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Distinct Synaptic Dynamics of Heterogeneous Pacemaker Neurons in an Oscillatory Network

Department of Mathematical Sciences, New Jersey Institute of Technology and Department of Biological Sciences, Rutgers University, Newark, New Jersey

Submitted 31 October 2006; accepted in final form 21 December 2006

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Many rhythmically active networks involve heterogeneous populations of pacemaker neurons with potentially distinct synaptic outputs that can be differentially targeted by extrinsic inputs or neuromodulators, thereby increasing possible network output patterns. To understand the roles of heterogeneous pacemaker neurons, we characterized differences in synaptic output from the anterior burster (AB) and pyloric dilator (PD) neurons in the lobster pyloric network. These intrinsically distinct neurons are strongly electrically coupled, coactive, and constitute the pyloric pacemaker ensemble. During pyloric oscillations, the pacemaker neurons produce compound inhibitory synaptic connections to the follower lateral pyloric (LP) and pyloric constrictor (PY) neurons, which fire out of phase with AB/PD and with different delay times. Using pharmacological blockers, we separated the synapses originating from the AB and PD neurons and investigated their temporal dynamics. These synapses exhibited distinct short-term dynamics, depending on the presynaptic neuron type, and had different relative contributions to the total synaptic output depending on waveform shape and cycle frequency. However, paired comparisons revealed that the amplitude or dynamics of synapses from either the AB or PD neuron did not depend on the postsynaptic neuron type, LP or PY. To address the functional implications of these findings, we examined the correlation between synaptic inputs from the pacemakers and the burst onset phase of the LP and PY neurons in the ongoing pyloric rhythm. These comparisons showed that the activity of the LP and PY neurons is influenced by the peak phase and amplitude of the synaptic inputs from the pacemaker neurons.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Many rhythmically active motor networks are driven by pacemaker neurons that act as core oscillators, synchronizing the activity of other network neurons into a coherent rhythmic pattern (Koshiya and Smith 1999; Marder and Bucher 2001; Smith et al. 1991; Tresch and Kiehn 2000; Wallen and Grillner 1987). In such pacemaker-driven pattern-generating networks, it is common that the pacemaker neurons use chemical synaptic inhibition to set the relative activity patterns of the remaining neurons (Eisen and Marder 1984; Tryba and Ritzmann 2000; Wolf 1992) and that the efficacy of these synapses can be altered by neuromodulators to reshape the network output (Harris-Warrick et al. 1998; Marder 1994). Moreover, heterogeneous populations of pacemaker neurons, as in the vertebrate respiratory system, can be differentially targeted by neuromodulators, thus increasing the number of possible network output patterns (Ramirez et al. 2004).

The rhythmically active pyloric central pattern generator of the spiny lobster, Panulirus interruptus, is driven by a pacemaker ensemble that consists of two intrinsically distinct neuron types: the anterior burster (AB) and two pyloric dilator (PD) neurons. The pacemaker neurons exhibit similar, but not identical, synaptic connections to all other follower neurons (Eisen and Marder 1982). During the ongoing pyloric rhythm (frequency 0.5–2 Hz), the AB and PD neurons are coactive because of their strong electrical coupling and thus produce a compound inhibitory chemical postsynaptic potential onto each of the follower neurons. In a recent study, we characterized the dynamics of the compound synapse from this pacemaker ensemble onto two classes of follower LP and PY neurons (Rabbah and Nadim 2005). The PY neurons always lag the LP neuron in their activity phase; thus we examined the hypothesis that this phase lag is partially explained by distinct synaptic inputs from the pacemaker ensemble. Surprisingly, our results showed that the total synaptic effect of the pacemaker ensemble on these two classes of follower neurons is identical.

Previous studies reported that the AB and PD neurons evoke inhibitory postsynaptic potentials (IPSPs) in the follower neurons that differ in their neurotransmitter type, time course of release, reversal potential, and ion selectivity (Eisen and Marder 1982, 1984; Rabbah et al. 2002). Furthermore, exogenous neuromodulators differentially affect the intrinsic and synaptic properties of these neuron types, subsequently altering their relative synaptic influence on follower neurons and thus on the pyloric oscillation pattern (Ayali and Harris-Warrick 1999; Harris-Warrick et al. 1998; Johnson and Harris-Warrick 1997). It is therefore important to characterize the individual synaptic outputs of the AB and PD neurons. In this study, we examined whether the synapses made by each of the pacemaker AB and PD neurons differ in their short-term plasticity, temporal dynamics, and dependency on presynaptic waveform shapes. We first characterized the dynamics of the synapses from the AB and PD neurons as a combined unit and then individually after blocking each synapse. We activated the synapses using various injection stimuli applied to the voltage-clamped presynaptic neurons and recorded simultaneously from the follower LP and PY neurons. We also examined the correlations between parameters describing the individual pacemaker synapses and the activity phase of the follower neurons. Our results show that the synapses to each of the two classes of follower neurons exhibit distinct dynamics depending on the presynaptic pacemaker neuron type, AB or PD. These results suggest that neuromodulatory inputs can differentially accentuate one set of dynamics over the other, allowing for selective control of the activity phase of the postsynaptic targets and thus leading to different network outputs.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Preparation and identification of the neurons

Adult male spiny lobsters (P. interruptus) were purchased from Don Tomlinson Fisheries (San Diego, CA) and maintained in artificial seawater tanks at 12–15°C until use. Before each dissection, the animals were anesthetized by cooling in ice for 30 min. The stomatogastric nervous system [STNS; including the stomatogastric ganglion (STG), the esophageal, and the commissural ganglia] was removed using standard methods (Harris-Warrick et al. 1992; Selverston et al. 1976). The STG was desheathed to allow penetration of the cell bodies and superfused using normal saline at 18°C, pH 7.35, containing (in mM): 12.8 KCl, 479 NaCl, 13.7 CaCl₂, 10.0 MgSO₄, 3.9 NaSO₄, 11.2 Trizma base, and 5.1 maleic acid.

For neuron impalement, glass microelectrodes were pulled using a Flaming-Brown micropipette puller (P87, Sutter Instruments, Novato, CA) and filled with 0.6 M K₂SO₄ + 0.02 M KCl (resistances of 8–13 M). Identification of the neurons was accomplished by matching intracellular action potential recordings to their corresponding extracellular recordings on motor nerves (Harris-Warrick et al. 1992; Selverston et al. 1976). Intracellular recordings were made from the soma of the neurons using Axoclamp 2B amplifiers (Molecular Devices, Foster City, CA) and extracellular recordings were amplified using a Differential AC amplifier model 1700 (A-M Systems, Carlsborg, WA).

Isolation of synapses from the AB and PD neurons to the LP and PY neurons

During the normal ongoing pyloric rhythm, the AB and PD neurons oscillate in synchrony as a result of their strong electrical coupling and so the follower neurons experience a compound inhibitory postsynaptic potential from both the AB and PD neurons (IPSP_AB/PD). Voltage-clamp stimulations of the two PD neurons in control saline are sufficient to elicit synaptic release from both AB and PD neurons (Rabbah and Nadim 2005). To characterize the synaptic dynamics of the AB and PD neurons separately, we stimulated the PD neurons while the preparation was superfused with 5 µM picrotoxin (PTX; Marder and Paupardin-Tritsch 1978). PTX blocks the glutamatergic synaptic release from the AB neuron and thus IPSPs recorded in the follower neurons were those elicited solely by the PD neurons (IPSP_PD). Because there are no known gap-junctional blockers in the STG, to isolate the component of the chemical synapse that was elicited by the AB neuron (IPSP_AB), IPSP_PD elicited in PTX conditions were digitally subtracted from IPSP_AB/PD elicited in control conditions. In separate experiments, 1 mM tetraethylammonium (TEA) was used to block the cholinergic PD synapses (Marder and Eisen 1984). The results obtained when using TEA were not quantitatively significantly different from the results obtained when the PTX traces were subtracted from the control traces (data not shown; n = 5). The data reported in this study are from the subtracted traces only. Note that the low concentration of TEA used is not sufficient to significantly block potassium currents in these neurons (Graubard and Hartline 1991; Kloppenburg et al. 1999; Peck et al. 2001).

The pacemaker neurons are connected to the ventricular dilator (VD) neuron by a mixed chemical and electrical synapse. VD also forms inhibitory chemical connections to both the LP and PY neurons (Eisen and Marder 1982). As a precautionary measure to eliminate any possible contamination of the AB/PD-induced IPSPs in the LP and PY neurons by the VD neuron, the VD neuron was photoinactivated in all experiments. The complete photoinactivation procedure is outlined in Eisen and Marder (1984), Miller and Selverston (1979), and Miller and Selverston (1982).

Comparison of IPSP_AB and IPSP_PD to the LP and PY neurons

The inhibitory synapses between the pacemaker neurons and the follower neurons use voltage-dependent (graded) release of neurotransmitter as the major form of transmission (Graubard 1978; Graubard et al. 1980; Johnson and Harris-Warrick 1990; Johnson et al. 1995; Maynard and Walton 1975). This form of synaptic release was isolated from the spike-mediated transmission by blocking the latter with Panulirus saline containing 10^–7 M tetrodotoxin (TTX; Biotium, Hayward, CA). Spontaneous rhythmic activity and modulatory inputs from anterior ganglia are also blocked by TTX (Raper 1979). The experiments were carried out in voltage clamp to better control the membrane potential of the presynaptic neurons. One PD neuron was voltage clamped with two electrodes to a holding membrane potential (V_hold) of –60 mV. The second PD neuron was impaled with one electrode. The current used to voltage clamp the first PD neuron was scaled up (using the specification of the amplifier) by a Brownlee Precision Amplifier (Santa Clara, CA) and injected into the second PD neuron to effectively voltage clamp the second PD neuron to the same V_hold with only one electrode (see Rabbah and Nadim 2005). The LP and PY neurons were impaled with one electrode each and the graded IPSPs, elicited by the stimulation of the PD neurons, were recorded in current clamp (see schematic in Fig. 1B). Only paired recordings of the LP and PY were used in this study.

View larger version (14K): [in this window] [in a new window]   FIG. 1. Pyloric network and experimental protocol. A: simultaneous intracellular recordings indicate that the anterior burster (AB) and pyloric dilator (PD) neurons oscillate in synchrony and inhibit the follower lateral pyloric (LP) and pyloric constrictor (PY) neurons, which burst out of phase with the AB/PD neurons and each other. PD burst onset was used a reference point (dashed line) to calculate Periodnatural and the LP and PY burst phases. Phase is calculated by dividing the time difference between burst onsets (t) by Periodnatural. Baselines for the membrane potential oscillations were –62 mV for AB, –57 mV for PD, –58 mV for LP, and –62 mV for PY. For simplicity, recording of only one of 2 PD neurons is shown. B: synaptic connectivity and the experimental paradigm represented schematically. All chemical synaptic connections (ball and stick symbols) are inhibitory; resistor symbols indicate gap-junctional couplings. All neurons were impaled with one electrode except for one PD neuron that was impaled with 2 for voltage clamp. Current used to voltage clamp that PD neuron was scaled and injected into the other PD neuron to voltage clamp the latter neuron to the same Vclamp value. Paired recordings of the LP and PY neurons were carried out in current clamp.

Whenever trains of pulses were used to activate the presynaptic neurons (square or realistic waveforms), the resulting IPSP maximal peak amplitudes were always measured from resting potential (V_rest) of the postsynaptic neuron as opposed to the respective baseline of each IPSP. This was done because at short interpulse intervals or periods, the IPSPs in each train did not have sufficient time to return to V_rest (see Fig. 3 for example).

View larger version (20K): [in this window] [in a new window]   FIG. 3. Synaptic dynamics of the IPSPs in response to a train of voltage pulses in the PD neurons. A: PD neurons were stimulated with a series of 5 pulses of 40-mV amplitude from Vhold = –60 mV with IPI 250 ms in control (red) and PTX (blue) while the response in the LP neuron was monitored. IPSPAB was approximated by subtracting the 2 traces (black trace). All IPSPs in response to the 5th pulse (horizontal arrow) were smaller in amplitude than the 1st pulse IPSPs (vertical arrow), indicating short-term synaptic depression. Note that we measure the amplitude of each IPSP from Vrest of the LP neuron. LP neuron had an initial membrane potential of –59 mV. B: extent of depression of the LP IPSP response was calculated as the ratio of the 5th peak amplitude to the amplitude of the 1st peak for all IPIs used (250–8,000 ms). Data were plotted vs. IPI (n = 6). Extent of depression of IPSPPD was similar to that of IPSPAB at all IPIs. C: same as in B using PY as the postsynaptic neuron (* indicates significant difference).

Recording, analysis, and statistics

An NI PCI-6070-E board (National Instruments, Austin, TX) was used for data acquisition and for current injection with the data-acquisition software Scope developed in the LabWindows/CVI software environment (National Instruments) on a Windows XP operating system. The acquired data were saved as individual binary files and were analyzed with the Readscope software. (Scope and Readscope are software developed in the Nadim laboratory and are available for download at http://stg.rutgers.edu/software/index.htm.) Digital subtraction of traces was done by custom-made programs written in LabWindows/CVI (National Instruments). Statistica (Statsoft, Tulsa, OK), SigmaStat (SPSS, San Rafael, CA), and Origin (OriginLab, Natick, MA) software packages were used for statistical and graphical analysis. Reported statistical significance indicated that the achieved significance level P was below the critical significance level = 0.05. When multiple comparisons were made, the alpha was corrected using the false discovery rate correction method (Curran-Everett 2000). In those cases, the value is given as B = corrected value. All error bars shown and error values reported denote SDs.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The electrically coupled AB and two PD neurons compose the pacemaker unit of the pyloric circuit. These neurons oscillate in synchrony (Fig. 1A; top two traces; for simplicity, only one PD neuron is shown) and, together, they drive the pyloric rhythmic activity. Each cycle of the triphasic pyloric rhythm is generated by a simultaneous burst in the pacemaker neurons, followed, with a short delay, by a burst in the LP neuron and then by a burst in the PY neurons (Fig. 1A). The pacemaker AB and PD neurons are each connected to the follower LP and PY neurons by chemical inhibitory synapses (Fig. 1B) and so, during normal ongoing rhythm, the AB and PD neurons produce a compound postsynaptic potential onto the LP and PY neurons.

In a recent publication (Rabbah and Nadim 2005), we compared the dynamics of the synapses between the pacemaker AB and PD neurons as a unit (IPSP_AB/PD) to the follower LP and PY neurons. Here, we examine instead the dynamics of the synaptic inputs the LP and PY neurons receive from the individual pacemaker AB (IPSP_AB) and PD (IPSP_PD) neurons. This analysis was done in three parts: First, we examine whether the intrinsically distinct AB and PD neurons exhibit distinct synaptic dynamics. Thus we start by characterizing various parameters of IPSP_AB and IPSP_PD onto the LP and PY neurons using paired LP and PY recordings and various injection stimuli into the pacemaker neurons. Second, we examine whether the synapses originating from the same presynaptic neuron (AB or PD) onto two different classes of target neurons (LP and PY) exhibit distinct dynamics. Third, we ask whether these synaptic parameters correlate with and thus help determine the activity phases of the follower LP and PY neurons in the normal ongoing pyloric activity.

Comparison of the dynamics of IPSP_AB and IPSP_PD to the LP and PY neurons

To measure synaptic strength, we activated the pacemaker synapses using single 2-s presynaptic square pulses of various amplitudes (Fig. 2). Figure 2A shows the synaptic response in the LP neuron when the PD neurons were stepped from –60 to –20 mV (top trace). The red trace represents the IPSPs elicited in the LP neuron in control (TTX) saline. This IPSP consists of the AB and PD components (IPSP_AB/PD). In the presence of PTX, the AB glutamatergic release is blocked and thus the LP neuron experiences synaptic release from the PD neurons only (IPSP_PD; blue trace). The AB component (IPSP_AB; black trace) was then approximated by digitally subtracting the PTX trace from the control trace (see METHODS). All three IPSPs showed a large early peak (Fig. 2A, dotted arrows) that decayed to a sustained value (Fig. 2A, dashed arrows) during the stimulation period. The peak component of IPSP_AB decayed faster than IPSP_AB/PD or IPSP_PD.

View larger version (21K): [in this window] [in a new window]   FIG. 2. Postsynaptic potentials of the LP and PY neurons in response to stimulations of the PD neurons with a single square pulse. A: membrane potentials of the PD neurons were stepped from –60 to –20 mV (top trace). Red trace: combined inhibitory postsynaptic potentials (IPSPs) from both the AB and PD neurons (IPSPAB/PD; red trace) in control [tetrodotoxin (TTX) saline]. In PTX, the AB synaptic output is blocked and the response in the LP neuron arises from the synapse from the PD neurons only (IPSPPD; blue trace). IPSPPD trace was subtracted from IPSPAB/PD to yield the synapse arising from the AB neuron only (IPSPAB; black trace). All IPSPs exhibited a transient peak (dotted arrows) that decayed to a sustained value (dashed arrows). LP neuron had an initial membrane potential of –59 mV. B: close-up of the IPSPs in A shows that the time courses of the synaptic response in all conditions were different from each other. Double-headed arrow: delay in onset of the synaptic response of the PD neurons. C: synaptic input–output (I/O) curve for square pulses of 10- to 40-mV amplitudes used to depolarize the presynaptic PD neurons (n = 6). Values were normalized to the amplitude IPSPAB/PD in response to a step to –20 mV. Amplitude of each IPSP is measured from resting potential (Vrest) to the maximal value of the IPSP. Results show that for presynaptic depolarization (Vpre) of –50 and –40 mV (from –60 mV), the compound synapse to the LP neuron was mostly from the AB neuron. IPSPPD required more positive PD potentials to activate compared with both IPSPAB/PD and IPSPAB onto LP. D: time to peak of the IPSPs was measured from the beginning of the presynaptic depolarization to the maximal peak of the IPSP (inset). IPSPPD onto the LP neuron reached its maximum peak value more slowly than IPSPAB. Moreover, both IPSPs showed a dependency on the presynaptic potential (n = 6). E: same as in C, but using the PY neuron as the postsynaptic target. Response is similar to that seen in the LP neuron. F: same as in D, but using the PY neuron as the postsynaptic target. Again, the results were similar to those of the LP neuron (* indicates significant difference).

The maximal peak amplitudes of the IPSPs (measure of synaptic strength) elicited in response to all presynaptic depolarization amplitudes (V_pre from –60 to –50, –40, –30, –20 mV) were normalized to the amplitude of IPSP_AB/PD recorded in response to a step to –20 mV in control conditions. The resulting normalized input–output (I/O) relationship is shown in Fig. 2C. Note that V_LP (and V_PY) values are plotted as negative in this and subsequent figures to indicate the inhibitory nature of the synapse from the pacemaker neurons onto the LP (and PY) neuron. The IPSP amplitudes elicited in the LP neuron increased with increasing presynaptic potentials as expected from a graded synapse. Interestingly, in response to depolarization steps that shifted the membrane potentials of the presynaptic neurons relatively little (V_pre values of –50 and –40 mV), we observed that the amplitudes of IPSP_AB/PD were mostly attributed to the AB neuron (compare red and black traces). In those presynaptic voltage ranges, IPSP_PD (blue trace) was significantly smaller in amplitude than IPSP_AB/PD (two-way ANOVA, P < 0.05; B = 1.67 x 10^–2; n = 6). With larger PD neuron depolarizations (V_pre to –30 and –20 mV), the chemical transmission from the PD neurons increased. In those voltage ranges, both IPSP_PD and IPSP_AB were significantly smaller than IPSP_AB/PD (two-way ANOVA, P < 0.05; B = 1.67 x 10^–2; n = 6) and not significantly different from each other, indicating that both AB and PD neurons were contributing to the compound synapse.

Blocking the AB to LP direct synapse in PTX removed the AB short time-to-peak component, revealing a slower PD component (see Fig. 2, A and B). To examine the time course of synaptic transmission, the time to peak of the IPSPs was quantified by calculating the t between the presynaptic pulse onset and the postsynaptic maximal peak hyperpolarization (Fig. 2D, inset). As the magnitude of presynaptic depolarization increased (V_pre values from –60 to –50, –40, –30, and –20 mV), the maximal postsynaptic responses in the LP neuron peaked earlier in time, independent of the identity of the presynaptic neuron (AB or PD) (Fig. 2D). However, the latency of the maximal peak hyperpolarization of IPSP_PD onto LP was greater than that of IPSP_AB (two-way ANOVA, post hoc Tukey analysis, P 0.048 for V_pre to –50, –40, and –30 mV; n = 6). Normalizing the t of the IPSP_PD and IPSP_AB to their respective t at the most depolarized presynaptic potential showed that the peak of the synapse from the PD neurons reached its maximal value 320% earlier as V_pre increased and 242% earlier for the synapse from the AB neuron (data not shown; one-way ANOVA, P 0.040 for both; n = 6).

The synaptic responses (IPSP_AB/PD, IPSP_PD, and IPSP_AB) recorded in the PY neuron during single square-pulse stimulation of the PD neurons were not significantly different in strength and time course compared with those elicited in the LP neuron. Moreover, the normalized I/O curve of the IPSPs in the PY neuron (Fig. 2E) showed a trend similar to that observed in the LP neuron (Fig. 2C). Specifically, the compound IPSP_AB/PD consisted mainly of the AB component at V_pre values of –50 and –40 mV and of both the AB and PD components at more depolarized presynaptic voltages. Plotting t_PY versus V_pre showed that IPSP_PD reached its maximal peak value significantly later at all presynaptic voltage values compared with IPSP_AB (Fig. 2F; two-way ANOVA, post hoc Tukey analysis, P 0.049 for all V_pre values; n = 6).

To characterize the parameters of the short-term dynamics of the synapses (extent of depression and time course of recovery) from the pacemaker neurons to the LP and PY neurons, we used a train of square pulses of fixed duration and different interpulse intervals (IPIs: 250–8,000 ms; Fig. 3). Figure 3A is an example showing the voltage traces of the LP neuron in response to a train of pulses with 500-ms IPI into the PD neurons. The synapses from IPSP_AB/PD (red trace) and from the individual AB (black trace) and PD (blue trace) components showed short-term depression: the second and subsequent presynaptic depolarization pulses elicited IPSPs (Fig. 3A, horizontal arrow) that were smaller in amplitude than those elicited by the first pulse (Fig. 3A, vertical arrow). Note that the amplitude of each IPSP was measured from the resting potential of the postsynaptic neuron (see METHODS).

The extent of synaptic depression was quantified as the ratio of the final (fifth) IPSP peak amplitude to the first plotted versus IPI (Mamiya et al. 2003; Rabbah and Nadim 2005) (Fig. 3B). At the shortest IPI, IPSP_PD and IPSP_AB exhibited the greatest depression and as the duration of the IPI increased, the magnitude of depression decreased (ratio of 1 indicates maximum recovery from depression). Even though IPSP_PD tended to show more depression compared with IPSP_AB, statistically, their degrees of depression (for IPSPs recorded in the LP neuron) were not significantly different from each other (two-way ANOVA, P > 0.05; n = 6). This trend was slightly more prominent in the responses recorded in the PY neuron (Fig. 3C; two-way ANOVA, post hoc Tukey analysis, P < 0.001 for IPI 250 ms; n = 6). However, for all IPIs tested, the degrees of depression of the synaptic responses (IPSP_AB/PD, IPSP_PD, and IPSP_AB) recorded in the PY neuron were not significantly different compared with those elicited in the LP neuron (two-way ANOVA, P > 0.05; n = 6).

To fully characterize the parameters of short-term synaptic dynamics, we measured the time constant of recovery (_rec) from depression by looking at the relationship between recovery and IPI. In each experiment, we graphed the ratio of the fifth peak/first peak versus IPI and fit it with a first-order exponential decay curve with the equation (1 – D_max)e^{–IPI/_rec (for example, see Fig. 4E; also Mamiya et al. 2003). D_max is the maximum amount of depression of a synapse as IPI tends to zero and _rec is the time constant of recovery. Average _rec values for the synaptic depression in the LP neuron showed that IPSP_PD (_rec = 311 ± 28.910 ms) tended to recover faster than IPSP_AB (_rec = 478 ± 103.501 ms), although this difference was not statistically significant (paired t-test, P = 0.192; n = 6; data not shown). Similarly, IPSP_PD to the PY neuron tended to recover from depression faster than IPSP_AB but not significantly (_rec values of 491 ± 110.692 and 576 ± 96.132 ms, respectively; data not shown). The _rec values for all IPSPs (IPSP_AB/PD, IPSP_PD, and IPSP_AB) recorded in the PY neuron were not significantly different compared with those recorded in the LP neuron (two-way ANOVA, P > 0.05; n = 6).}

View larger version (26K): [in this window] [in a new window]   FIG. 4. IPSPs in the LP neuron in response to a train of 2 realistic PD waveforms. A: PD neurons were stimulated with 2 types of realistic waveforms: duty cycle (DC) 46% (top trace) and DC 17% (bottom trace). Both waveforms were prerecorded in the PD neuron from 2 different preps, low-pass filtered (at 10 Hz), and amplified to 40-mV amplitudes. They were injected from Vh = –60 mV in a train of 5 pulses with frequencies ranging from 0.5 to 4 Hz. B: LP response to DC 46% (top) and DC 17% (bottom) with cycle period 500 ms. IPSPs elicited by the individual AB (black trace) and PD neurons (blue trace) and by the compound AB/PD unit (red trace) are superimposed for comparison. C: extent of depression of the IPSPs (5th/1st peak) in response to DC 46% was plotted vs. cycle periods (n = 6). D: extent of depression of the IPSPs in response to DC 17% plotted vs. cycle periods showed that IPSPAB exhibited significantly less depression (marked by *) than IPSPPD for cycle periods 250–1,000 ms (n = 6). E: time constant of recovery from depression of the IPSPs was calculated by fitting the 5th/1st ratio vs. cycle periods for each experiment with a single exponential fit (dashed line). F: average time constant of recovery (rec) values. For DC 17%, IPSPAB recovered significantly faster than IPSPPD (n = 6) (* indicates significant difference).

The realistic waveforms were applied into the two voltage-clamped PD neurons from a baseline of –60 mV with a fixed 40-mV (trough-to-peak) amplitude and with cycle periods 250 to 2,000 ms (frequency 4 to 0.5 Hz). Figure 4B shows an example of IPSP_AB/PD (red traces), IPSP_PD (blue traces), and IPSP_AB (black traces) elicited in the LP neuron in response to a train of waveform stimulations corresponding to DC 46% (top traces) and DC 17% (bottom traces) and cycle period of 500 ms. As with trains of voltage pulses, all IPSPs showed short-term depression and recovered from depression as the cycle period was increased (Fig. 4, C and D). Also, similar to trains of voltage pulses, the degrees of depression of IPSP_PD and IPSP_AB in response to DC 46% (Fig. 4C) were statistically similar (two-way ANOVA, P = 0.836; n = 6). Interestingly, however, in response to DC 17%, IPSP_AB exhibited significantly less depression compared with IPSP_PD at the shortest IPIs tested (Fig. 4D; two-way ANOVA, post hoc Tukey analysis, P 0.041 for periods 250, 500, and 1,000 ms; n = 6).

We measured the _rec of the IPSPs in response to the two realistic waveforms as previously described for Fig. 3 (see also Fig. 4E). Figure 4F shows that, in response to DC 46%, the PD component tended to recover from depression faster than the AB component but this tendency was not statistically significant (_rec values of 390 ± 46.303 and 536 ± 98 ms, respectively; Student's t-test, P = 0.198; n = 6). This trend was similar to that seen with the square-pulse protocol. Interestingly, however, when DC 17% was used, the trend of recovery from depression was reversed: The IPSP that the LP neuron received from the PD neuron recovered significantly more slowly from depression than the IPSP from the AB neuron (_rec values of 510 ± 76.903 and 344 ± 34 ms, respectively; Student's t-test, P = 0.029; n = 6).

We also activated the synapse between the pacemaker neurons and the PY neuron with the two presynaptic realistic waveforms in control and PTX conditions. Figure 5A shows an example of IPSP_AB/PD (red traces; control), IPSP_PD (blue traces; PTX), and IPSP_AB (black traces) recorded in the PY neuron in response to a train of waveform stimulations corresponding to DC 46% (top traces) and DC 17% (bottom traces) and cycle period of 500 ms. All IPSPs showed short-term depression as the cycle period was increased (Fig. 5, B and C). Unlike what was seen in the LP neuron, in response to both PD realistic waveforms, IPSP_AB exhibited significantly less depression compared with IPSP_PD (two-way ANOVA, post hoc Tukey analysis, P 0.042 for periods 250, 500, 1,000, 1,500, and 2,000 ms; n = 6). However, comparison of the degrees of depression in the LP versus PY neurons attributed to IPSP_PD and IPSP_AB were not significantly different from each other (two-way ANOVA, P > 0.05; n = 6).

View larger version (25K): [in this window] [in a new window]   FIG. 5. IPSPs in the PY neuron in response to a train of 2 realistic PD waveforms. A: PY response to the stimulation of the PD neurons with DC 46% (top) and DC 17% (bottom) corresponding to amplitude 40 mV and cycle period 500 ms. B: ratio 5th/1st peak in response to DC 46% plotted vs. cycle periods showed that IPSPAB depressed significantly less than IPSPPD (n = 6). Again, the amplitudes of the IPSPs were measured from Vrest. C: similar to B, the extent of depression of IPSPAB in response to DC 17% was significantly less than IPSPPD. D: average rec values of the IPSPs calculated as in Fig. 4E (n = 6) (* indicates significant difference).

The maximal peak amplitude of the IPSP in the LP neuron elicited by either AB or PD components appeared sensitive to the slope of the rising phase of the presynaptic depolarizations (Fig. 6). We focus on the IPSPs in response to the fifth waveform (see Fig. 5A) because it carries information about the dynamics of the "stationary" synapses after transient changes are over. Figure 6A presents a comparison of the amplitudes of the fifth IPSPs elicited with DC 46% (left) with those elicited with DC 17% (right) corresponding to the 500-ms cycle period. We observed that, in response to DC 46%, IPSP_AB was smaller in amplitude than IPSP_PD (Fig. 6A, left; compare black and blue traces). Similar to square pulses, we measure the amplitude of each IPSP from V_rest. However, in response to DC 17%, IPSP_AB and IPSP_PD appeared equal in amplitude (Fig. 6A, right; compare black and blue traces). To illustrate the relative contribution of the AB and PD components to the total synaptic transmission onto the LP neuron (Fig. 6A, red traces), we normalized the steady-state amplitudes of IPSP_PD and IPSP_AB to the steady-state amplitude of IPSP_AB/PD at frequency 0.67 Hz and plotted the values as a function of presynaptic frequency (Fig. 6, B for DC 46% and C for DC 17%). The results of the data presented in these two panels are twofold. First, IPSP_AB showed no dependency on presynaptic frequency irrespective of the presynaptic waveform used (one-way ANOVA, P = 0.151 for DC 46% and P = 0.438 for DC 17%; n = 6), indicating that the AB synapse is activated to the same degree for all presynaptic frequencies and duty cycles tested. In contrast, the steady-state amplitudes of IPSP_PD significantly decreased as the frequency was increased (one-way ANOVA, P < 0.001 for both DC 46% and DC 17%; n = 6). Second, the relative contribution of the PD neurons compared with the AB neuron was dependent on the shape of the presynaptic waveform: When the presynaptic waveform with DC 46% was used, IPSP_PD recorded in the LP neuron at all frequencies (except 4 Hz) was significantly larger in amplitude than IPSP_AB (Fig. 6B; two-way ANOVA, post hoc Tukey analysis, P 0.004 for frequencies 0.5, 0.67, 1, and 2 Hz; n = 6). In contrast, when DC 17% was used, IPSP_PD and IPSP_AB were recruited to similar levels for most frequencies tested (Fig. 6C; two-way ANOVA, post hoc Tukey analysis, P 0.026 for frequencies 0.5 and 0.67 Hz only; n = 6). These results suggest that, at low frequencies, the synapse from the pacemaker unit onto the LP neuron is dictated mostly by the PD neurons for both presynaptic duty cycles tested. As the frequency of the presynaptic depolarization is increased, the PD neuron contribution decreases and approaches the (unchanging) AB neuron contribution but to a different degree, depending on the duty cycle of the presynaptic waveform.

View larger version (15K): [in this window] [in a new window]   FIG. 6. Peak amplitude of the IPSPs in the LP neuron appeared sensitive to the slope of the rising phase of the presynaptic depolarizations. A: IPSPs in response to the 5th waveform (see Fig. 5A) elicited with DC 46% (left) and DC 17% (right) corresponding to cycle period 500 ms. Amplitude of IPSPAB is smaller than that of IPSPPD for DC 46% only. B: steady-state amplitudes of IPSPPD and IPSPAB were normalized to the steady-state amplitudes of IPSPAB/PD at frequency 0.67 Hz for DC 46%. Values were plotted as a function of presynaptic frequency. Relative contribution of IPSPPD was significantly larger for the lowest frequencies (n = 6). C: amplitudes of the IPSPs in response to DC 17% were normalized as in B and plotted vs. frequency (n = 6). (* indicates significant difference).

View larger version (15K): [in this window] [in a new window]   FIG. 7. Peak amplitude of the IPSPs in the PY neuron appeared sensitive to the slope of the rising phase of the presynaptic depolarizations. A: IPSPs in response to the 5th waveform elicited with DC 46% (left) and DC 17% (right) corresponding to cycle period 500 ms. IPSPAB is smaller in amplitude than IPSPPD for DC 46% and equal in amplitude for DC 17%. B: steady-state amplitudes of IPSPPD and IPSPAB in the PY neurons were normalized and plotted as a function of presynaptic frequency as in Fig. 6. Relative contributions of both the AB and PD components were not significantly different from each other at all frequencies tested (n = 6). C: amplitudes of the IPSPs in response to DC 17% were normalized as in Fig. 6 and plotted vs. frequency. Again, no significant difference between the amplitudes at all frequencies tested (n = 6) (* indicates significant difference).

View larger version (13K): [in this window] [in a new window]   FIG. 8. Time course of synaptic transmission of the IPSPs in the LP neuron in response to the realistic waveform stimulations in the PD neurons and its effect on the LP IPSP peak phase. A: IPSPs in response to the 5th waveform elicited with DC 46% (top) and DC 17% (bottom) at cycle frequency 2 Hz showed that IPSPPD (blue open circles) reached its maximum peak significantly later in time than IPSPAB (black open circles) independent of the duty cycle of the waveform used. Double-headed arrows are meant to represent only the delay between the start of hyperpolarization in the LP neuron arising from the AB vs. the PD synapse. B: LP IPSP peak phase was calculated as t (measured from the peak of the presynaptic waveform to the peak IPSP) divided by Periodapplied (inset) and plotted vs. frequency (n = 6). In response to DC 46%, the LP IPSP peak phase was significantly delayed because of the synapse from the PD neurons (blue trace). C: similar results were obtained for DC 17% (n = 6) (* indicates significant difference).

View larger version (12K): [in this window] [in a new window]   FIG. 9. Time course of synaptic transmission of the IPSPs in the PY neuron in response to the realistic waveform stimulations and its effect on the IPSP peak phase. A: IPSPs in response to the 5th waveform elicited with DC 46% (top) and DC 17% (bottom) at cycle frequency 2 Hz showed that IPSPPD (blue open circles) reached its maximum peak significantly later in time than IPSPAB (black open circles) independent of the duty cycle of the waveform used. Double-headed arrows are meant to represent only the delay between the start of hyperpolarization in the PY neuron attributed to the AB vs. the PD synapse. B and C: PY IPSP peak phase was calculated as in Fig. 8B and plotted vs. frequency (n = 6). Results were similar to those reported for the LP neuron for both DC 46% (B, n = 6) and DC 17% (C, n = 6) (* indicates significant difference).

Correlation between the dynamics of IPSP_AB and IPSP_PD on the phase onset of the LP and PY neurons

Thus far, we have shown that the synaptic dynamics of the pacemaker AB and PD neurons exhibit distinct dynamics dependent on the frequency and shape of the presynaptic depolarizations. Specifically, the synaptic transmission from the PD neurons is a relatively slow process that serves to delay the peak of the hyperpolarization in the postsynaptic LP and PY neurons (and thus their IPSP peak phase) at all presynaptic frequencies. The opposite is true for the synaptic transmission from the AB neuron. Moreover, the relative contributions of the AB and PD neurons to the total synaptic output onto the LP and PY neurons change as a function of presynaptic depolarization, frequency, and duty cycle of the injected stimuli. We have also shown that the dynamics of the synapses originating from either presynaptic pacemaker neuron (AB or PD) to the LP neuron versus the PY neurons are not distinct from each other. We now investigate the functional importance of synaptic dynamics for the burst phase of postsynaptic neurons in a network driven by strongly electrically coupled pacemaker neurons that evoke distinct synaptic dynamics onto the same subset of neurons.

We begin by examining the relationship between the amplitudes of IPSP_PD and IPSP_AB and the activity phase of the LP neuron during the ongoing rhythm. Figure 10A presents a correlation of the LP burst phase during the ongoing rhythm to the amplitudes of the steady-state IPSPs measured in the LP neuron when the PD neurons were activated with DC 46% (left) and DC 17% (right). The LP burst phase was calculated using the natural pyloric period and the PD neuron as a reference, before the application of TTX (see METHODS). As representative, we chose the stationary amplitudes of the IPSPs at a cycle period of 1 s (frequency 1 Hz). The correlations made using the 0.5-s cycle period produced similar results (data not shown). Each point on the graph represents one preparation.

View larger version (15K): [in this window] [in a new window]   FIG. 10. LP burst phase in the ongoing rhythm is positively correlated with the amplitude of the AB and PD synapses but not with their peak phase. LP burst phase in the ongoing rhythm was measured using the natural pyloric period and the PD neuron as a reference point. IPSP values chosen for this figure are those corresponding to cycle Periodapplied of 1 s. Each point in the graph represents one preparation. A: LP burst phase during the ongoing rhythm showed a strong positive correlation to the IPSP amplitudes attributed to AB and PD neurons, for both DC 46% (left) and DC 17% (right). B: LP burst phase plotted vs. the IPSP amplitudes attributed to the AB/PD unit for DC 46% and DC 17%. C: IPSP peak phase was calculated using the time to peak with respect to the peak of the presynaptic waveform (see example in Fig. 8B, inset) and plotted vs. the LP burst phase during the ongoing rhythm for DC 46% (left) and DC 17% (right). D: scatterplot of IPSPAB/PD peak phase in DC 46% vs. DC 17% showed no correlation to the LP burst phase. Solid diagonal lines in A and B are best linear fits.

The correlation between the strength of the IPSPs and the LP burst phase does not account for the distinct temporal dynamics of IPSP_PD and IPSP_AB. Thus we calculated the IPSP time-to-peak with respect to the presynaptic stimuli (see example in Fig. 8B, inset), divided the values by the applied period of 1 s (Period_applied), and examined their correlation to the LP burst phase during the natural pyloric rhythm (Mamiya et al. 2003). The right and left panels of Fig. 10C are scatterplots of these results using DC 46% and DC 17%, respectively. Our results show that when the presynaptic stimulus has a duty cycle of 46 or 17%, the burst phase of the LP neuron is not correlated to the IPSP peak phase it receives from either the PD (blue stars) or AB (black stars) neuron. In addition, the peak phase of IPSP_AB/PD was not correlated to the LP burst phase with either waveform (Fig. 10D).

In contrast to what was seen in the LP neuron, in response to either presynaptic waveform, the PY burst phase (during the normal ongoing rhythm) showed a weak negative correlation to the amplitudes of both IPSP_PD and IPSP_AB; i.e., as the IPSP amplitudes increased, the PY phase was delayed (Fig. 11A, left for DC 46%: IPSP_PD: y = –0.014x + 0.601, r = –0.601, P = 0.251; IPSP_AB: y = –0.01x + 0.651, r = –0.450, P = 0.371; Fig. 11A, right for DC 17%: IPSP_PD: y = –0.015x + 0.592, r = –0.931, P = 0.021; IPSP_AB: y = –0.015x + 0.612, r = –0.611, P = 0.111). In response to both waveforms, the amplitude of the compound synapse IPSP_AB/PD was also weakly negatively correlated to the burst phase of the PY neuron (Fig. 11B; for DC 46%: y = –0.01x + 0.592, r = –0.791, P = 0.112; for DC 17%: y = –0.015x + 0.572, r = –0.821, P = 0.081).

View larger version (15K): [in this window] [in a new window]   FIG. 11. PY burst phase in the ongoing rhythm is negatively correlated with some dynamic parameters of the AB and PD synapses. PY burst phase in the ongoing rhythm is calculated as in Fig. 10. A: PY burst phase during the ongoing rhythm tended to be negatively correlated to the amplitudes of IPSPAB and IPSPPD for DC 17% only (right). B: amplitudes of IPSPAB/PD for DC 46% and DC 17% also tended to be negatively correlated to the PY burst phase. C: IPSP peak phase was calculated as in Fig. 10 and plotted vs. the PY burst phase during the ongoing rhythm for DC 46% (left) and DC 17% (right). D: correlation of IPSPAB/PD peak phase in DC 46% vs. DC 17% to the PY burst phase. Solid diagonal lines in all panels are best linear fits.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
In a pacemaker-driven oscillatory network, the synaptic interactions between the pacemaker neurons and the follower neurons are of crucial importance. These synapses can play a role in setting the activity phase of the follower neurons and the frequency of the rhythmic output (Eisen and Marder 1984; Tryba and Ritzmann 2000; Wolf 1992). However, when neural networks use a heterogeneous population of pacemaker neurons, a greater level of complexity is added to the effect of pacemaker synapses on the relative phase relationships of member neurons. In the lobster pyloric network, the AB and PD pacemaker neurons produce compound chemical synapses to the follower LP and PY neurons. The compound synapses from these pacemaker neurons to the LP and PY neurons are identical in strength and dynamics and thus do not play a role in determining the relative activity phase of these follower neurons (Rabbah and Nadim 2005). However, neuromodulatory inputs to the pyloric network can differentially affect the intrinsic and synaptic properties of the AB and PD neurons as well as the strength of the electrical coupling between them (Harris-Warrick et al. 1998; Johnson and Harris-Warrick 1997; Johnson et al. 1993). In this manner, neuromodulators can emphasize one type of pacemaker neurons (and their synaptic connections) over the other, thus leading to a different functional network output. It is thus important to investigate the dynamics of the synapses originating from the AB and PD neurons separately and not only as a compound synapse.

We report a detailed examination of the temporal dynamics of the AB and PD synapses. Our work builds on previous studies of Eisen and Marder who reported on the neurotransmitter content, reversal potential, and ion selectivities of these two synapses, as well as their time course of release (Eisen and Marder 1982, 1984; Marder and Eisen 1984). We investigated the temporal dynamics of the synapses from the AB and PD synapses separately, in the absence of network neuromodulation, and addressed the functional implications of these synaptic dynamics in predicting the time of activity of the follower neurons.

Synaptic efficacy depends on presynaptic waveform shape and frequency

Our results show that the relative contributions of IPSP_AB and IPSP_PD to the compound AB/PD synapse in the LP and PY neurons are dependent on the shape of presynaptic waveform and frequency. We activated the PD neurons with two distinct realistic waveforms, with short (DC 17%) and long (DC 46%) duty cycles at the same amplitude and frequency, and recorded the postsynaptic responses (Fig. 6 for LP and Fig. 7 for PY). For all frequencies tested except 4 Hz, in response to DC 46%, IPSP_PD recorded in the LP and PY neurons was significantly larger than IPSP_AB. However, in response to DC 17%, IPSP_PD to the LP neuron was significantly larger at low frequencies (0.5 and 0.67 Hz) only. Taken together, these results indicate that the strength of the AB/PD synapse to the LP and PY neurons can be dictated either mostly by the PD neurons (as in the case of DC 46%) or by both the AB and PD neurons (as in the case of DC 17%), in a manner dependent on cycle frequency. Changes in cycle frequency are commonly induced in the pyloric circuit by network neuromodulators (Marder and Thirumalai 2002). In this manner, depending on the state of the network (i.e., which neuromodulator is present), the frequency (and duty cycle) can be altered, allowing either one or both of the pacemaker AB and PD neurons, and the temporal dynamics of their synaptic outputs, to be important determinants of network output.

Short-term depression of the synapses is dependent on the membrane potential oscillation waveform of the PD neurons

Short-term depression allows for the dynamic control of synaptic strength between network neurons as a function of cycle period (Manor et al. 2003; Nadim and Manor 2000). Our results indicate that the short-term depression of IPSP_AB and IPSP_PD in the LP and PY neurons depends on the membrane potential waveforms of pacemaker neurons. Specifically, in response to waveforms with the sharpest rise time (square pulses) and longest duty cycle (DC 46%) activated with cycle periods comparable to the range of periods of the pyloric rhythm in vivo (Rezer and Moulins 1983; Turrigiano and Heinzel 1992), the synapses from the AB and PD neurons to the LP neuron depressed and recovered from depression to the same extent. However, in response to DC 17%, IPSP_AB depressed significantly less and recovered significantly faster. In the case of the PY neuron, the short-term dynamics observed for IPSP_AB and IPSP_PD was similar for the square pulses but different for both realistic waveforms used. These results indicate that the kinetics of both depression and recovery of the pyloric synapses is tuned so that the synaptic transmission is sensitive to period and duty cycle in the range over which the pyloric network normally operates (Mamiya and Nadim 2004, 2005; Manor et al. 1997). For example, in response to perturbations that increase cycle period and decrease duty cycle of the pacemaker neurons, the amplitudes of the pacemaker synapses to the LP and PY neurons will exhibit less depression and thus grow stronger. Such perturbations, elicited by neuromodulatory or synaptic inputs, can potentially modify the influence of the pacemaker neurons on the activity patterns of the follower neurons merely by changing the pacemaker membrane potential waveforms.

Measured IPSP peak amplitude as a predictive measure of the burst phase of the follower neurons in the normal ongoing rhythm

We were interested in investigating whether the relative amplitudes of the AB and PD synapses onto the LP and PY neurons measured in TTX conditions serve as predictive measurements of the burst phase of the LP and PY neurons in the ongoing rhythm. We found that the amplitude of the LP IPSP arising from either the AB or PD neurons was strongly positively correlated to the LP burst phase in the ongoing rhythm; the larger the IPSP, the earlier the LP burst phase. This is consistent with activating a hyperpolarization-activated inward current I_h, a current that is very prominent in the LP neuron and is responsible for the advancement of its burst phase following hyperpolarization (Harris-Warrick et al. 1995a,b; Tierney and Harris-Warrick 1992). In contrast to the findings in the LP neuron, but similar to the findings of Mamiya et al. (2003), a larger IPSP in the PY neuron (evoked by either AB or PD) resulted in a negative correlation to the PY burst phase in the ongoing rhythm. In this case, the larger IPSP most likely serves to activate larger amounts of the fast transient potassium current I_A, a subthreshold current involved in the postinhibitory property of the pyloric neurons (Graubard and Hartline 1984; Harris-Warrick 1989; Tierney and Harris-Warrick 1992). Hyperpolarization removes inactivation from this current and, when activated, it can delay the burst onset of the postsynaptic neuron (Harris-Warrick et al. 1995a). A clear understanding of the functional effects of the pacemaker IPSPs on the activity phases of the follower LP and PY neurons, however, requires further experiments that also take into account the synaptic connectivity between the follower neurons (Mamiya and Nadim 2005; Mamiya et al. 2003).

Measured IPSP peak phase as a predictive measure of the burst phase of the follower neurons in the normal ongoing rhythm

Synaptic outputs from the PD neurons are relatively slow, producing delayed peaks in the postsynaptic potentials of the LP and PY neurons at all presynaptic frequencies. The opposite is true for synaptic transmission from the AB neuron. It is expected that a delayed IPSP peak phase produce a delayed burst in the postsynaptic neuron. However, when we examined the relationship between the IPSP peak phase at various presynaptic periods and the phase of the LP neuron in the ongoing rhythm, we found no correlation between the two (Fig. 10, C and D). In contrast, when PY was the postsynaptic neuron, the burst phase tended to be (weakly) negatively correlated to the phase of IPSP_AB. This latter result is consistent with faster removal of inactivation of an outward current in the PY neuron. The PY neuron, and not the LP neuron, possesses appreciable amounts of I_A (Baro et al. 2000; Graubard and Hartline 1984; Harris-Warrick 1989; Tierney and Harris-Warrick 1992). It is plausible that if the IPSP in the PY neuron peaks earlier, a larger outward current is produced on rebound from inhibition, thus delaying the PY burst onset.

The absence of very strong correlations between the measured synaptic parameters and the burst phase of the follower LP and PY neurons in the ongoing rhythm is not completely unexpected. The pyloric neurons form a multitude of synaptic connections with each other, including mixed electrical and chemical synaptic connections between the LP neuron and PY neurons (Mamiya et al. 2003). Moreover, the pyloric neurons have a variety of nonlinear intrinsic ionic currents (Golowasch and Marder 1992; Johnson et al. 2003; Kloppenburg et al. 1999). Thus in an ongoing rhythm, the activity patterns of the LP and PY neurons are probably determined, not only by inputs from the pacemaker neurons, but by a dynamic combination of their intrinsic properties and various parameters of all their synaptic inputs, including strength, rate of transmission, and extent of depression and recovery (Golowasch et al. 1992; Harris-Warrick et al. 1995a; Hartline and Gassie 1979; Mamiya et al. 2003