Neuromodulator-Evoked Synaptic Metaplasticity within a Central Pattern Generator Network

Transcription

1 Articles in PresS. J Neurophysiol (August 29, 2012). doi: /jn Neuromodulator-Evoked Synaptic Metaplasticity within a Central Pattern Generator Network 2 3 Mark D. Kvarta 1,2, Ronald M. Harris-Warrick 1 and Bruce R. Johnson Department of Neurobiology and Behavior, S.G. Mudd Hall, Cornell University, Ithaca, NY USA 2 Current Address: Department of Physiology, Program in Neuroscience, Medical Scientist Training Program, University of Maryland School of Medicine, 655 W. Baltimore Street Baltimore MD Abbreviated Title: Metaplasticity at a graded chemical synapse Corresponding Author: Bruce R. Johnson Department of Neurobiology and Behavior, S.G. Mudd Hall, Cornell University Ithaca, NY USA Telephone: FAX: brj1@cornell.edu Copyright 2012 by the American Physiological Society.

2 Abstract Synapses show short-term activity-dependent dynamics that alter the strength of neuronal interactions. This synaptic plasticity can be tuned by neuromodulation as a form of metaplasticity. We examined neuromodulator-induced metaplasticity at a graded chemical synapse in a model central pattern generator (CPG), the pyloric network of the spiny lobster stomatogastric ganglion. Dopamine, serotonin and octopamine each produce a unique motor pattern from the pyloric network, partially through their modulation of synaptic strength in the network. We characterized synaptic depression and its amine modulation at the graded PD LP synapse, driving the PD neuron with both long square pulses and trains of realistic waveforms over a range of presynaptic voltages. We show that the three amines can differentially affect the amplitude of graded synaptic transmission independently of the synaptic dynamics. Low concentrations of dopamine had weak and variable effects on the strength of the graded synaptic potentials (gipsps), but reliably accelerated the onset of synaptic depression and recovery from depression independently of gipsp amplitude. Octopamine enhanced gipsp amplitude but decreased the amount of synaptic depression; it slowed the onset of depression and accelerated its recovery during square pulse stimulation. Serotonin reduced gipsp amplitude but increased the amount of synaptic depression, and accelerated the onset of depression. These results suggest that amine-induced metaplasticity at graded chemical synapses can alter the parameters of synaptic dynamics in multiple and independent ways. Key words: graded synaptic transmission; synaptic depression; lobster pyloric neurons; dopamine, serotonin, octopamine 2

3 Introduction Short-term synaptic dynamics, such as facilitation and depression, alter synaptic strength in activity-dependent ways (Abbott and Regehr 2004). Synaptic dynamics have been studied most extensively at synapses using action potential-evoked transmitter release (Fioravante and Regehr 2011; Fisher et al. 1997; Thomson, 2000; Zucker and Regehr 2002). Transient, activitydependent changes in synaptic strength can alter synaptic transmission to increase the response to novel stimuli, enhance directional sensitivity, adapt to repeated sensory stimuli, and detect bursts of activity in sensory networks (Chacron et al. 2009; Fortune and Rose 2001; Klug 2011). They can also contribute to gain control and network stabilization in central networks (Abbott and Regehr 2004), and help regulate motor network activity (Gelman et al. 2011; Li et al. 2007; Nadim and Manor 2000; Parker and Griller 2000; Sanchez and Kirk 2002). Chemical synapses using graded transmitter release, where release is a continuous function of presynaptic membrane potential, also show short-term dynamics (Goutman and Glowatzki 2007; Jackman et al. 2009). The roles of graded synaptic dynamics have been analyzed in the pyloric network of the lobster stomatogastric ganglion (STG). In this 14 neuron central pattern generator (CPG) network, graded chemical synapses help determine the phasing of pyloric neurons to produce coordinated motor patterns (Hartline et al. 1988). The synaptic strength in this network dynamically tracks activity levels, and thus alters network connectivity as a function of cycle frequency (Nadim and Manor 2000; Mouser et al. 2008). Synaptic depression during pyloric network activity is thought to control the transition to different oscillatory modes of the network (Nadim et al. 1999), and to promote phase maintenance over different cycle frequencies (Nadim et al. 2003; Manor et al. 2003). 3

4 Short-term synaptic plasticity can be regulated through neuromodulation. This is a form of metaplasticity (Philpot et al. 1999), where neuromodulators alter the characteristics of activity-dependent changes in synaptic strength (Fischer et al. 1997; Kreitzer and Regehr 2000; Parker 2001; Parker and Grillner 1999; Qian and Delaney 1997). In the pyloric network, we previously demonstrated that monoamines such as dopamine and octopamine can alter the steady state synaptic depression at pyloric synapses (Johnson et al. 2005, 2011). In addition, the peptide proctolin can change the sign of synaptic plasticity from depression to facilitation at a pyloric graded synapse to help stabilize the cycle period (Zhao et al. 2011). In this paper we examine amine modulation of synaptic dynamics at the pyloric PD LP synapse. This synapse provides an important output from the pacemaker kernel, which sets the cycle frequency of its follower cells, and thus helps regulate network activity. The PD LP graded synapse shows synaptic depression when the PD neuron is driven with presynaptic square pulses (Graubard et al. 1983; Johnson and Harris-Warrick 1990), and when the PD neuron is artificially driven with realistic voltage waveforms that resemble its normal activity (Rabbah and Nadim 2007). Graded synaptic strength at non-depressed PD LP synapses is reduced by dopamine and serotonin, and enhanced by octopamine (Johnson and Harris Warrick 1990), but the effects of amines on PD LP synaptic strength under conditions of realistic rhythmic activity are unknown. Here we show that the three amines can have independent effects on synaptic strength, the magnitude and temporal dynamics of synaptic depression and the recovery from depression Materials and Methods General procedures. California spiny lobsters (Panulirus interruptus) were supplied by Don Tomlinson Commercial Fishing (San Diego, CA) and maintained in marine aquaria at 16 C. 4

5 After lobsters were anesthetized in ice, the stomatogastric nervous system (STNS) was removed and pinned in a Sylgard coated petri dish in chilled Panulirus saline of the following composition (mm): 479 NaCl, 12.8 KCl, 13.7 CaCl 2, 3.9 Na 2 SO 4, 10.0 MgSO 4, 2 glucose, 11.1 Tris base, ph 7.35 (Mulloney and Selverston 1974). The STG was desheathed and enclosed in a 1 ml pool walled with Vaseline and superfused at 5 ml/min with oxygenated Panulirus saline. Experiments were performed at 19 C to enhance the strength of graded synaptic transmission (Johnson et al. 1991). Dopamine (DA:10-5 M), octopamine (Oct: 10-5 M) and serotonin (5HT: 10-5 M), were dissolved in saline just before application (amine conditions). If the amine s effects did not reverse after a min wash, the experiment s results were discarded. Amines were purchased from Sigma Chemical Co. Electrophysiological recording and cell identification. Pyloric neuron activity was monitored using pin electrodes to record extracellular action potentials from appropriate motor roots (differential AC amplifier, model 1700, A-M Systems) and intracellular electrodes (3 M KCl, MΩ) to record from cell bodies in the STG. We identified pyloric neuron somata during ongoing rhythmic activity by matching the extracellularly recorded action potentials with intracellularly recorded action potentials, by the characteristic shape and amplitude of membrane potential oscillations and action potentials of pyloric neurons, and by the known synaptic connections between pyloric neurons (Johnson et al. 2011). Isolation of graded PD LP synapse Inhibitory glutamatergic synapses in the pyloric network were blocked by 5x10-6 M picrotoxin (PTX, Sigma Chemical Co.) to pharmacologically isolate the PD LP synapse (Bidaut 1980). The AB neuron, which is electrically coupled to the PD neurons, was photoinactivated by intracellular iontophoresis of 5, 6-carboxyflourescein and illumination with 5

6 bright blue light (Miller and Selverston 1979). This eliminated any amine-induced AB activity spreading to the PD neuron through the AB PD electrical synapse. We waited at least 1 hour after AB photoinactivation to start our experiments, to allow the preparation to recover from the acute effects of the light exposure (Flamm and Harris-Warrick 1986). Dynamics of PD LP graded synaptic transmission LP graded inhibitory post-synaptic potentials (gipsps) were recorded after adding 10-7 M tetrodotoxin (TTX, Tocris) to Panulirus saline to block action potentials. We studied the PD LP graded chemical synapse using two different presynaptic stimulation protocols. The first used single, 3 sec square pulses as voltage clamp commands to drive the PD neuron, while the second used a train of 12 linked realistic PD waveforms as voltage clamp commands to drive PD oscillations (see Johnson et al. 2005, 2011 for realistic waveform construction). Averaged and filtered PD waveforms from pyloric recordings had a mean amplitude of 15.5 ± 3.5 mv and a mean cycle period of 688 ± 199 ms (n = 10); we used this shape and cycle period as presynaptic stimuli. Although realistic presynaptic waveforms are more physiologically relevant than square pulse waveforms, we used both stimulation regimes for two reasons. First, some basic characteristics of synaptic depression and its modulation at the PD LP synapse 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 transmission between pyloric neurons have often used square pulse stimulation (for example, Graubard et al. 1983; Johnson et al. 1995; Manor et al. 1997; Zhao et al. 2011), and we wished to compare our results with these earlier studies and determine whether postsynaptic responses differed between the two stimulus forms. Both stimulation protocols used two electrode voltage clamp to drive the PD from a V hold of -55 mv in increasing amplitudes of 5 mv increments up to -25 mv. 6

7 Stepping the PD up to -25 mv evokes near-maximum gipsps in the LP neuron (Johnson and Harris-Warrick 1990; Rabbah and Nadim 2007). We separated stimulation runs by a minimum of thirty seconds, which was sufficient to eliminate all effects of the previous stimulation. We used two electrode current clamp to hold the post-synaptic LP membrane potential at -50 mv; postsynaptic electrodes were filled with 0.6M K 2 SO M KCl and presynaptic electrodes with 3M KCl. In these experiments we used Axoclamp-2A and 2B amplifiers (Molecular Devices). Under control and amine conditions (at least 5 min amine superfusion), we measured the initial peak and steady state amplitudes of the gipsps to calculate input-output (I/O) curves and dynamics of synaptic depression (Figs. 1A, B). The steady state amplitude was measured as the gipsp plateau level at the end of the square pulse stimulation, or as the mean amplitude of the last 5 gipsps in response to the realistic PD waveform train. A synaptic depression index (DI) was calculated as the fractional gipsp decrease from the initial peak response at steady state: DI= (initial peak amplitude steady state amplitude)/initial peak amplitude. Thus, a larger DI corresponds to greater synaptic depression. The time constant (τ Dep ) of the gipsp depression was measured during presynaptic square pulse stimulation by fitting the voltage decay from the initial peak to the steady state amplitude with a single exponential function (Fig. 1A). The rate of synaptic depression was rapid enough that it could not be measured accurately from the separated peaks of the realistic waveform series. In a separate measurement, we quantified the time constant of recovery from synaptic depression, τ Rec, for both realistic and square waveform stimulation under control and amine conditions. We evoked steady state depression by stimulating the PD neuron (V hold, -55 mv) as described above, followed by a single square pulse or realistic oscillation test stimulation at 7

8 increasing times after the end of the last conditioning stimulus. The percent recovery of the test pulse back to the initial peak value over time was fitted with a single exponential in each experiment to calculate τ Rec (see Figs. 2B, C). Data acquisition and analysis. Electrophysiological recordings were digitized at 4KHz using a PCI-6070-E board (National Instruments), and stored on a PC using custom-made recording software (Scope) written in Lab Windows/CVI (National Instruments). This software also controlled the injection of oscillatory and square pulse waveforms as voltage clamp commands into the PD neuron (Johnson et al. 2005, 2011). All data were analyzed with the related custommade software program ReadScope, also written in Lab Windows/CVI; these programs were kindly provided to us by Dr. F. Nadim ( Data were exported to Clampfit software (Molecular Devices) to calculate τ Dep and τ Rec. For statistical comparisons, we used JMP software (SAS Institute, Inc.) to run multivariate analysis followed by post-hoc tests to determine the statistical significance of differences between individual data groups. Statistical differences between mean values were accepted with p < 0.05 (2-tailed probability) for F and t values. Mean measured values and percentages are reported ± SD Results Synaptic dynamics of the PD LP graded synapse under control conditions We first characterized the baseline synaptic dynamics of the PD LP graded synapse under control conditions, using single 3 sec square pulses (n = 10) and repeated presynaptic activation with trains of 12 realistic oscillations (n= 7). As described previously 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 PD transmitter release (approximately -50 8

9 mv), the LP response is a rapid initial peak hyperpolarization which rapidly depresses to a significantly smaller steady state hyperpolarization (p < across PD depolarizations; Fig 1A, C). The LP responds to a train of repeated PD oscillations with an initial peak gipsp followed by subsequent gipsps whose amplitudes depress with repetition to a significantly smaller steady state value over several oscillations (p < across PD depolarizations; Figs. 1B, D; see also Rabbah and Nadim 2007). Peak and steady state gipsp amplitudes increased with increasing PD voltage commands from -40 to -25 mv under both stimulation protocols (Figs. 1C, D). The amplitude of the initial peak LP gipsp tended to be larger when elicited with PD square pulse stimulation than with realistic waveform stimulation (compare Figs. 1C and D open and closed circles), and this difference was significant during PD depolarizations to -30 and -35 mv (p = 0.03 and 0.05, respectively). However, the mean steady state gipsp amplitudes were significantly greater when elicited with oscillations compared to square steps at strong PD depolarizations (to -30 and -25 mv: compare Figs. 1C and D open and closed squares; p = 0.05 and 0.03, respectively). Since square pulse stimulation evoked larger peak initial gipsps and smaller steady-state amplitudes than oscillation stimulation, it also evoked greater synaptic depression at all PD stimulation voltages (Fig. 1E; p < at all PD voltages). For example, the mean Depression Index (DI, defined as (Peak Steady State)/Peak) was twice as large, at PD depolarization to -25 mv, using square waves compared to oscillations (Fig. 1E). This probably results from the greater depression during continued presynaptic stimulation than during trains of oscillations, where the synapse could partially recover between oscillations (see Discussion). There was no significant effect of PD voltage on the magnitude of the depression index over the voltage range of either PD stimulation protocol (Fig. 1E; p = 0.23 and 0.21 for square pulse and oscillations, respectively). 9

10 We also characterized the time constants of synaptic depression and recovery from depression at the PD LP graded synapse using the square pulse series. The decay of the LP response to PD stimulation (Fig. 1A) was well fit by a single exponential (fit correlation r = 0.99 ± 0.001, n = 11). The mean time constant of synaptic depression, τ Dep, was 400 ± 200 ms during PD square pulse steps to -25 mv. There was no voltage dependence of τ Dep during square step stimulation (p = 0.95; Fig. 2A). Due to the comparatively slow cycle frequency of the oscillation stimulation (cycle period 688 msec), we were unable to calculate the faster τ Dep from the oscillation data. In a separate measurement, we characterized the time constant of recovery from depression, τ Rec, using both stimulation protocols (PD steps to -25 mv only), and fitting the gipsps at increasing post-stimulation intervals with a single exponential (Figs. 2B, C; square pulse: exponential fit correlation r = 0.95 ± 0.03; oscillation: fit correlation r = 0.98 ± 0.01). Recovery from synaptic depression was 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 of the recovery process during maintained presynaptic depolarization (see Discussion) Amines change the synaptic dynamics of the PD LP graded synapse Dopamine, 5HT and Oct affect synapses in the pyloric network by a complex set of pre- and post-synaptic actions (Harris-Warrick and Johnson 2010, Johnson and Harris-Warrick 1997). In addition, they can alter synaptic strength indirectly by modifying the waveforms of the pre-or post-synaptic neuronal oscillations (Johnson et al. 2005). In order to focus on synaptic metaplasticity, we attempted to limit the other consequences of amine modulation. For square 10

11 pulse stimulations, we held the pre-synaptic neuron at -55 mv by voltage clamp both before and during amine application. For oscillation stimulation, we used the realistic waveforms obtained from PD oscillations during the normal pyloric rhythm for both control and amine conditions. Thus, the effects of amine-evoked changes in PD oscillations were eliminated. The postsynaptic LP neuron was held at -55 mv under all conditions, to prevent indirect effects of neuronal membrane potential changes evoked by the amines (Flamm and Harris-Warrick 1986). Dopamine The physiological DA concentrations normally achieved in vivo will depend on the spike frequency of the dopaminergic neurons, and are not known. We demonstrated previously that 10-4 M DA greatly reduces or abolishes the LP gipsp in response to square pulse PD stimulation (Johnson and Harris-Warrick 1990). This also occurs using realistic waveform PD stimulation (data not shown), so we decided instead to use a lower concentration of 10-5 M DA, which does not eliminate PD LP transmission (Fig. 3), to examine DA s effects on PD LP graded synaptic dynamics. At this lower concentration, DA had weak and highly variable effects on the LP gipsps in different preparations. Using square pulse PD stimulation, DA reversibly increased initial gipsp amplitudes in about half the preparations (Fig. 3A), and reduced or had little effect on gipsp amplitudes in the other half (Figs. 3B, C); all of these effects reversed upon washout of DA. We suggest that this variability is caused by the known opposing effects of DA to decrease pre-synaptic PD transmitter release and increase post-synaptic LP input resistance (Harris-Warrick and Johnson 2010), but to differing amounts in different preparations. On average, 10-5 M DA did not significantly change the mean amplitude of the peak and steady state components of the LP gipsp during PD square pulse stimulation (Fig. 3D, n = 5; p > 0.3 for both). In contrast, during oscillation stimulation, DA significantly increased the mean first 11

12 gipsp amplitude when compared across all PD depolarizations (example shown in Fig. 3E; p = 0.02; n = 16). However, this effect was weak, and only produced a trend to increase initial peak gipsp amplitude at single PD voltage step amplitudes (Fig. 3D; p = 0.08; n = 4 each). DA did not significantly change the steady state gipsp amplitude across PD oscillation steps (Fig 3D; p = 0.26). With regard to synaptic depression, there was no significant effect of DA on the amplitude of synaptic depression, as measured by the DI, for either square pulses (Control DI: 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 ± 0.17 at -25 mv) at any PD voltage (p > 0.10 for both). Despite these weak and variable effects on the amplitudes of LP gipsps, DA consistently accelerated both the onset of, and the recovery from, synaptic depression. Figure 3C shows a typical square wave measurement, where τ Dep was accelerated by DA compared to the control gipsp response, with little change in gipsp amplitude. The mean τ Dep was significantly decreased over the entire PD voltage range (Fig. 4A; n = 5, p < 0.005), but the DA effect showed no significant voltage dependence (p = 0.78). Recovery from depression was also faster with both square pulse and realistic waveform stimulation (Fig. 4A). This is seen in the example using oscillation stimulation in Figure 4B, which shows more complete recovery of the depressed gipsp 1 sec after the stimulus train during DA (89% recovery) than under control conditions (72% recovery). The mean τ Rec at -25 mv PD depolarization was significantly faster in DA for both square pulse PD stimulation (Fig. 4A, 39 ± 23% τ Rec decrease; p < 0.001, n = 5) and realistic waveform PD stimulation (Fig. 4A, 43 ± 19% τ Rec decrease; p = 0.03, n = 4). This acceleration of onset and recovery from depression did not depend on the sign of the rather variable DA effect on gipsp amplitudes described above. As summarized in Figure 4C, in all 12

13 but 1 experiment the τ Dep and τ Rec values were smaller while DA either increased or decreased gipsp amplitude. Thus, while DA (10-5 M) had highly variable effects on gipsp amplitude, it had reliable, significant and independent effects to accelerate the time course of synaptic depression onset and recovery from synaptic depression Octopamine We previously demonstrated that 10-5 M Oct strengthens the PD LP synapse when tested with square wave PD stimulation (Johnson and Harris-Warrick 1990). In our current experiments, Oct consistently increased the amplitude of gipsps over the full range of presynaptic voltages using both square pulse (n = 5) and realistic (n= 4) PD waveforms (Figs. 5A, B). The peak and steady state responses to square pulse stimulation were significantly larger during Oct application at all PD depolarizations (p < and p = 0.004, respectively): during a PD step to-25 mv, the mean peak gipsp increased by 55 ± 29% while the steady state gipsp increased by 157 ± 32% (Fig. 5C). As can be seen in Figure 5C, Oct increased the steady state gipsp amplitude significantly more than the initial peak amplitude across the voltage steps (p = 0.04). Using the oscillation protocol, Oct also increased the first peak and steady state gipsp amplitudes across all PD stimulation voltages (p < for both); for example, at -25 mv, the peak amplitudes increased by 57 ± 33% while the steady state amplitudes increased by 48 ± 37% (Fig. 5C). Unlike the results using square pulse stimulation, during oscillation stimulation there was no significant difference between Oct s enhancement of the peak and the steady state gipsp amplitudes at any PD voltage (p = 0.15; Fig. 5C). There was no voltage dependence of the Oct effect on gipsp amplitude. 13

14 During square pulse stimulation, the greater Oct enhancement of the steady state over the peak gipsp amplitude resulted in a significant decrease in the depression index across PD depolarizations (16 ± 6% decrease at -25 mv, p = ; Fig. 5D). However, there was no equivalent DI decrease using oscillation stimulation, because Oct did not differentially increase the first peak and steady state amplitudes of the gipsp (Fig. 5C, D; p = 0.15). Octopamine significantly altered the onset and recovery of synaptic depression in response to square pulse PD stimulation, in a way that is quite different from DA. Octopamine significantly slowed τ Dep across PD steps (increase by 80 ± 41 % at -25 mv; n = 5, p < ; Fig. 6A). This slowing of synaptic depression was not caused simply by the larger gipsps evoked during Oct. When gipsp amplitudes were matched in the presence and absence of Oct using different amplitude pre-synaptic PD steps in the same preparation, τ Dep remained slower during Oct (Fig. 6B). In contrast, Oct significantly accelerated the recovery from depression using square pulse PD stimulation (Fig. 6C), by an average of 47 ± 24% using -25 mv steps (n = 4; p = 0.026, Fig. 6A). However, when measured with oscillation stimulation, Oct had highly variable effects on τ Rec which could either accelerate or slow down, and were not significant overall (Control: 1120 ± 610 ms, Oct: 1510 ± 1510 ms; p= 0.63). Thus the main effect of Oct was to increase PD LP synaptic strength using both PD stimulation protocols. Using square pulse PD stimulation, Oct reduced the depression amplitude and slowed the onset of synaptic depression, while speeding up the rate of recovery from depression. Serotonin We previously showed that 10-5 M 5HT weakens the PD LP synapse when tested with square wave PD stimulation (Johnson and Harris-Warrick 1990). In our recent experiments, we have reproduced these results using both square pulse (n = 4) and oscillation (n= 6) PD 14

15 waveforms (Figs. 7A, B). Using square pulse stimulation, the mean initial peak and steady state responses were consistently and significantly smaller during 5HT application at all PD depolarizations (p < for initial peak and p = for steady state). This effect did not show any voltage dependence: with -25 mv steps, the peak gipsps fell by 28 ± 17% and the steady state gipsps fell by 40 ± 12% (Fig. 7C). Similar results were seen using oscillation PD stimulation at all voltages (p = and < for initial peak and steady state responses, respectively): at -25 mv PD depolarization, the peak gipsps fell by 20 ± 12% and the steady state gipsps by 27 ± 10% (Fig. 7C). During 5HT application, synaptic depression was significantly greater across all PD depolarizations using square pulse (12 ± 8% greater at -25 mv; p = ; Fig. 7D), and oscillation stimulation (32 ± 38% greater at -25 mv; p = 0.004; Fig 7D). Serotonin significantly accelerated the time course of synaptic depression measured with square pulse PD stimulation, and tended to accelerate the time course of recovery from depression (Fig. 8A), though this effect did not reach statistical significance. The acceleration in τ Dep was not caused by a smaller gipsp during 5HT. For example, the trace in Fig. 8B shows that when sized matched (using different pre-synaptic voltage steps), the gipsp recorded during 5HT depressed more quickly than in control conditions. Serotonin significantly accelerated τ Dep with square pulses across PD voltages (p = 0.02); at -25 mv PD depolarization, τ Dep was reduced by 25 ± 34 % (Fig. 8A; n = 4). In 3 of 3 experiments, 5HT decreased τ Rec by 28 ± 14%, but this did not reach statistical significance with the small sample size and variability, (p = 0.10; Fig. 8A). Serotonin also decreased τ Rec in 5 of 5 experiments by 26 ± 27 %, measured by oscillation stimulation to -25 mv (see example in Fig. 8C), but this also did not reach our criterion for statistical significance (p = 0.07; Fig. 8A). Thus, 5HT reduces PD LP synaptic strength, 15

16 increases the rate of onset of synaptic depression and the amount of synaptic depression, while tending to weakly accelerate the recovery from depression

17 Discussion Amine-induced metaplasticity at the PD LP graded synapse Graded chemical synapses of the pyloric and gastric CPG networks of the lobster STG show marked synaptic depression with prolonged voltage steps or repeated trains of presynaptic activation; all pyloric synapses are tonically depressed at normal pyloric cycle frequencies (reviewed by Hartline and Graubard, 1992; see also Johnson et al. 2005, 2011; Mamiya and Nadim 2004; Manor et al. 1997). Synaptic depression is commonly seen at graded chemical synapses, such as those between retinal neurons and from hair cells to afferent fibers (Edmonds et al. 2004; Wan and Heidelberger 2011), although not all graded chemical synapses show short term activity-dependent changes in synaptic strength (Narayan et al. 2011; Simmons 1981). Aside from studies with pyloric network synapses, little previous work has examined neuromodulation of synaptic dynamics at graded chemical synapses. We have previously described amine effects on the strength of the PD LP graded synapse (Johnson and Harris- Warrick 1990), and here we demonstrate that the magnitude and kinetics of short-term synaptic dynamics are also modulated by amines at this synapse. The most surprising conclusion from our results is that the synaptic strength and the magnitude of synaptic depression, as well as the kinetics of its onset and recovery, can be independently modulated by each amine. We suggest that this may occur through multiple pre and post-synaptic mechanisms of neuromodulatory actions that shape synaptic strength in a functioning network. Dopamine We have previously demonstrated that DA (10-4 M) increases graded synaptic strength at the LP PY and LP PD synapses, but only changes (increases) the amount of synaptic depression at the LP PY synapse (Johnson et al. 2005, 2011). Thus DA can have differential effects on 17

18 synaptic depression and synaptic strength depending on the postsynaptic target. We used 10-5 M DA in the present study because 10-4 M DA abolishes functional synaptic transmission at the PD LP graded synapse (Johnson and Harris Warrick 1990). At the lower concentration, DA consistently accelerated τ Dep and τ Rec despite small and variable effects on PD LP synaptic strength and on the magnitude of synaptic depression. This demonstrates that the kinetics of synaptic depression can be modulated independently of synaptic strength and the magnitude of synaptic depression. At present, we cannot correlate 10-5 M DA s variable actions on synaptic strength with its effects on other cellular properties. None of the known effects of DA on pre- or postsynaptic ionic currents described previously (Harris-Warrick and Johnson 2010) can account for the DA-induced changes in depression kinetics. DA may be acting postsynaptically to accelerate the kinetics of transmitter receptor desensitization and recovery from desensitization (Papke et al. 2011). Motor patterns produced by the lobster pyloric network are qualitatively different when treated with 10-4 and 10-5 M DA (Flamm and Harris-Warrick 1986). Concentration-dependent DA effects on the onset and recovery of synaptic depression may contribute to shaping distinct DA-induced network activity. Octopamine Octopamine s enhancement of synaptic strength was accompanied by reduced synaptic depression, slowed τ Dep and accelerated τ Rec for PD square pulse stimulation, but not PD oscillation stimulation. Part of the synaptic strength increase by Oct may be due to its increase in postsynaptic LP input resistance (Johnson et al. 1993), but the change in synaptic depression suggests more dynamic effects on synaptic transmission. AP-evoked synaptic transmission at crustacean and vertebrate central synapses depresses more at synapses with high transmitter output than with low transmitter output and depresses less with low transmitter output (Miller 18

19 and Atwood 2004; Thomson 2000). Reduced synaptic depression coupled with increased synaptic strength during Oct does not fit this pattern, which would directly link transmitter release probability with short term synaptic dynamics. Instead, at the PD LP graded synapse, enhancement of presynaptic I Ca by Oct could lead to both greater transmitter release and more rapid mobilization of vesicles into the readily releasable pool, thus leading to reduced depression and accelerated recovery from depression (Babai et al. 2010; Gomis et al. 1999; see below). However, the effects of Oct on PD ionic currents are not known. Serotonin Serotonin reduced PD LP graded synaptic strength but increased synaptic depression; again, this does not fit a pattern directly linking synaptic dynamics with the amount of transmitter release. Serotonin s effects could reflect pre-synaptic mechanisms such as a reduction in I Ca(V), which would reduce release and slow the rate of vesicle mobilization. Serotonin may have additional postsynaptic effects besides decreasing LP input resistance (Johnson et al. 1993), as described above for DA. Serotonin showed a trend to accelerate both the onset and recovery from depression, which would allow greater flexibility of the synapse as the cycle frequency changes; the accelerated recovery may limit the reduction in synaptic strength during the normal oscillating activity of the neurons in the pyloric rhythm. More work is needed to clarify the mechanisms for the metaplastic effects of amines, but it is clear that amines have complex and sometimes opposing effects on short term dynamics of graded synaptic transmission in the pyloric network. Characteristics of synaptic dynamics at the PD LP graded synapse The use of both square pulse and oscillation stimulation protocols allowed us to probe characteristics of synaptic depression and its modulation at the PD LP graded synapse of the 19

20 pyloric that might not be evident with either stimulation protocol alone. First, we found that the magnitude of the synaptic depression was greater using square pulse stimulation of PD than with trains of PD oscillations, emphasizing that realistic oscillation stimulation allows a more physiological characterization of pyloric synapses (Manor et al. 1997). Weaker synaptic depression with realistic oscillation stimulation may be explained simply by the partial recovery from depression that occurs between the PD oscillations. Second, we could more accurately determine the time course of onset of depression with long square pulse PD stimulation. The onset of depression, measured with square pulse PD stimulation, had a time constant of ~400 ms, and could not be accurately measured with oscillations occurring every 688 msec. Values for τ Dep vary from ms to tens of seconds at other AP-evoked and graded chemical synapses (Cho et al. 2011; Wang and Manis 2008; Zucker and Regehr 2002). The recovery from depression was over twice as fast using square pulse PD stimulation (τ Rec ~600 ms) compared to PD oscillations (τ Rec ~ 1600 ms). Our τ Rec times are longer that those previously reported by Rabbah and Nadim (2007) at the PD LP graded synapse, but the stimulation protocols differed between the two studies. Time constants for recovery from depression range from 10s of msec at hair cell synapses to seconds at other graded synapses, again depending on experimental conditions (Rabl et al. 2006; Wan and Heidelberger 2011). Finally, amines sometimes affected synaptic depression and its onset and recovery kinetics differentially (see discussion above), suggesting more complex actions on synaptic dynamics than would be seen with PD oscillation stimulation alone. The most common mechanism of synaptic depression at both action potential evoked and graded chemical synapses is transmitter depletion during prolonged or repeated synaptic activity (von Gersdorff and Matthews 1997; Zucker and Regher 2002); other reported pre-synaptic 20

21 mechanisms include calcium channel inactivation (Xu et al. 2007), autoreceptor activation (Davies et al. 1990), and accumulating refractoriness of the transmitter release mechanism (Waldeck et al. 2000). Our experiments were not designed to determine the site of depression, but they suggest a mixed mechanism for depression at the PD LP graded synapse. Changing the length of the pre-synaptic square pulse, or the duration of the PD oscillations, altered the level of depression (Johnson et al. 2005). In addition, the faster τ Rec using square pulse PD stimulation, which would accumulate more intracellular calcium, is consistent with a calciumdependent replenishment of the readily releasable transmitter pool, seen at AP-evoked (Neher and Sakaba 2008) and graded synapses (Babai et al. 2010; Gomis et al. 1999). We also found that the magnitude of PD LP depression and its onset kinetics were not sensitive to presynaptic voltage stimulation, suggesting that depression was independent of the amount of transmitter release. The pre-synaptic voltage independence of both the depression magnitude and τ Dep is inconsistent with a simple pre-synaptic transmitter depletion model of synaptic depression. The lack of correlation between the amount of synaptic depression and gipsp amplitude at the PD LP graded synapse is also seen at AP-evoked synapses from other preparations where synaptic depression is clearly due to a presynaptic mechanism, but its magnitude is independent of PSP amplitude (Hefft et al. 2002; Wu et al. 2005). There may also be a postsynaptic contribution at our synapse, such as receptor desensitization (Jonas and Westbrook 1995; Papke et al. 2011; Trussell et al. 1993). Possible functional importance of metaplasticity in the pyloric network Short term synaptic depression plays an important role in organizing a flexible pyloric motor pattern. The magnitude of depression and its onset and recovery kinetics dynamically determine the synaptic strength at different pyloric cycle periods, and when pyloric rhythm 21

22 periods are perturbed by interactions with linked networks such as from the cardiac sac and gastric mill motor networks (Ayali and Harris Warrick 1998; Russell and Hartline 1981; Thurma and Hooper 2002, 2003). Synaptic depression promotes phase maintenance as the pyloric network changes its cycle frequency (Greenburg and Manor 2005; Manor et al. 2003; Nadim et al. 2003), and contributes to stabilization of the rhythm period (Mamiya and Nadim 2004). It can enable a switch between different forms of network bistability (Nadim and Manor 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 maintain or reset synaptic strengths between network neurons to adjust the post-synaptic neurons firing phase. For example, the 10-5 M DA effects to accelerate both τ Dep and τ Rec with no change in gipsp amplitude or depression could stabilize PD LP synaptic strength as the cycle frequency changes in DA (Johnson et al. 2011). Octopamine enhances LP excitability, but does not change the LP firing onset phase (Johnson et al. 2011). The greater PD LP synaptic strength in Oct, with reduced depression and faster onset and recovery kinetics, could balance enhanced LP excitability to maintain LP phasing at its pre-oct values (Johnson et al. 2011). There is also no change in LP onset firing phase during 5HT despite a reduction in LP excitability (Johnson et al. 2011). A faster τ Dep might contribute to weakening the PD LP gipsp and to enhanced depression, and this may be mitigated by the accelerated τ Rec to maintain the LP firing onset phase. At the crab LP PY graded synapse, the peptide proctolin changes the sign of synaptic plasticity from depression to facilitation at certain presynaptic voltage levels, and functions to help stabilize the cycle period (Zhao et al. 2011). 22

23 Metaplasticity through the actions of many different neuromodulators is a common feature of many neural networks (Barriere et al. 2008; Carey et al. 2011; Kreitzer and Regehr 2000; Parker 2001; Sakurai and Katz 2009), and should be considered one of the building blocks of network operation. Although well studied at AP-evoked synapses, there is relatively little known about metamodulation at graded chemical synapses. The pyloric network of crustaceans provides an excellent model system to explore the ranges of expression of metaplasticity at graded synapses, and its functional role in organizing network operation and plasticity (Johnson et al. 2005; 2011; Zhao et al. 2011)

24 Acknowledgements We thank Françoise Vermeylen from the Cornell Statistical Consulting Unit for help with the statistical analysis. Grants Research supported by NIH NS17323 to R.M. Harris-Warrick Disclosures No conflicts of interest financial or otherwise, are declared by the authors Author Contributions B.R.J., R.M.H.-W. and M.D.K. designed the research; M.D.K. performed experiments; M.D.K. and B.R.J. analyzed data, and interpreted results with R.M.H.-W.; B.R.J. and M.D.K. prepared figures; B.R.J., R.M.H.-W., and M.D.K. wrote and edited the manuscript

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33 Figure legends Figure 1. Synaptic dynamics of graded synaptic transmission at the PD LP chemical synapse. A. LP gipsps in response to 3 second PD square pulse depolarizations. Initial peak and steady state IPSP amplitudes and the time constant (τ Dep ) for synaptic depression were measured for all PD depolarizations from V hold = -55 mv. B. LP gipsps during a train of 12 realistic PD oscillations of increasing amplitude from V hold = -55 mv. The first gipsp amplitude (initial peak) and the mean of the last 5 gipsps amplitudes (steady state) were measured for all PD depolarizations. C. Mean input/output relationship for the amplitude of the initial peak (open circles) and steady state (open squares) gipsps using square pulse PD stimulation (n = 11). D. Mean input/output relationship for initial (closed circles) and steady state (closed squares) LP gipsp amplitudes using PD oscillation stimulation (n = 7). E. Mean depression index for square pulse (open bars) and oscillation (closed bars) stimulation across PD stimulation voltages. * indicates that the depression index for PD square pulse stimulation is significantly greater than for PD oscillation stimulation (p < for all PD voltages). The depression index did not show voltage dependence with either stimulation protocol (p > 0.20 for both). Figure 2. Synaptic depression and recovery at the PD LP graded synapse. A. Mean τ Dep across PD square pulse stimulation voltages (n = 11). τ Dep did not show any voltage dependence (p = 0.95). B. Measurement of the time constant of LP gipsp recovery from depression (τ Rec ) in response to PD square pulse depolarization to -25 mv. C. Measurement of τ Rec using PD oscillation depolarization to -25 mv. D. Mean τ Rec for LP gipsps elicited by PD square pulse (n = 13) and oscillation (n = 10) stimulation. * indicates significantly longer τ Rec with PD oscillation stimulation (p = 0.003). Figure 3. Dopamine (10-5 M) modulation of synaptic strength at the PD LP synapse. Examples of variable effects of DA to reversibly enhance (A), reduce (B), or have no effect (C) on the initial LP gipsp amplitude with PD square pulse stimulation. D. Mean effects of DA on amplitudes of initial peak and steady state LP gipsps elicited by PD square pulse (initial peak, open bars; steady state, light gray bars) and oscillation stimulation (initial peak, closed bars; 33

34 steady state, dark gray bars) at -25 mv PD depolarization. E. Example showing DA enhancement of the initial peak gipsp during PD oscillation stimulation. Figure 4. Dopamine acceleration of synaptic depression and recovery at the PD LP synapse. A. Mean effects of DA on τ Dep (left open bars; n = 5), and τ Rec (middle open bars; n = 5) for PD square pulse stimulation, and for τ Rec during PD oscillation stimulation (right closed bars; n = 4). * indicates significantly shorter τ in DA compared to control conditions (p < for all). B. Example showing more rapid recovery of gipsp amplitude during DA after a 1 sec interval following PD oscillation stimuli, and no effect of DA on the initial peak gipsp. Gray lines mark the amplitude of the initial peak gipsp. C. Lack of correlation between DA effects on initial peak LP gipsp and its effects on depression and recovery in individual experiments. Open squares, τ Dep and open circles, τ Rec for PD square pulse stimulation; closed circles, τ Rec for PD oscillation stimulation. Figure 5. Octopamine (10-5 M) enhances synaptic strength and reduces synaptic depression at the PD LP synapse. Examples showing Oct enhancement of initial peak and steady state gipsp amplitudes using PD square pulse (A) and oscillation stimulation (B). Black traces, control conditions; gray traces, Oct. C. Oct enhances mean absolute amplitudes of initial peak and steady state LP gipsps elicited by PD square pulse (initial peak, open bars; steady state, light gray bars; n = 5) and oscillation stimulation (initial peak, closed bars; steady state, dark gray bars; n = 4) at -25 mv PD depolarization. *: significant increase (p < for all); #: Oct enhances the initial peak gipsp amplitude significantly less than the steady state gipsp during PD square pulse stimulation (p = 0.04). D. Oct significantly decreases synaptic depression with PD square pulse but not with oscillation stimulation at -25 mv PD depolarization. *: significant reduction (p < ). Figure 6. Octopamine modulation of synaptic depression and recovery at the PD LP synapse. A. Octopamine increases τ Dep (left open bars; n = 5) and decreases τ Rec (right open bars; n = 4) at -25 mv PD square pulse depolarization. *: significant effects (p < 0.03 for both). B. Example using PD square pulse depolarizations of differing amplitudes to adjust the LP gipsp to the same amplitude under control and Oct conditions, showing the Oct-induced increase in τ Dep in size 34

35 matched LP gipsps (control, black traces; Oct, gray traces). C. Example of normalized initial peak gipsps in control (black traces) and Oct (gray traces), with more rapid gipsp recovery in Oct during PD square pulse depolarizations to -25 mv. Figure 7. Serotonin (10-5 M) decreases synaptic strength and enhances synaptic depression at the PD LP synapse. Examples showing 5HT reduction of initial peak and steady state gipsp amplitudes using PD square pulse (A) and oscillation stimulation (B). Black traces, control conditions; gray traces, 5HT. C. 5HT reduces the amplitudes of initial peak and steady state LP gipsps elicited by PD square pulse (initial peak, open bars; steady state, light gray bars; n = 4) and oscillation stimulation (initial peak, closed bars; steady state, dark gray bars; n = 6) at -25 mv PD depolarization. *: significant at p < for both stimulation protocols. D. 5HT increases synaptic depression. *: significant at p < for both stimulation protocols. Figure 8. Serotonin modulation of kinetics of synaptic depression and recovery at the PD LP synapse. A. 5HT reduces τ Dep (left open bars; n = 4) and has only weak effects on τ Rec (middle open bars; n = 3) using -25 mv PD square pulse depolarization, and τ Rec (right closed bars; n = 5) using -25 mv PD oscillation depolarization. *: significant at p = B. Example with PD square pulse depolarizations adjusted in control and 5HT conditions to show the 5HT-induced acceleration of τ Dep in size matched LP gipsps (control, black traces; 5HT, gray traces). C. Example showing normalized initial peak gipsps in control (black traces) and 5HT (gray traces), and more rapid gipsp recovery in 5HT 1 sec after the end of PD oscillations. Gray lines mark the amplitudes of initial peak gipsps

36 A B PD -55 mv 10 mv -50 mv C LP 8 τ steady state Dep 1s PD Square Pulse 2mV D 8 initial peak PD Oscillation steady state 1s 2mV LP gipsp (mv) initial peak steady state E Depression Index PD Potential (mv) PD Square * PD Oscillation * * * PD Potential (mv) PD Potential (mv)

37 A gipsp τ Dep (ms) C PD -55 mv -50 mv LP PD Potential (mv) τ Rec = 2900 ms B PD -55 mv -50 mv LP 20 mv 1mV 1s D gipsp τ Rec (ms) τ Rec = 516 ms PD Square * 1s PD Oscillation 10 mv 1mV

38 A PD -55 mv LP -50 mv B DA DA -50 mv Ctl Wash -50 mv DA Ctl C Ctl Wash 10 mv 5mV D E LP gipsp (mv) Ctl DA PD Square Ctl DA PD Oscillation 2mV PD -55 mv 10 mv -50 mv 2mV LP 1mV 1s DA 1s

39 A 2500 gipsp τ (ms) τ Dep * τ Rec * τ Rec * B PD -55 mv -50 mv LP 0 Ctl DA Ctl DA Ctl DA PD Square PD Square PD Oscillation 10 mv Ctl 1mV -50 mv C 25 DA 1mV 1s 0 % Change τ τ Dep τ RecSq τ RecOsc % Change Initial Peak gipsp

40 A B PD -55 mv -50 mv LP C LP gipsp (mv) mv Ctl 5mV Oct 1s Oct Ctl Oct Ctl Oct 0.8 Ctl Oct Ctl PD Square Pulse * # * * * initial peak steady state Depression Index D * 10 mv 5mV 1s Oct 0 PD Square PD Oscillation 0 PD Square PD Oscillation

41 A 1200 τ Dep τ Rec gipsp τ (ms) * * 300 B C 0 PD -55 mv -50 mv LP PD -55 mv -50 mv LP Ctl Oct Ctl Oct PD Square PD Square Ctl Oct 10 mv Ctl Oct 5mV 1s 10 mv 1mV 1s 1mV