driven with presynaptic square 83

pulses (Graubard et al. 1983; Johnson and Harris-Warrick 1990), and when the PD neuron is 84

artificially driven with realistic voltage waveforms that resemble its normal activity (Rabbah and 85

Nadim 2007). Graded synaptic strength at non-depressed PDLP synapses is reduced by 86

dopamine and serotonin, and enhanced by octopamine (Johnson and Harris Warrick 1990), but 87

the effects of amines on PDLP synaptic strength under conditions of realistic rhythmic activity 88

are unknown. Here we show that the three amines can have independent effects on synaptic 89

strength, the magnitude and temporal dynamics of synaptic depression and the recovery from 90

depression. 91

Materials and Methods 92

General procedures. California spiny lobsters (Panulirus interruptus) were supplied by Don 93

Tomlinson Commercial Fishing (San Diego, CA) and maintained in marine aquaria at 16° C. 94

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After lobsters were anesthetized in ice, the stomatogastric nervous system (STNS) was removed 95

and pinned in a Sylgard–coated petri dish in chilled Panulirus saline of the following 96

composition (mM): 479 NaCl, 12.8 KCl, 13.7 CaCl2, 3.9 Na2SO4, 10.0 MgSO4, 2 glucose, 11.1 97

Tris base, pH 7.35 (Mulloney and Selverston 1974). The STG was desheathed and enclosed in a 98

1 ml pool walled with Vaseline and superfused at 5 ml/min with oxygenated Panulirus saline. 99

Experiments were performed at 19°C to enhance the strength of graded synaptic transmission 100

(Johnson et al. 1991). Dopamine (DA:10-5 M), octopamine (Oct: 10-5 M) and serotonin (5HT: 101

10-5 M), were dissolved in saline just before application (amine conditions). If the amine’s 102

effects did not reverse after a 15-60 min wash, the experiment’s results were discarded. Amines 103

were purchased from Sigma Chemical Co. 104

Electrophysiological recording and cell identification. Pyloric neuron activity was monitored 105

using pin electrodes to record extracellular action potentials from appropriate motor roots 106

(differential AC amplifier, model 1700, A-M Systems) and intracellular electrodes (3 M KCl, 10-107

15 MΩ) to record from cell bodies in the STG. We identified pyloric neuron somata during 108

ongoing rhythmic activity by matching the extracellularly recorded action potentials with 109

intracellularly recorded action potentials, by the characteristic shape and amplitude of membrane 110

potential oscillations and action potentials of pyloric neurons, and by the known synaptic 111

connections between pyloric neurons (Johnson et al. 2011). 112

Isolation of graded PDLP synapse 113

Inhibitory glutamatergic synapses in the pyloric network were blocked by 5x10-6M 114

picrotoxin (PTX, Sigma Chemical Co.) to pharmacologically isolate the PDLP synapse 115

(Bidaut 1980). The AB neuron, which is electrically coupled to the PD neurons, was 116

photoinactivated by intracellular iontophoresis of 5, 6-carboxyflourescein and illumination with 117

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bright blue light (Miller and Selverston 1979). This eliminated any amine-induced AB activity 118

spreading to the PD neuron through the ABPD electrical synapse. We waited at least 1 hour 119

after AB photoinactivation to start our experiments, to allow the preparation to recover from the 120

acute effects of the light exposure (Flamm and Harris-Warrick 1986). 121

Dynamics of PDLP graded synaptic transmission 122

LP graded inhibitory post-synaptic potentials (gIPSPs) were recorded after adding 10-7 M 123

tetrodotoxin (TTX, Tocris) to Panulirus saline to block action potentials. We studied the 124

PDLP graded chemical synapse using two different presynaptic stimulation protocols. The 125

first used single, 3 sec square pulses as voltage clamp commands to drive the PD neuron, while 126

the second used a train of 12 linked realistic PD waveforms as voltage clamp commands to drive 127

PD oscillations (see Johnson et al. 2005, 2011 for realistic waveform construction). Averaged 128

and filtered PD waveforms from pyloric recordings had a mean amplitude of 15.5 ± 3.5 mV and 129

a mean cycle period of 688 ± 199 ms (n = 10); we used this shape and cycle period as pre-130

synaptic stimuli. Although realistic presynaptic waveforms are more physiologically relevant 131

than square pulse waveforms, we used both stimulation regimes for two reasons. First, some 132

basic characteristics of synaptic depression and its modulation at the PDLP synapse can only 133

be measured with square pulse stimulation, for example, accurate measurements of the onset of 134

synaptic depression (see below). Second, previous studies of graded synaptic transmission 135

between pyloric neurons have often used square pulse stimulation (for example, Graubard et al. 136

1983; Johnson et al. 1995; Manor et al. 1997; Zhao et al. 2011), and we wished to compare our 137

results with these earlier studies and determine whether postsynaptic responses differed between 138

the two stimulus forms. Both stimulation protocols used two electrode voltage clamp to drive 139

the PD from a Vhold of -55 mV in increasing amplitudes of 5 mV increments up to -25 mV. 140

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Stepping the PD up to -25 mV evokes near-maximum gIPSPs in the LP neuron (Johnson and 141

Harris-Warrick 1990; Rabbah and Nadim 2007). We separated stimulation runs by a minimum of 142

thirty seconds, which was sufficient to eliminate all effects of the previous stimulation. We used 143

two electrode current clamp to hold the post-synaptic LP membrane potential at -50 mV; 144

postsynaptic electrodes were filled with 0.6M K2SO4 + 0.02 M KCl and presynaptic electrodes 145

with 3M KCl. In these experiments we used Axoclamp-2A and 2B amplifiers (Molecular 146

Devices). 147

Under control and amine conditions (at least 5 min amine superfusion), we measured the 148

initial peak and steady state amplitudes of the gIPSPs to calculate input-output (I/O) curves and 149

dynamics of synaptic depression (Figs. 1A, B). The steady state amplitude was measured as the 150

gIPSP plateau level at the end of the square pulse stimulation, or as the mean amplitude of the 151

last 5 gIPSPs in response to the realistic PD waveform train. A synaptic depression index (DI) 152

was calculated as the fractional gIPSP decrease from the initial peak response at steady state: 153

DI= (initial peak amplitude– steady state amplitude)/initial peak amplitude. Thus, a larger DI 154

corresponds to greater synaptic depression. The time constant (τDep) of the gIPSP depression was 155

measured during presynaptic square pulse stimulation by fitting the voltage decay from the initial 156

peak to the steady state amplitude with a single exponential function (Fig. 1A). The rate of 157

synaptic depression was rapid enough that it could not be measured accurately from the 158

separated peaks of the realistic waveform series. 159

In a separate measurement, we quantified the time constant of recovery from synaptic 160

depression, τRec, for both realistic and square waveform stimulation under control and amine 161

conditions. We evoked steady state depression by stimulating the PD neuron (Vhold, -55 mV) as 162

described above, followed by a single square pulse or realistic oscillation test stimulation at 163

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increasing times after the end of the last conditioning stimulus. The percent recovery of the test 164

pulse back to the initial peak value over time was fitted with a single exponential in each 165

experiment to calculate τRec (see Figs. 2B, C). 166

Data acquisition and analysis. Electrophysiological recordings were digitized at 4KHz using a 167

PCI-6070-E board (National Instruments), and stored on a PC using custom-made recording 168

software (Scope) written in Lab Windows/CVI (National Instruments). This software also 169

controlled the injection of oscillatory and square pulse waveforms as voltage clamp commands 170

into the PD neuron (Johnson et al. 2005, 2011). All data were analyzed with the related custom-171

made software program ReadScope, also written in Lab Windows/CVI; these programs were 172

kindly provided to us by Dr. F. Nadim (http://www.stg.rutgers.edu/software/index.html). Data 173

were exported to Clampfit software (Molecular Devices) to calculate τDep and τRec. For statistical 174

comparisons, we used JMP software (SAS Institute, Inc.) to run multivariate analysis followed 175

by post-hoc tests to determine the statistical significance of differences between individual data 176

groups. Statistical differences between mean values were accepted with p < 0.05 (2-tailed 177

probability) for F and t values. Mean measured values and percentages are reported ± SD. 178

179

Results 180

Synaptic dynamics of the PDLP graded synapse under control conditions 181

We first characterized the baseline synaptic dynamics of the PDLP graded synapse 182

under control conditions, using single 3 sec square pulses (n = 10) and repeated presynaptic 183

activation with trains of 12 realistic oscillations (n= 7). As described previously for square pulse 184

stimulation of the PD neuron (Graubard et al. 1983; Johnson and Harris-Warrick 1990; Rabbah 185

and Nadim 2007), above the threshold for graded PD transmitter release (approximately -50 186

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mV), the LP response is a rapid initial peak hyperpolarization which rapidly depresses to a 187

significantly smaller steady state hyperpolarization (p < 0.001 across PD depolarizations; Fig 1A, 188

C). The LP responds to a train of repeated PD oscillations with an initial peak gIPSP followed 189

by subsequent gIPSPs whose amplitudes depress with repetition to a significantly smaller steady 190

state value over several oscillations (p < 0.001 across PD depolarizations; Figs. 1B, D; see also 191

Rabbah and Nadim 2007). Peak and steady state gIPSP amplitudes increased with increasing PD 192

voltage commands from -40 to -25 mV under both stimulation protocols (Figs. 1C, D). The 193

amplitude of the initial peak LP gIPSP tended to be larger when elicited with PD square pulse 194

stimulation than with realistic waveform stimulation (compare Figs. 1C and D open and closed 195

circles), and this difference was significant during PD depolarizations to -30 and -35 mV (p = 196

0.03 and 0.05, respectively). However, the mean steady state gIPSP amplitudes were 197

significantly greater when elicited with oscillations compared to square steps at strong PD 198

depolarizations (to -30 and -25 mV: compare Figs. 1C and D open and closed squares; p = 0.05 199

and 0.03, respectively). Since square pulse stimulation evoked larger peak initial gIPSPs and 200

smaller steady-state amplitudes than oscillation stimulation, it also evoked greater synaptic 201

depression at all PD stimulation voltages (Fig. 1E; p < 0.001 at all PD voltages). For example, 202

the mean Depression Index (DI, defined as (Peak – Steady State)/Peak) was twice as large, at PD 203

depolarization to -25 mV, using square waves compared to oscillations (Fig. 1E). This probably 204

results from the greater depression during continued presynaptic stimulation than during trains of 205

oscillations, where the synapse could partially recover between oscillations (see Discussion). 206

There was no significant effect of PD voltage on the magnitude of the depression index over the 207

voltage range of either PD stimulation protocol (Fig. 1E; p = 0.23 and 0.21 for square pulse and 208

oscillations, respectively). 209

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We also characterized the time constants of synaptic depression and recovery from 210

depression at the PDLP graded synapse using the square pulse series. The decay of the LP 211

response to PD stimulation (Fig. 1A) was well fit by a single exponential (fit correlation r = 0.99 212

± 0.001, n = 11). The mean time constant of synaptic depression, τDep, was 400 ± 200 ms during 213

PD square pulse steps to -25 mV. There was no voltage dependence of τDep during square step 214

stimulation (p = 0.95; Fig. 2A). Due to the comparatively slow cycle frequency of the oscillation 215

stimulation (cycle period 688 msec), we were unable to calculate the faster τDep from the 216

oscillation data. 217

In a separate measurement, we characterized the time constant of recovery from 218

depression, τRec, using both stimulation protocols (PD steps to -25 mV only), and fitting the 219

gIPSPs at increasing post-stimulation intervals with a single exponential (Figs. 2B, C; square 220

pulse: exponential fit correlation r = 0.95 ± 0.03; oscillation: fit correlation r = 0.98 ± 0.01). 221

Recovery from synaptic depression was over twice as fast with square pulse PD stimulation (n = 222

13) as with oscillation stimulation (n = 12; Fig. 2D; p = 0.003). This may result from increased 223

mobilization of the recovery process during maintained presynaptic depolarization (see 224

Discussion). 225

226

Amines change the synaptic dynamics of the PDLP graded synapse 227

Dopamine, 5HT and Oct affect synapses in the pyloric network by a complex set of pre- and 228

post-synaptic actions (Harris-Warrick and Johnson 2010, Johnson and Harris-Warrick 1997). In 229

addition, they can alter synaptic strength indirectly by modifying the waveforms of the pre-or 230

post-synaptic neuronal oscillations (Johnson et al. 2005). In order to focus on synaptic 231

metaplasticity, we attempted to limit the other consequences of amine modulation. For square 232

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pulse stimulations, we held the pre-synaptic neuron at -55 mV by voltage clamp both before and 233

during amine application. For oscillation stimulation, we used the realistic waveforms obtained 234

from PD oscillations during the normal pyloric rhythm for both control and amine conditions. 235

Thus, the effects of amine-evoked changes in PD oscillations were eliminated. The post-236

synaptic LP neuron was held at -55 mV under all conditions, to prevent indirect effects of 237

neuronal membrane potential changes evoked by the amines (Flamm and Harris-Warrick 1986). 238

Dopamine 239

The physiological DA concentrations normally achieved in vivo will depend on the spike 240

frequency of the dopaminergic neurons, and are not known. We demonstrated previously that 10-241

4M DA greatly reduces or abolishes the LP gIPSP in response to square pulse PD stimulation 242

(Johnson and Harris-Warrick 1990). This also occurs using realistic waveform PD stimulation 243

(data not shown), so we decided instead to use a lower concentration of 10-5M DA, which does 244

not eliminate PDLP transmission (Fig. 3), to examine DA’s effects on PDLP graded 245

synaptic dynamics. At this lower concentration, DA had weak and highly variable effects on the 246

LP gIPSPs in different preparations. Using square pulse PD stimulation, DA reversibly 247

increased initial gIPSP amplitudes in about half the preparations (Fig. 3A), and reduced or had 248

little effect on gIPSP amplitudes in the other half (Figs. 3B, C); all of these effects reversed upon 249

washout of DA. We suggest that this variability is caused by the known opposing effects of DA 250

to decrease pre-synaptic PD transmitter release and increase post-synaptic LP input resistance 251

(Harris-Warrick and Johnson 2010), but to differing amounts in different preparations. On 252

average, 10-5M DA did not significantly change the mean amplitude of the peak and steady state 253

components of the LP gIPSP during PD square pulse stimulation (Fig. 3D, n = 5; p > 0.3 for 254

both). In contrast, during oscillation stimulation, DA significantly increased the mean first 255

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gIPSP amplitude when compared across all PD depolarizations (example shown in Fig. 3E; p = 256

0.02; n = 16). However, this effect was weak, and only produced a trend to increase initial peak 257

gIPSP amplitude at single PD voltage step amplitudes (Fig. 3D; p = 0.08; n = 4 each). DA did 258

not significantly change the steady state gIPSP amplitude across PD oscillation steps (Fig 3D; p 259

= 0.26). 260

With regard to synaptic depression, there was no significant effect of DA on the 261

amplitude of synaptic depression, as measured by the DI, for either square pulses (Control DI: 262

0.68 ± 0.07, DA DI: 0.70 ± 0.10 at -25 mV) or oscillations (Control DI: 0.32 ± 0.06, DA DI: 0.37 263

± 0.17 at -25 mV) at any PD voltage (p > 0.10 for both). 264

Despite these weak and variable effects on the amplitudes of LP gIPSPs, DA consistently 265

accelerated both the onset of, and the recovery from, synaptic depression. Figure 3C shows a 266

typical square wave measurement, where τDep was accelerated by DA compared to the control 267

gIPSP response, with little change in gIPSP amplitude. The mean τDep was significantly 268

decreased over the entire PD voltage range (Fig. 4A; n = 5, p < 0.005), but the DA effect showed 269

no significant voltage dependence (p = 0.78). Recovery from depression was also faster with 270

both square pulse and realistic waveform stimulation (Fig. 4A). This is seen in the example 271

using oscillation stimulation in Figure 4B, which shows more complete recovery of the 272

depressed gIPSP 1 sec after the stimulus train during DA (89% recovery) than under control 273

conditions (72% recovery). The mean τRec at -25 mV PD depolarization was significantly faster 274

in DA for both square pulse PD stimulation (Fig. 4A, 39 ± 23% τRec decrease; p < 0.001, n = 5) 275

and realistic waveform PD stimulation (Fig. 4A, 43 ± 19% τRec decrease; p = 0.03, n = 4). This 276

acceleration of onset and recovery from depression did not depend on the sign of the rather 277

variable DA effect on gIPSP amplitudes described above. As summarized in Figure 4C, in all 278

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but 1 experiment the τDep and τRec values were smaller while DA either increased or decreased 279

gIPSP amplitude. Thus, while DA (10-5 M) had highly variable effects on gIPSP amplitude, it 280

had reliable, significant and independent effects to accelerate the time course of synaptic 281

depression onset and recovery from synaptic depression. 282

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Octopamine 284

We previously demonstrated that 10-5M Oct strengthens the PDLP synapse when 285

tested with square wave PD stimulation (Johnson and Harris-Warrick 1990). In our current 286

experiments, Oct consistently increased the amplitude of gIPSPs over the full range of 287

presynaptic voltages using both square pulse (n = 5) and realistic (n= 4) PD waveforms (Figs. 288

5A, B). The peak and steady state responses to square pulse stimulation were significantly larger 289

during Oct application at all PD depolarizations (p < 0.0001 and p = 0.004, respectively): during 290

a PD step to-25 mV, the mean peak gIPSP increased by 55 ± 29% while the steady state gIPSP 291

increased by 157 ± 32% (Fig. 5C). As can be seen in Figure 5C, Oct increased the steady state 292

gIPSP amplitude significantly more than the initial peak amplitude across the voltage steps (p = 293

0.04). Using the oscillation protocol, Oct also increased the first peak and steady state gIPSP 294

amplitudes across all PD stimulation voltages (p <0.0001 for both); for example, at -25 mV, the 295

peak amplitudes increased by 57 ± 33% while the steady state amplitudes increased by 48 ± 37% 296

(Fig. 5C). Unlike the results using square pulse stimulation, during oscillation stimulation there 297

was no significant difference between Oct’s enhancement of the peak and the steady state gIPSP 298

amplitudes at any PD voltage (p = 0.15; Fig. 5C). There was no voltage dependence of the Oct 299

effect on gIPSP amplitude. 300

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During square pulse stimulation, the greater Oct enhancement of the steady state over the 301

peak gIPSP amplitude resulted in a significant decrease in the depression index across PD 302

depolarizations (16 ± 6% decrease at -25 mV, p = 0.0001; Fig. 5D). However, there was no 303

equivalent DI decrease using oscillation stimulation, because Oct did not differentially increase 304

the first peak and steady state amplitudes of the gIPSP (Fig. 5C, D; p = 0.15). 305

Octopamine significantly altered the onset and recovery of synaptic depression in 306

response to square pulse PD stimulation, in a way that is quite different from DA. Octopamine 307

significantly slowed τDep across PD steps (increase by 80 ± 41 % at -25 mV; n = 5, p < 0.0001; 308

Fig. 6A). This slowing of synaptic depression was not caused simply by the larger gIPSPs 309

evoked during Oct. When gIPSP amplitudes were matched in the presence and absence of Oct 310

using different amplitude pre-synaptic PD steps in the same preparation, τDep remained slower 311

during Oct (Fig. 6B). In contrast, Oct significantly accelerated the recovery from depression 312

using square pulse PD stimulation (Fig. 6C), by an average of 47 ± 24% using -25 mV steps (n = 313

4; p = 0.026, Fig. 6A). However, when measured with oscillation stimulation, Oct had highly 314

variable effects on τRec which could either accelerate or slow down, and were not significant 315

overall (Control: 1120 ± 610 ms, Oct: 1510 ± 1510 ms; p= 0.63). Thus the main effect of Oct 316

was to increase PDLP synaptic strength using both PD stimulation protocols. Using square 317

pulse PD stimulation, Oct reduced the depression amplitude and slowed the onset of synaptic 318

depression, while speeding up the rate of recovery from depression. 319

Serotonin 320

We previously showed that 10-5M 5HT weakens the PDLP synapse when tested with 321

square wave PD stimulation (Johnson and Harris-Warrick 1990). In our recent experiments, we 322

have reproduced these results using both square pulse (n = 4) and oscillation (n= 6) PD 323

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waveforms (Figs. 7A, B). Using square pulse stimulation, the mean initial peak and steady state 324

responses were consistently and significantly smaller during 5HT application at all PD 325

depolarizations (p < 0.0001 for initial peak and p = 0.004 for steady state). This effect did not 326

show any voltage dependence: with -25 mV steps, the peak gIPSPs fell by 28 ± 17% and the 327

steady state gIPSPs fell by 40 ± 12% (Fig. 7C). Similar results were seen using oscillation PD 328

stimulation at all voltages (p = 0.0035 and < 0.0001 for initial peak and steady state responses, 329

respectively): at -25 mV PD depolarization, the peak gIPSPs fell by 20 ± 12% and the steady 330

state gIPSPs by 27 ± 10% (Fig. 7C). During 5HT application, synaptic depression was 331

significantly greater across all PD depolarizations using square pulse (12 ± 8% greater at -25 332

mV; p = 0.0015; Fig. 7D), and oscillation stimulation (32 ± 38% greater at -25 mV; p = 0.004; 333

Fig 7D). 334

Serotonin significantly accelerated the time course of synaptic depression measured with 335

square pulse PD stimulation, and tended to accelerate the time course of recovery from 336

depression (Fig. 8A), though this effect did not reach statistical significance. The acceleration in 337

τDep was not caused by a smaller gIPSP during 5HT. For example, the trace in Fig. 8B shows 338

that when sized matched (using different pre-synaptic voltage steps), the gIPSP recorded during 339

5HT depressed more quickly than in control conditions. Serotonin significantly accelerated τDep 340

with square pulses across PD voltages (p = 0.02); at -25 mV PD depolarization, τDep was reduced 341

by 25 ± 34 % (Fig. 8A; n = 4). In 3 of 3 experiments, 5HT decreased τRec by 28 ± 14%, but this 342

did not reach statistical significance with the small sample size and variability, (p = 0.10; Fig. 343

8A). Serotonin also decreased τRec in 5 of 5 experiments by 26 ± 27 %, measured by oscillation 344

stimulation to -25 mV (see example in Fig. 8C), but this also did not reach our criterion for 345

statistical significance (p = 0.07; Fig. 8A). Thus, 5HT reduces PDLP synaptic strength, 346

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increases the rate of onset of synaptic depression and the amount of synaptic depression, while 347

tending to weakly accelerate the recovery from depression. 348

349

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Discussion 350

Amine-induced metaplasticity at the PDLP graded synapse 351

Graded chemical synapses of the pyloric and gastric CPG networks of the lobster STG show 352

marked synaptic depression with prolonged voltage steps or repeated trains of presynaptic 353

activation; all pyloric synapses are tonically depressed at normal pyloric cycle frequencies 354

(reviewed by Hartline and Graubard, 1992; see also Johnson et al. 2005, 2011; Mamiya and 355

Nadim 2004; Manor et al. 1997). Synaptic depression is commonly seen at graded chemical 356

synapses, such as those between retinal neurons and from hair cells to afferent fibers (Edmonds 357

et al. 2004; Wan and Heidelberger 2011), although not all graded chemical synapses show short 358

term activity-dependent changes in synaptic strength (Narayan et al. 2011; Simmons 1981). 359

Aside from studies with pyloric network synapses, little previous work has examined 360

neuromodulation of synaptic dynamics at graded chemical synapses. 361

We have previously described amine effects on the strength of the PDLP graded 362

synapse (Johnson and Harris- Warrick 1990), and here we demonstrate that the magnitude and 363

kinetics of short-term synaptic dynamics are also modulated by amines at this synapse. The most 364

surprising conclusion from our results is that the synaptic strength and the magnitude of synaptic 365

depression, as well as the kinetics of its onset and recovery, can be independently modulated by 366

each amine. We suggest that this may occur through multiple pre and post-synaptic mechanisms 367

of neuromodulatory actions that shape synaptic strength in a functioning network. 368

Dopamine 369

We have previously demonstrated that DA (10-4M) increases graded synaptic strength at the 370

LPPY and LPPD synapses, but only changes (increases) the amount of synaptic depression 371

at the LPPY synapse (Johnson et al. 2005, 2011). Thus DA can have differential effects on 372

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synaptic depression and synaptic strength depending on the postsynaptic target. We used 10-5M 373

DA in the present study because 10-4M DA abolishes functional synaptic transmission at the 374

PDLP graded synapse (Johnson and Harris Warrick 1990). At the lower concentration, DA 375

consistently accelerated τDep and τRec despite small and variable effects on PDLP synaptic 376

strength and on the magnitude of synaptic depression. This demonstrates that the kinetics of 377

synaptic depression can be modulated independently of synaptic strength and the magnitude of 378

synaptic depression. At present, we cannot correlate 10-5 M DA’s variable actions on synaptic 379

strength with its effects on other cellular properties. None of the known effects of DA on pre- or 380

postsynaptic ionic currents described previously (Harris-Warrick and Johnson 2010) can account 381

for the DA-induced changes in depression kinetics. DA may be acting postsynaptically to 382

accelerate the kinetics of transmitter receptor desensitization and recovery from desensitization 383

(Papke et al. 2011). Motor patterns produced by the lobster pyloric network are qualitatively 384

different when treated with 10-4 and 10-5M DA (Flamm and Harris-Warrick 1986). 385

Concentration-dependent DA effects on the onset and recovery of synaptic depression may 386

contribute to shaping distinct DA-induced network activity. 387

Octopamine 388

Octopamine’s enhancement of synaptic strength was accompanied by reduced synaptic 389

depression, slowed τDep and accelerated τRec for PD square pulse stimulation, but not PD 390

oscillation stimulation. Part of the synaptic strength increase by Oct may be due to its increase in 391

postsynaptic LP input resistance (Johnson et al. 1993), but the change in synaptic depression 392

suggests more dynamic effects on synaptic transmission. AP-evoked synaptic transmission at 393

crustacean and vertebrate central synapses depresses more at synapses with high transmitter 394

output than with low transmitter output and depresses less with low transmitter output (Miller 395

19

and Atwood 2004; Thomson 2000). Reduced synaptic depression coupled with increased 396

synaptic strength during Oct does not fit this pattern, which would directly link transmitter 397

release probability with short term synaptic dynamics. Instead, at the PDLP graded synapse, 398

enhancement of presynaptic ICa by Oct could lead to both greater transmitter release and more 399

rapid mobilization of vesicles into the readily releasable pool, thus leading to reduced depression 400

and accelerated recovery from depression (Babai et al. 2010; Gomis et al. 1999; see below). 401

However, the effects of Oct on PD ionic currents are not known. 402

Serotonin 403

Serotonin reduced PDLP graded synaptic strength but increased synaptic depression; again, 404

this does not fit a pattern directly linking synaptic dynamics with the amount of transmitter 405

release. Serotonin’s effects could reflect pre-synaptic mechanisms such as a reduction in ICa(V), 406

which would reduce release and slow the rate of vesicle mobilization. Serotonin may have 407

additional postsynaptic effects besides decreasing LP input resistance (Johnson et al. 1993), as 408

described above for DA. Serotonin showed a trend to accelerate both the onset and recovery 409

from depression, which would allow greater flexibility of the synapse as the cycle frequency 410

changes; the accelerated recovery may limit the reduction in synaptic strength during the normal 411

oscillating activity of the neurons in the pyloric rhythm. More work is needed to clarify the 412

mechanisms for the metaplastic effects of amines, but it is clear that amines have complex and 413

sometimes opposing effects on short term dynamics of graded synaptic transmission in the 414

pyloric network. 415

Characteristics of synaptic dynamics at the PDLP graded synapse 416

The use of both square pulse and oscillation stimulation protocols allowed us to probe 417

characteristics of synaptic depression and its modulation at the PDLP graded synapse of the 418

20

pyloric that might not be evident with either stimulation protocol alone. First, we found that the 419

magnitude of the synaptic depression was greater using square pulse stimulation of PD than with 420

trains of PD oscillations, emphasizing that realistic oscillation stimulation allows a more 421

physiological characterization of pyloric synapses (Manor et al. 1997). Weaker synaptic 422

depression with realistic oscillation stimulation may be explained simply by the partial recovery 423

from depression that occurs between the PD oscillations. Second, we could more accurately 424

determine the time course of onset of depression with long square pulse PD stimulation. The 425

onset of depression, measured with square pulse PD stimulation, had a time constant of ~400 ms, 426

and could not be accurately measured with oscillations occurring every 688 msec. Values for 427

τDep vary from ms to tens of seconds at other AP-evoked and graded chemical synapses (Cho et 428

al. 2011; Wang and Manis 2008; Zucker and Regehr 2002). The recovery from depression was 429

over twice as fast using square pulse PD stimulation (τRec~600 ms) compared to PD oscillations 430

(τRec~ 1600 ms). Our τRec times are longer that those previously reported by Rabbah and Nadim 431

(2007) at the PDLP graded synapse, but the stimulation protocols differed between the two 432

studies. Time constants for recovery from depression range from 10s of msec at hair cell 433

synapses to seconds at other graded synapses, again depending on experimental conditions (Rabl 434

et al. 2006; Wan and Heidelberger 2011). Finally, amines sometimes affected synaptic 435

depression and its onset and recovery kinetics differentially (see discussion above), suggesting 436

more complex actions on synaptic dynamics than would be seen with PD oscillation stimulation 437

alone. 438

The most common mechanism of synaptic depression at both action potential evoked and 439

graded chemical synapses is transmitter depletion during prolonged or repeated synaptic activity 440

(von Gersdorff and Matthews 1997; Zucker and Regher 2002); other reported pre-synaptic 441

21

mechanisms include calcium channel inactivation (Xu et al. 2007), autoreceptor activation 442

(Davies et al. 1990), and accumulating refractoriness of the transmitter release mechanism 443

(Waldeck et al. 2000). Our experiments were not designed to determine the site of depression, 444

but they suggest a mixed mechanism for depression at the PDLP graded synapse. Changing 445

the length of the pre-synaptic square pulse, or the duration of the PD oscillations, altered the 446

level of depression (Johnson et al. 2005). In addition, the faster τRec using square pulse PD 447

stimulation, which would accumulate more intracellular calcium, is consistent with a calcium-448

dependent replenishment of the readily releasable transmitter pool, seen at AP-evoked (Neher 449

and Sakaba 2008) and graded synapses (Babai et al. 2010; Gomis et al. 1999). We also found 450

that the magnitude of PDLP depression and its onset kinetics were not sensitive to presynaptic 451

voltage stimulation, suggesting that depression was independent of the amount of transmitter 452

release. The pre-synaptic voltage independence of both the depression magnitude and τDep is 453

inconsistent with a simple pre-synaptic transmitter depletion model of synaptic depression. The 454

lack of correlation between the amount of synaptic depression and gIPSP amplitude at the PD 455

LP graded synapse is also seen at AP-evoked synapses from other preparations where synaptic 456

depression is clearly due to a presynaptic mechanism, but its magnitude is independent of PSP 457

amplitude (Hefft et al. 2002; Wu et al. 2005). There may also be a postsynaptic contribution at 458

our synapse, such as receptor desensitization (Jonas and Westbrook 1995; Papke et al. 2011; 459

Trussell et al. 1993). 460

Possible functional importance of metaplasticity in the pyloric network 461

Short term synaptic depression plays an important role in organizing a flexible pyloric 462

motor pattern. The magnitude of depression and its onset and recovery kinetics dynamically 463

determine the synaptic strength at different pyloric cycle periods, and when pyloric rhythm 464

22

periods are perturbed by interactions with linked networks such as from the cardiac sac and 465

gastric mill motor networks (Ayali and Harris Warrick 1998; Russell and Hartline 1981; Thurma 466

and Hooper 2002, 2003). Synaptic depression promotes phase maintenance as the pyloric 467

network changes its cycle frequency (Greenburg and Manor 2005; Manor et al. 2003; Nadim et 468

al. 2003), and contributes to stabilization of the rhythm period (Mamiya and Nadim 2004). It 469

can enable a switch between different forms of network bistability (Nadim and Manor 2001; 470

Nadim et al. 1999), and can even determine the sign of a synaptic interaction (Johnson et al. 471

2005; Mamiya et al. 2003). 472

Neuromodulator-induced metaplasticity could help maintain or reset synaptic strengths 473

between network neurons to adjust the post-synaptic neurons’ firing phase. For example, the 10-474

5M DA effects to accelerate both τDep and τRec with no change in gIPSP amplitude or depression 475

could stabilize PDLP synaptic strength as the cycle frequency changes in DA (Johnson et al. 476

2011). Octopamine enhances LP excitability, but does not change the LP firing onset phase 477

(Johnson et al. 2011). The greater PDLP synaptic strength in Oct, with reduced depression 478

and faster onset and recovery kinetics, could balance enhanced LP excitability to maintain LP 479

phasing at its pre-Oct values (Johnson et al. 2011). There is also no change in LP onset firing 480

phase during 5HT despite a reduction in LP excitability (Johnson et al. 2011). A faster τDep 481

might contribute to weakening the PDLP gIPSP and to enhanced depression, and this may be 482

mitigated by the accelerated τRec to maintain the LP firing onset phase. At the crab LPPY 483

graded synapse, the peptide proctolin changes the sign of synaptic plasticity from depression to 484

facilitation at certain presynaptic voltage levels, and functions to help stabilize the cycle period 485

(Zhao et al. 2011). 486

23

Metaplasticity through the actions of many different neuromodulators is a common 487

feature of many neural networks (Barriere et al. 2008; Carey et al. 2011; Kreitzer and Regehr 488

2000; Parker 2001; Sakurai and Katz 2009), and should be considered one of the building blocks 489

of network operation. Although well studied at AP-evoked synapses, there is relatively little 490

known about metamodulation at graded chemical synapses. The pyloric network of crustaceans 491

provides an excellent model system to explore the ranges of expression of metaplasticity at 492

graded synapses, and its functional role in organizing network operation and plasticity (Johnson 493

et al. 2005; 2011; Zhao et al. 2011). 494

495

496

24

497 Acknowledgements 498

We thank Françoise Vermeylen from the Cornell Statistical Consulting Unit for help with the 499

statistical analysis. 500

501

Grants 502

Research supported by NIH NS17323 to R.M. Harris-Warrick 503

504

Disclosures 505

No conflicts of interest financial or otherwise, are declared by the authors. 506

507

Author Contributions 508

B.R.J., R.M.H.-W. and M.D.K. designed the research; M.D.K. performed experiments; M.D.K. 509

and B.R.J. analyzed data, and interpreted results with R.M.H.-W.; B.R.J. and M.D.K. prepared 510

figures; B.R.J., R.M.H.-W., and M.D.K. wrote and edited the manuscript. 511

512

25

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748 Figure legends 749 750

Figure 1. Synaptic dynamics of graded synaptic transmission at the PDLP chemical synapse. 751

A. LP gIPSPs in response to 3 second PD square pulse depolarizations. Initial peak and steady 752

state IPSP amplitudes and the time constant (τDep) for synaptic depression were measured for all 753

PD depolarizations from Vhold = -55 mV. B. LP gIPSPs during a train of 12 realistic PD 754

oscillations of increasing amplitude from Vhold = -55 mV. The first gIPSP amplitude (initial 755

peak) and the mean of the last 5 gIPSPs amplitudes (steady state) were measured for all PD 756

depolarizations. C. Mean input/output relationship for the amplitude of the initial peak (open 757

circles) and steady state (open squares) gIPSPs using square pulse PD stimulation (n = 11). D. 758

Mean input/output relationship for initial (closed circles) and steady state (closed squares) LP 759

gIPSP amplitudes using PD oscillation stimulation (n = 7). E. Mean depression index for square 760

pulse (open bars) and oscillation (closed bars) stimulation across PD stimulation voltages. * 761

indicates that the depression index for PD square pulse stimulation is significantly greater than 762

for PD oscillation stimulation (p < 0.001 for all PD voltages). The depression index did not 763

show voltage dependence with either stimulation protocol (p > 0.20 for both). 764

765

Figure 2. Synaptic depression and recovery at the PDLP graded synapse. A. Mean τDep 766

across PD square pulse stimulation voltages (n = 11). τDep did not show any voltage dependence 767

(p = 0.95). B. Measurement of the time constant of LP gIPSP recovery from depression (τRec) in 768

response to PD square pulse depolarization to -25 mV. C. Measurement of τRec using PD 769

oscillation depolarization to -25 mV. D. Mean τRec for LP gIPSPs elicited by PD square pulse (n 770

= 13) and oscillation (n = 10) stimulation. * indicates significantly longer τRec with PD 771

oscillation stimulation (p = 0.003). 772

773

Figure 3. Dopamine (10-5M) modulation of synaptic strength at the PDLP synapse. 774

Examples of variable effects of DA to reversibly enhance (A), reduce (B), or have no effect (C) 775

on the initial LP gIPSP amplitude with PD square pulse stimulation. D. Mean effects of DA on 776

amplitudes of initial peak and steady state LP gIPSPs elicited by PD square pulse (initial peak, 777

open bars; steady state, light gray bars) and oscillation stimulation (initial peak, closed bars; 778

34

steady state, dark gray bars) at -25 mV PD depolarization. E. Example showing DA 779

enhancement of the initial peak gIPSP during PD oscillation stimulation. 780

781

Figure 4. Dopamine acceleration of synaptic depression and recovery at the PDLP synapse. 782

A. Mean effects of DA on τDep (left open bars; n = 5), and τRec (middle open bars; n = 5) for PD 783

square pulse stimulation, and for τRec during PD oscillation stimulation (right closed bars; n = 4). 784

* indicates significantly shorter τ in DA compared to control conditions (p < 0.005 for all). B. 785

Example showing more rapid recovery of gIPSP amplitude during DA after a 1 sec interval 786

following PD oscillation stimuli, and no effect of DA on the initial peak gIPSP. Gray lines mark 787

the amplitude of the initial peak gIPSP. C. Lack of correlation between DA effects on initial 788

peak LP gIPSP and its effects on depression and recovery in individual experiments. Open 789

squares, τDep and open circles, τRec for PD square pulse stimulation; closed circles, τRec for PD 790

oscillation stimulation. 791

792

Figure 5. Octopamine (10-5M) enhances synaptic strength and reduces synaptic depression at 793

the PDLP synapse. Examples showing Oct enhancement of initial peak and steady state gIPSP 794

amplitudes using PD square pulse (A) and oscillation stimulation (B). Black traces, control 795

conditions; gray traces, Oct. C. Oct enhances mean absolute amplitudes of initial peak and 796

steady state LP gIPSPs elicited by PD square pulse (initial peak, open bars; steady state, light 797

gray bars; n = 5) and oscillation stimulation (initial peak, closed bars; steady state, dark gray 798

bars; n = 4) at -25 mV PD depolarization. *: significant increase (p < 0.004 for all); #: Oct 799

enhances the initial peak gIPSP amplitude significantly less than the steady state gIPSP during 800

PD square pulse stimulation (p = 0.04). D. Oct significantly decreases synaptic depression with 801

PD square pulse but not with oscillation stimulation at -25 mV PD depolarization. *: significant 802

reduction (p < 0.0001). 803

804

Figure 6. Octopamine modulation of synaptic depression and recovery at the PDLP synapse. 805

A. Octopamine increases τDep (left open bars; n = 5) and decreases τRec (right open bars; n = 4) at 806

-25 mV PD square pulse depolarization. *: significant effects (p < 0.03 for both). B. Example 807

using PD square pulse depolarizations of differing amplitudes to adjust the LP gIPSP to the same 808

amplitude under control and Oct conditions, showing the Oct-induced increase in τDep in size 809

35

matched LP gIPSPs (control, black traces; Oct, gray traces). C. Example of normalized initial 810

peak gIPSPs in control (black traces) and Oct (gray traces), with more rapid gIPSP recovery in 811

Oct during PD square pulse depolarizations to -25 mV. 812

813

Figure 7. Serotonin (10-5M) decreases synaptic strength and enhances synaptic depression at the 814

PDLP synapse. Examples showing 5HT reduction of initial peak and steady state gIPSP 815

amplitudes using PD square pulse (A) and oscillation stimulation (B). Black traces, control 816

conditions; gray traces, 5HT. C. 5HT reduces the amplitudes of initial peak and steady state LP 817

gIPSPs elicited by PD square pulse (initial peak, open bars; steady state, light gray bars; n = 4) 818

and oscillation stimulation (initial peak, closed bars; steady state, dark gray bars; n = 6) at -25 819

mV PD depolarization. *: significant at p < 0.005 for both stimulation protocols. D. 5HT 820

increases synaptic depression. *: significant at p < 0.004 for both stimulation protocols. 821

822

Figure 8. Serotonin modulation of kinetics of synaptic depression and recovery at the PDLP 823

synapse. A. 5HT reduces τDep (left open bars; n = 4) and has only weak effects on τRec (middle 824

open bars; n = 3) using -25 mV PD square pulse depolarization, and τRec (right closed bars; n = 825

5) using -25 mV PD oscillation depolarization. *: significant at p = 0.002. B. Example with PD 826

square pulse depolarizations adjusted in control and 5HT conditions to show the 5HT-induced 827

acceleration of τDep in size matched LP gIPSPs (control, black traces; 5HT, gray traces). C. 828

Example showing normalized initial peak gIPSPs in control (black traces) and 5HT (gray traces), 829

and more rapid gIPSP recovery in 5HT 1 sec after the end of PD oscillations. Gray lines mark 830

the amplitudes of initial peak gIPSPs. 831

832

833

834

835

PD

LP

-55 mV

-50 mV

10 mV

steady state

1 s

2 mVτDep

A

2 mV1 s

steady stateinitial peak

B

D

-45 -40 -35 -30 -25

0

2

4

6

8

PD Potential (mV)

PD OscillationC

LPgIPSP(mV)

-45 -40 -35 -30 -25

0

2

4

6

8

steady state

initial peak

PD Square Pulse

PD Potential (mV)

E

-40 -35 -30 -250

0.8

0.4

0.6

0.2DepressionIndex

* * **PD Square

PD Oscillation

PD Potential (mV)

A

C D

PD

LP

τRec = 2900 ms

-55 mV-50 mV

PDSquare

3000

1500

1000

500

0

2000

2500

gIPSP

τ Rec(ms)

*

1 mV

1 s

20 mV

PDOscillation

B

PD Potential (mV)

PD

LP

τRec = 516 ms

-55 mV

-50 mV

-40 -35 -30 -25

800

600

400

200

0

gIPSP

τ Dep(ms)

10 mV

1 mV

1 s

Ctl

DAWash

PD

-55 mV

LP

-50 mV

B

C-50 mV DA

-50 mV

DA

Ctl

WashCtl

LPgIPSP(mV)

PD Oscillation

DA CtlCtl DA

PD Square

10

8

6

4

2

0

DA

EPD

LP

10 mV

2 mV

5 mV

2 mV

1 s DA 1 s

1 mV

-50 mV-55 mV

10 mV

25

0

-25

-50

-75

-100100 150500-50

%Change

τ

% Change Initial Peak gIPSP

C

τDepτRecSqτRecOsc

BPD

LP

-55 mV

-50 mV

-50 mV

1 s

1 mV

1 mV

Ctl

DA

10 mV

A

gIPSP

τ(ms)

PDSquare

PDSquare

2500

1500

1000

500

0

2000

Ctl DA Ctl CtlDA DA

*

**

τRecτRecτDep

PDOscillation

BAPD

LP5 mV

1 s 1 s5 mV

-50 mV

-55 mV

Oct

Ctl

10 mV

Oct

10 mV

DC

LPgIPSP(mV)

PD Square Pulse

0

6

9

12

15

3 steady state

initial peak

PD OscillationPD Square PD OscillationPD Square

DepressionIndex

0

0.4

0.6

0.8

0.2

**

*

*

*#

Oct CtlCtl Oct Oct CtlCtl Oct

CPD

LP

-55 mV

-50 mV

10 mV

1 mV

1 s1 mV

B

A

gIPSP

τ(ms)

PD Square PD Square

900

600

300

0

1200

Ctl Oct Ctl Oct

PD

LP

-55 mV

-50 mV

1 s

5 mV

Ctl

Oct10 mV

**

Ctl

Oct

τRecτDep

BA

2 mV

1 s

10 mV

DC

LPgIPSP(mV)

PD Square Pulse

0

4

6

8

2

PD OscillationPD Square

**

*

PD OscillationPD Square

DepressionIndex

0

0.4

0.6

0.8

0.2

*

-50 mV

-55 mV

PD

LP Ctl

5HT

Ctl

10 mV

1 s

5 mV

*

*

*

5HT CtlCtl 5HT 5HT CtlCtl 5HT

1 s

2 mV

Ctl

5HT

5HT

Ctl

-50 mV

-55 mV

PD

LP

10 mV

C

B

A

gIPSP

τ(ms)

PDSquare

PDSquare

1800

1200

600

0

2400

Ctl 5HT Ctl 5HT

*

*

Ctl 5HTPD

Oscillation

τDep τRecτRec

*

PD

LP

-55 mV-50 mV

10 mV

1 s1 mV

1 mV

5HT

Ctl