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1Department of Neurobiology and Behavior, Cornell University, Ithaca, New York; and 2Department of Mathematical Sciences, New Jersey Institute of Technology, and Department of Biological Science, Rutgers University, Newark, New Jersey
Submitted 2 May 2005; accepted in final form 23 June 2005
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ABSTRACT |
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PY synapses, driving the LP neuron with trains of realistic waveforms constructed from prerecorded control and DA LP oscillations, which differed in shape and duration. Under control conditions, chemical inhibition underwent severe depression and disappeared; in the mixed synapses, electrical coupling dominated. Switching between control and DA LP waveforms (with or without DA present) caused only subtle changes in synaptic transmission. DA markedly enhanced synaptic inhibition, reduced synaptic depression and weakened electrical coupling, reversing the sign of the mixed synapses. Despite this, removal of the LP from the intact network still had only weak effects on PY firing. DA also enhances PY intrinsic rebound properties, which still control the onset of PY firing. Thus in a rhythmic network, the functional importance of synaptic modulation can only be understood in the context of parallel modulation of intrinsic properties. |
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INTRODUCTION |
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; Harris-Warrick et al. 1992; Orlovsky et al. 1999), as well as the pattern of sensory feedback (Pearson 2004; Pearson and Ramirez 1997). In many CPGs, the timing of onset and offset of neuronal firing in the networks is determined by a dynamic interaction between the intrinsic firing properties of the neurons (such as postinhibitory rebound, bistability, and bursting) and the pattern of synaptic interactions within the network (Cymbalyuk et al. 2002; Get-ting 1989; Harris-Warrick et al. 1992; Hooper 1997; Katz 1999; Ramirez et al. 2004; Stein et al. 1997). The phasing of neurons in the cycle is not fixed, but can vary, generating different motor patterns, because of sensory input or the actions of neuromodulators such as amines and peptides. Plasticity in phasing can arise from the recruitment of new neurons that provide new synaptic inhibition or excitation (Jing and Weiss 2001; Jing et al. 2003; Selverston et al. 1997), modulation of existing synapses (Johnson et al. 1995; Marder and Thirumalai 2002; Sillar et al. 1998), and modulation of the intrinsic properties of the CPG neurons (Harris-Warrick et al. 1995a,b, 1998; Kloppenburg et al. 1999; Marder and Thirumalai 2002; Matsushima et al. 1993). While the involvement of these parameters is widely accepted, their relative contributions in determining changes in phasing are not well understood in any system.
In this paper, we look at a specific example of modulation of phasing, how dopamine (DA) modifies the phasing of the eight pyloric (PY) neurons in the pyloric network of the crustacean stomatogastric ganglion (STG). The pyloric network contains 14 neurons and generates a triphasic rhythmic motor pattern driven by a 3-neuron pacemaker kernel (Ayali and Harris-Warrick 1999; Johnson and Hooper 1992). Under control conditions, the pacemaker anterior burster (AB)/pyloric dilator (PD) neuron group fires first and inhibits all other neurons; then the lateral pyloric (LP) neuron fires, followed by the PY neurons. The firing phase of the PY neurons is thought to be established by the combined actions of intrinsic postinhibitory rebound (PIR) after pacemaker inhibition and by synaptic input from the LP neuron (Hartline and Gassie 1979; Selverston et al. 1998). The LP chemically inhibits the PY neurons through graded transmission and is additionally electrically coupled to a subset of them (Fig. 1A) (Johnson et al. 1994). Under control conditions, the chemical inhibition is weak and undergoes significant synaptic depression during normal LP oscillations, leaving the electrotonic coupling between LP and PY as the dominant synaptic interaction (Mamiya et al. 2003). Removal of the LP neuron has only slight effects on the phasing of PY firing in the intact network (Mamiya et al. 2003; Weaver and Hooper 2003b).
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). At the same time, it enhances LPPY chemical inhibition and weakens LPPY electrical coupling, reversingthe sign of the LPPY interaction from depolarizing to hyperpolarizing (Johnson et al. 1993b, 1994). In theory, these changes should cause the LPPY synapse to oppose and constrain the advance in PY onset phase caused by PY PIR. However, two factors might lessen its impact. First, the LP neuron appears to oscillate with a significantly narrower waveform in DA compared with control (Flamm and Harris-Warrick 1986a), potentially reducing synaptic transmission. Second, the degree of synaptic depression of the DA-modified LPPY synapse is unknown, because previous studies were done with single square pulse depolarizations of the LP neuron (Johnson et al. 1994). Here, we address these two factors and examine the relative role of the LPPY synapse in determining the onset phase of PY firing in the DA-modified pyloric rhythm. Using prerecorded realistic waveforms to drive synaptic transmission, we show that the LP waveform shape plays a surprisingly small role in determining the strength of the LPPY synapse. Even though the synapse undergoes synaptic depression, it still becomes strongly inhibitory in the presence of DA. Despite this sign reversal, the LPPY synapse continues to play only a minor role in setting PY onset phase, which is dominated by DA's enhancement of the PY neurons' intrinsic PIR properties. |
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METHODS |
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) and pinned in a Sylgard-coated petri dish in chilled Panulirus saline of the following composition (in mM): 479 NaCl, 12.8 KCl, 13.7 CaCl2, 3.9 Na2SO4, 10.0 MgSO4, 2 glucose, and 11.1 Tris base, pH 7.35 (Mulloney and Selverston 1974). The STG was desheathed, enclosed in a 1-ml pool walled with Vaseline, and superfused at 5 ml/min with oxygenated Panulirus saline (18–19°C). In all experiments, DA was prepared in the appropriate control solution to a final concentration of 10–4 M just before application (DA conditions). All measurements were taken after a 5-min perfusion with DA. Results were discarded if the effects of DA did not show reversal after a 30-min wash. All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Electrophysiological recording and cell identification
Pyloric neuron activity was monitored using extracellular pin electrodes and standard intracellular recording techniques. We identified pyloric neuron somata during ongoing rhythmic activity 1) by matching extracellularly recorded action potentials from the appropriate motor roots with intracellularly recorded action potentials, 2) by the characteristic shape and amplitude of membrane potential oscillations and action potentials, and 3) by the neuron's synaptic connectivity (Johnson and Harris-Warrick 1997; Johnson et al. 1994). We examined the LPPY synaptic interaction using two-electrode voltage clamp of the presynaptic LP neuron and two-electrode current clamp to maintain the postsynaptic PY membrane potential at the desired level (3 M KCl filled electrodes, 10–15 M
resistance) using Axoclamp-2A and 2B amplifiers (Axon Instruments) as previously described (Johnson et al. 1994; Mamiya et al. 2003).
LP waveform construction and LPPY synaptic transmission
We constructed artificial, realistic LP waveforms from prerecorded LP activity in control and DA conditions to use as presynaptic voltage-clamp commands. LP recordings were low-pass filtered at 30 Hz to preserve the slope of LP rebound from pacemaker inhibition and to filter out spike transients. An averaged, normalized LP waveform, sampled at 1,000 points with the first and last points corresponding to the beginning and ending midpoint voltage values of a single oscillation, was constructed from the average of 10 consecutive oscillation cycles in a preparation. The original average period and amplitude of these cycles were preserved as separate values. The waveforms from six different preparations were then averaged and adjusted for the appropriate averaged period. Because LP waveform amplitudes were not significantly different in control and 10–4 M DA conditions (see RESULTS), both control and DA waveforms were scaled to 30-mV amplitude. This waveform amplitude drove the LP neuron from a holding value of –55 mV, near the resting potential of silent LP neurons (Johnson et al. 1992), to a peak of approximately –25 mV, a value that evokes the largest chemical synaptic response in PY neurons (Johnson et al. 1994).
Pyloric cells release transmitter as a continuous function of presynaptic voltage by a process called graded synaptic transmission (Hartline and Graubard 1992). These graded synaptic interactions shape the pyloric pattern in the lobster (Hartline et al. 1988). To record PY graded inhibitory postsynaptic potentials (IPSPs), we added 10–7 M TTX to the saline to block spiking activity. In these experiments, the AB neuron was killed by intracellular iontophoresis of 5,6-carboxyflourescein and illumination with bright blue light (Miller and Selverston 1979). In control-TTX conditions, the PY neuron was held at –55 mV with current injection in current clamp, while 10 linked control or DA waveforms were injected as voltage-clamp commands into LP. This was repeated in the presence of 10–4 M DA; in separate runs, the PY was either held at –55 mV or allowed to depolarize to its DA-induced value. We measured the PY peak response to the first LP oscillation and the mean steady-state response to repeated LP oscillations, as calculated from the average amplitudes of the last five PY IPSPs. A synaptic depression index (DI) was calculated as the steady-state peak response divided by the initial peak response. In addition, we examined the voltage dependence of the electrotonic synapse between LP and PY in control-TTX conditions after adding 5 x 10–6 M picrotoxin (PTX) to block the LP glutamatergic chemical transmission (Bidaut 1980; Eisen and Marder 1982). In one experiment, we also added 20 mM TEA to block voltage-gated K+ currents to ensure that shunting alone did not reduce electrical coupling during PY depolarization.
Firing properties of PY neurons during rhythmic activity
To examine the functional importance of DA modulation of LPPY synaptic dynamics on PY onset phasing during rhythmic pyloric activity, we measured the delay between the onset of LP and PY spiking in control and DA conditions; these experiments were done with intact descending modulatory inputs activating the pyloric network. In addition, we hyperpolarized the LP neuron to temporarily remove it from network activity and measured the timing of PY firing onset relative to the AB pacemaker. In these experiments, we also characterized the following PY parameters in control and 10–4 M DA conditions: number of spikes/burst, burst duration, duty cycle, and PY onset phase relative to AB onset. For each PY neuron, we averaged burst and firing measurements from 5 to 10 oscillation cycles in control and DA conditions.
Data acquisition and analysis
Electrophysiological recordings were digitized at 4 KHz using a PCI-6070-E board (National Instruments) and stored on a PC using custom-made recording software written in Lab Windows/CVI (National Instruments). The same software was also used to inject artificial control and DA waveforms as voltage-clamp commands into the LP neuron. All data were analyzed using another custom-made software program also written in Lab Windows/CVI (software available on request). For statistical comparisons, we used Statview, SAS, and SPSS software to run paired and unpaired t-tests, two-way repeated-measures ANOVA, and Levene's test for equality of variances, as appropriate. ANOVA tests were followed by a post hoc t-test to determine specific statistical differences between individual data groups. Statistical differences between mean values were accepted with P < 0.05 (2-tailed probability) for F or t values. Mean measured values and percentages are reported ±SD.
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RESULTS |
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The LP neuron displays quite different waveform shapes during control and DA-modulated pyloric rhythms (Fig. 1B). The control LP waveform is monophasic: it rebounds from AB/PD pacemaker inhibition to fire a burst of spikes, which is terminated by synaptic inhibition from the PY and VD neurons (Fig. 1A) before the next round of pacemaker inhibition (Fig. 1B, top left). During modulation by 10–4 M DA, the LP waveform is biphasic (Fig. 1B, top right) (Flamm and Harris-Warrick 1986a). Its rebound from pacemaker inhibition is accelerated by DA (Flamm and Harris-Warrick 1986a; Harris-Warrick et al. 1995b), but its firing is quickly terminated by DA-enhanced PY inhibition (Fig. 1B, PY, right) (Johnson et al. 1995). The second, brief depolarizing phase in DA is caused by its release from tonic PY inhibition as the AB/PD pacemaker group inhibits the PY cells (Johnson and Harris-Warrick 1997). This second depolarizing phase is terminated by pacemaker inhibition, which is also enhanced by DA (Johnson et al. 1995).
We generated realistic waveforms for the LP neuron in control and 10–4 M DA conditions. Because synaptic transmission between pyloric neurons is primarily graded, we filtered (30 Hz) and averaged recordings from six LP neurons in control and in DA; these averaged waveforms (Fig. 2A, top) reflected the shapes of the LP slow wave oscillations in Fig. 1B. The control and DA waveforms did not differ significantly in amplitude (12.2 ± 1.84 and 13.7 ± 1.79 mV, respectively; n = 6, paired t-test; P = 0.27) or period (645 ± 40.62 and 692 ± 57.9 ms, respectively; n = 6, paired t-test; P = 0.53). The trend to a longer mean period of the DA waveform reflects the tendency of DA to slow down the rhythm slightly (Ayali and Harris-Warrick 1999). However, the half-durations of the depolarizing phase of the control and the first depolarizing component of the DA waveforms were significantly different (389.6 ± 57.45 vs. 211.3 ± 17.54 ms; n = 6, paired t-test; P = 0.03). These two waveforms were applied periodically in trains of 10 as presynaptic voltage commands in the LP neuron to examine DA modulation of LPPY graded synaptic dynamics. We drove the LP neuron with both the control and the DA waveforms under both control conditions and in the presence of 10–4 M DA; this allowed us to discriminate between the direct effects of DA on the LPPY synapse and its indirect effects caused by changes in the LP waveform.
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PY synapses driven by realistic LP waveforms
In the pyloric network, a subset of PY neurons is both chemically inhibited by and electrically coupled to the LP neuron. The remaining PY neurons only receive chemical inhibition from the LP neuron. We first looked at the DA effects on the mixed chemical-electrical LPPY synapses with TTX added to the saline to block spontaneous activity and spike-evoked transmitter release (Fig. 2). In response to both control and DA presynaptic LP waveforms, the steady-state PY responses in five LPPY synapses from different preparations under control conditions were weakly depolarizing. However, in 10–4 M DA, the synaptic response to both LP waveforms reversed in sign at four of these synapses to become strongly hyperpolarizing. In the example of Fig. 2, which is the same cell pair shown in Fig. 1B, the first LP waveform in the series elicited weak, biphasic PY responses, consisting of an electrotonic depolarization that outweighed the weak chemical inhibition (Fig. 2A, middle). This response occurred with both the control and the DA LP waveforms. By the second or third waveform in the series, only depolarizing electrotonic responses were obvious, because of marked synaptic depression of the chemical component (Fig. 2A, middle; note PY responses above dashed line marking the resting potential; see also Mamiya et al. 2003). Application of DA depolarized this PY by 15 mV. Both control and DA LP waveforms elicited large, hyperpolarizing graded chemical synaptic potentials, which depressed to a steady-state hyperpolarized value by the forth or fifth repeated LP waveform (Fig. 2A, bottom; note PY IPSPs below dashed line marking the resting potential and the 10-fold reduced voltage scale). In between IPSPs in DA, the PY neuron depolarized above the initial resting potential, reflecting the marked enhancement of postinhibitory rebound that DA evokes in these neurons (Harris-Warrick et al. 1995a).
In theory, the DA-induced depolarization of the postsynaptic PY neuron could by itself explain the enhanced chemical IPSP in Fig. 2A by increasing the driving force on the inhibitory synapse. At the four mixed synapses where synaptic sign was reversed, DA depolarized the PY neurons an average of 14 ± 1.5 mV. However, when the PY neuron was depolarized to the same extent under control conditions without DA, only a small, initial hyperpolarization was seen, and this initial response depressed into the noise level with repeated LP oscillations (Fig. 2B; same synapse as shown in Fig. 2A, note the dashed line marking the resting potential and the expanded voltage scale). We could not analyze PY IPSPs at –55 mV in the presence of DA because these PY neurons generated slow rhythmic membrane potential oscillations when hyperpolarized by current injection.
Figure 3 shows the mean peak PY responses to the first LP waveform and the steady-state PY responses at the end of the LP train at these four mixed synapses, using both control and DA waveforms under both control and DA conditions. DA had significant main effects on the initial and steady-state PY responses, using either the control or the DA LP waveforms (repeated-measures, 2-way ANOVA, P = 0.005 and 0.003, respectively). DA caused significant increases in both peak initial and steady-state PY responses using either the control (post hoc t-test; P = 0.02, and 0.007, respectively) or DA waveforms (post hoc t-test; P = 0.03, and 0.02, respectively). The initial peak PY response in DA was also significantly larger than the initial response under control conditions when the PY neuron was depolarized to the same membrane potential as seen during DA application (n = 4; paired t-test, P = 0.03). Surprisingly, the different LP waveform shapes (control and DA) did not have any significant main effect on the peak amplitudes of either the initial or steady-state PY responses in either control or DA conditions (repeated-measures, 2-way ANOVA, P = 0.77 and 0.33, respectively). Notably, in DA, the second depolarizing component of the DA waveform had no detectable effect on the PY neuron. These experiments suggest that the rather dramatic change in LP waveform evoked by DA has only subtle effects on its synaptic output. A minority of PY neurons across different preparations do not respond to DA (Johnson, Schneider, and Harris-Warrick, unpublished observations), and at a fifth mixed LPPY synapse, DA did not depolarize the PY neuron. The chemical synapse onto this PY was undetectable under control conditions and became apparent only during the first LP oscillation in DA. Like the other mixed synapses we studied in control conditions, the chemical component of this synapse depressed with repeated LP oscillations to leave a predominantly electrotonic, steady-state component (data not shown). Thus at all of these synapses, DA enhanced chemical inhibition, and in the majority of the mixed synapses (4 of 5 from different preparations), this reversed the synaptic sign from depolarizing to hyperpolarizing. This predominant hyperpolarization was maintained during repeated LP oscillations despite relatively strong chemical synaptic depression.
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). This is a rectifying electrical junction where depolarization of the LP is transferred to the PY neuron as a function of the difference between their membrane potentials. Thus DA-induced PY depolarization, by lessening the differential voltage between the LP and PY neurons, could further contribute to weakening the electrotonic component of the mixed synapse. This is shown in Fig. 4, which shows the isolated electrotonic component of the LPPY synapse after PTX was added to block the chemical component and 20 mM TEA was added to partially block voltage-gated K+ conductances and enhance control of the electrotonic component. Under control conditions (with no DA), LP depolarization drove a relatively large electrotonic potential (1.9 mV) in the PY neuron (Fig. 4, left). Depolarization of the PY neuron by 15 mV (to mimic the effect of DA) reduced the electrotonic potential amplitude to 0.62 mV (Fig. 4, middle). This diminished PY electrotonic response could be partially restored when the LP waveform amplitude was in turn increased by 10 mV (Fig. 4A, right). In four experiments, the mean PY electrotonic response at –55 mV to a 30-mV LP depolarization (1.3 ± 0.63 mV) was significantly greater than the PY response at –40 mV (0.43 ± 0.16 mV; paired t-test; P = 0.048). This shows that LPPY rectifying electrical coupling depends on the voltage difference between the two neurons.
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PY synapses during realistic network activity
A subset of the PY neurons are not electrically connected to the LP and show only chemical inhibition on LP stimulation (Johnson et al. 1994). At these purely chemical LPPY synapses, the PY neuron displayed a hyperpolarizing response to the first LP waveform in control conditions, but this depressed to little or no postsynaptic response with repeated LP oscillations using either the control or the DA LP waveforms (Fig. 5A, middle; note dashed line marking the resting potential). DA enhanced synaptic inhibition in response to the first control and DA waveforms (Fig. 5A, bottom). Although these synapses depressed strongly, there remained a small hyperpolarizing response at steady state during the LP train using both pulse types (Fig. 5A, bottom; note PY responses below dashed line marking the resting potential). In these neurons, DA evoked a depolarization in the PY neurons that was weaker than in the PYs receiving mixed synapses (4 ± 2.6 mV; n = 4); we could eliminate this slight depolarization with current injection, but the IPSP was still enhanced (Fig. 5B; same synapse as in Fig. 5A). Figure 6 shows the mean peak PY responses to the first LP waveform and the steady-state PY responses at the end of the LP train at these chemical synapses, using both control and DA waveforms under both control and DA conditions. In three of four experiments, under control conditions, synaptic depression was strong enough to completely eliminate the PY steady-state response. Again, DA had significant main effects on the initial and steady-state PY responses, using either the control or the DA LP waveforms (repeated-measures, 2-way ANOVA, P = 0.007 and 0.01, respectively). DA significantly increased the peak initial and steady-state PY responses to the control (post hoc t-test; P = 0.03, and 0.04, respectively) and DA waveforms (post hoc t-test; P = 0.01, and 0.03, respectively). When the PYs were hyperpolarized back to the control resting potential in DA conditions, the initial PY IPSP was still significantly larger in response to the control LP waveform (n = 4; paired t-test, P = 0.046). Again, switching between control and DA LP waveform shapes had no significant main effect on the initial PY responses or the steady-state PY responses under control conditions or during DA application (repeated-measures, 2-way ANOVA, P = 0.52 and 0.62, respectively; Fig. 6). Thus during realistic network activity, DA enhanced the purely chemical LPPY synapses strongly enough to maintain chemical inhibition that depressed to silence in most cases under control conditions.
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We were surprised that changing the presynaptic LP waveform from the control to the DA shape did not cause significant differences in the amplitudes of the initial and steady state PY IPSPs (Figs. 3 and 6). We looked for more subtle effects of waveform shape and found two small, but statistically significant, differences in the duration and depression of PY inhibitory responses to the two types of waveforms during DA application. We combined the IPSP measurements that were enhanced by DA from both mixed and purely chemical LPPY synapses (n = 8) because these IPSPs were large enough to measure accurately, and because DA-enhanced steady-state IPSPs did not depress completely, allowing calculation of the depression index (DI). In the presence of DA, the mean half-duration of the first PY IPSP in response to the control LP waveform was significantly longer (225 ± 71.6 ms) than the response to the DA waveform (199 ± 63.3 ms; paired t-test; P = 0.05), correlating with the different durations of the two LP waveforms (Fig. 7A, bottom).
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LPPY firing delay in control and DA conditions
Under control conditions, the LPPY synapse seems to have little effect on the onset of PY firing in the rhythmic pyloric motor pattern, in that LP hyperpolarization does not strongly advance the onset of PY spiking (Mamiya et al. 2003; Weaver and Hooper 2003a). We hypothesized that application of DA, by weakening electrical coupling and very significantly strengthening the functional LPPY inhibition, may increase the LP control of PY firing onset. We first examined the onset time delay between LP and PY (1st LP spike to 1st PY spike) in experiments where the cycling network activity of both neuron types was recorded simultaneously in the absence and presence of DA. Apparently consistent with our hypothesis, the mean LP-PY delay across our population of 21 cell pairs from 14 preparations was 42% greater in DA than in control conditions (Fig. 8A). This effect was not, however, statistically significant (paired t-test; P = 0.10). During DA application, the pyloric cycle period also became slightly longer, as described above (Ayali and Harris-Warrick 1999), so when the LP-PY delay was converted into a fraction of the period to take this into account, the phasing between LP and PY was unchanged by DA (Fig. 8B; paired t-test; P = 0.734).
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PY synaptic connection for every cell pair, the PY firing delay did not seem to correlate with the type of synaptic connection with the LP (purely chemical vs. mixed chemical/electrical). Thus for most PY neurons, DA appeared to make the onset of their firing more uniform relative to the LP neuron. LP influence on the AB to PY firing delay and PY burst properties
To further examine the LP influence on PY firing onset, we examined the effect of removing its synaptic input by hyperpolarizing the LP neuron and measuring the change in the delay of PY firing relative to the AB pacemaker during network activity in the presence and absence of DA. If the LP neuron exerted more control over PY firing during DA application because of its stronger synaptic inhibition, its temporary removal from the network should advance PY firing relative to the pacemaker AB to a greater extent in DA than under control conditions. Figure 9A shows an experiment to test this hypothesis in a cycling preparation; the LP neuron was hyperpolarized under control conditions and during application of DA. Here, block of LP activity weakly accelerated the cycle frequency in both control (8% decrease in period; see also Hooper and Marder 1987; Selverston and Miller 1980; Weaver and Hooper 2003b) and DA conditions (10% decrease in period), because of the removal of LP inhibitory feedback to the pacemaker kernel (Figs. 9A and 1A). When the LP neuron was released from hyperpolarization, it fired a strong postinhibitory rebound burst which inhibited AB firing in control (Fig. 9A, left) and delayed AB firing in DA (Fig. 9A, right). This showed that LP chemical synapses were active, and their steady-state depression was removed by the hyperpolarization.
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PY synaptic inhibition was enhanced by DA (17.5 ± 18.1 ms; Fig. 9B, DA; no main effect of LP hyperpolarization on AB to PY delays; repeated-measures, 2-way ANOVA; P = 0.27). Because of the acceleration of the pyloric rhythm, there was no main effect of LP hyperpolarization on the phase of PY onset relative to AB during LP hyperpolarization in either control or DA conditions (Fig. 9C; 2-way ANOVA; P = 0.39). These results suggest that, contrary to our earlier hypothesis, the addition of DA did not increase LP control of PY firing onset.
These small changes in AB to PY delay during LP hyperpolarization cannot account for the tendency of the LP to PY time delay to increase during DA application (Fig. 8A); if this increase was caused by stronger LP inhibition of the PY neurons, it would also delay PY onset relative to the AB neuron. We further compared the AB to LP and AB to PY time delays during control and DA network activity. These experiments showed that, in fact, DA decreases the AB to LP firing delay more (32 ± 4.1%) than the AB to PY firing delay (16 ± 6.5%; post hoc t-test, P = 0.002); that is, the increases in LP to PY delay (Fig. 8C) are caused by the LP advancing more, relative to the AB, than the PY, rather than the LP delaying the PY (Fig. 9D). The slightly longer cycle period during DA application neutralized the slightly longer time delay between LP and PY onset; indeed, in these experiments, the DA-evoked phase advances for LP (0.25 ± 0.02) and PY (0.26 ± 0.09) relative to AB were similar (post hoc t-test; P = 0.48; Fig. 9E), consistent with the constancy of LP to PY phase under control and DA conditions (Fig. 8B). These results did not depend on the LPPY synaptic connection; the LP neuron made mixed synapses with one-half of these PY neurons and purely chemical synapses with the rest.
LP hyperpolarization also had no significant main effects on other aspects of PY activity in the presence or absence of DA, including the burst duration (Fig. 10A), the number of APs per burst (Fig. 10B), or the duty cycle (Fig. 10C; repeated-measures ANOVAs; P > 0.42 for all comparisons). Note, however, that DA did have a significant main effect to enhance excitability in these PY neurons, as indicated by the increases in burst duration, number of action potentials per burst, and duty cycle (Fig. 10, A–C; repeated-measures ANOVAs; P < 0.003 for all comparisons). We have previously documented the significant enhancement of DA of postinhibitory rebound in the PY neurons (Harris-Warrick et al. 1995a).
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PY inhibition (and even its sign reversal in isolated conditions), this synapse is still not strong enough to significantly delay PY firing onset in an intact, rhythmic network. Thus intrinsic PY firing properties and their enhancement by DA determine PY firing under both control and DA conditions. |
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DISCUSSION |
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PY synaptic transmission
Using single square pulses, we previously showed that DA strengthens LPPY chemical inhibition (Johnson et al. 1994) and weakens electrical coupling between the neurons (Johnson et al. 1993a). However, these graded chemical synapses show marked depression during normal membrane potential oscillations (Mamiya and Nadim 2004; Mamiya et al. 2003; Manor et al. 1997), and it was not clear whether DA could still enhance a depressed synapse. Here we have shown that under partially depressed conditions using realistic LP waveforms, DA can still dramatically strengthen the inhibitory component and weaken the electrotonic component of the mixed LPPY synapse, reversing the sign of most LPPY synapses from depolarizing to strongly hyperpolarizing. DA increases chemical inhibition by both presynaptic enhancement of ICa (Johnson et al. 2003) to enhance transmitter release from LP terminals (Johnson and Harris-Warrick 1997) and by postsynaptic enhancement of the PY neurons' response to glutamate, the LP neuron's transmitter (Johnson and Harris-Warrick 1997), accompanied by depolarization and increased input resistance (Harris-Warrick et al. 1995a; Johnson et al. 1993a). These postsynaptic actions are partially caused by a decrease in IA (Harris-Warrick et al. 1995a) and perhaps to a small extent by an increase in Ih (J. Peck and R. Harris-Warrick, unpublished observations). In addition, electrical coupling at mixed LPPY synapses is weakened directly by DA (Johnson et al. 1993a), and as we show here, also indirectly as a result of postsynaptic depolarization that decreases the voltage difference between the two neurons at this rectifying synapse.
DA might also reduce the steady-state level of synaptic depression at the LPPY synapse. In our experiments, for both mixed and purely chemical synapses, the chemical inhibition was almost completely depressed at steady state under control conditions. In control conditions similar to ours, Mamiya and Nadim (2005) estimated that the chemical component of LPPY mixed synapses depresses by 
90%. In contrast, the chemical inhibition was only depressed by 65% at steady state in the presence of DA. DA has been shown to prevent the development of synaptic depression in other systems (Baimoukhametova et al. 2004).
Effect of control and DA LP waveform shape on LPPY dynamics
The amplitude and time-courses of graded IPSPs have been shown to depend on the shape of the presynaptic waveform (Mamiya and Nadim 2004; Manor et al. 1997; Olsen and Calabrese 1996; Simmons 2002). Our ability to drive the LP cell with either control or DA waveforms in the absence or presence of DA allows us to distinguish direct DA modulation of the presynaptic release process itself from indirect modulation caused by DA-induced changes in the LP presynaptic waveform. Despite the very marked differences in LP waveform shape under control and DA conditions (Figs. 1A and 2A), we saw no significant effect of LP waveforms on the amplitudes of the initial or steady-state PY IPSPs, when tested under the same conditions (control or during application of DA). Thus DA enhancement of the peak LPPY inhibition arises from direct actions of DA at the synapse and not from indirect changes in presynaptic waveform shape. The different waveform shapes did cause subtle changes in the duration of the IPSP and on the depression index, but these effects are probably too small to have significant functional consequences for the network, especially because the enhanced LPPY inhibition did not contribute much to the onset time of PY firing.
Intrinsic rebound properties outweigh LP synaptic inhibition in determining the onset of PY firing
The control of patterned firing in motor networks is normally thought to be achieved through a balance between synaptic interactions and the intrinsic firing properties of the network neurons. Under control conditions of ongoing rhythmic network activity, we found, as others had before (Mamiya et al. 2003; Weaver and Hooper 2003b), that removal of the LP neuron had only a small effect to advance the onset of PY firing. It seems that the PY neurons are primarily responding to the very strong periodic inhibition from the pacemaker kernel (AB-PD), and their intrinsic PIR from this pacemaker inhibition plays the dominant role in setting their onset time under control conditions. However, we expected that after DA strongly enhanced LPPY inhibition, this synapse would assume a more important role in delaying PY onset. This was not observed: removal of LP had equally small effects on PY onset times in the presence and absence of DA (Fig. 9, A and B) and had no effect on PY phasing relative to the pacemaker kernel (Fig. 9C). DA strongly enhances the PY neurons' intrinsic postinhibitory rebound after pacemaker inhibition (Harris-Warrick et al. 1995a), and this still outweighs the effects of the LPPY synapse in setting PY onset times. While the overall effect of DA was to phase advance the PY neurons relative to the pacemaker kernel (AB/PD; Fig. 9C), the LP neuron was time-advanced even more than the PY, relative to the pacemaker AB neuron (Fig. 9D); this led to the small average increase in timing delay of PY relative to LP.
A more important effect of DA is to make the delay between LP and PY onset more uniform (Fig. 8B). PY neurons that had short delays relative to LP had them prolonged by DA, whereas PY neurons with long delays had them shortened, so the variance of the LP to PY delay was significantly smaller in DA. Thus the PY neurons fire more synchronously as a group in DA than under control conditions. Because the PY neurons poly-innervate nonspiking muscles, this more synchronized and strengthened PY activity should evoke stronger PY contractions in DA than under control conditions. It is likely that this enhanced regularity of PY firing onset arises primarily from intrinsic changes in their rebound from AB/PD inhibition, with LPPY inhibition playing a minor role.
These reset experiments were performed with an actively cycling pyloric preparation, driven by mixed modulatory inputs from higher ganglia. It is thus possible that DA failed to enhance the effect of hyperpolarizing the LP on PY onset because these synapses are already fully modulated by the inputs from higher centers. DA is a natural neuromodulator in crustaceans (Nusbaum and Beenhakker 2002; Tierney et al. 2003), and DA and other modulators may already be affecting the LPPY synapses during pyloric activity, occluding the effects of additional DA. However, we think this unlikely. Under the same conditions, bath-applied DA has a number of other effects similar to those seen in the absence of other modulators, including exciting PY firing (Fig. 10; Flamm and Harris-Warrick 1986b; Harris-Warrick et al. 1995a), and strengthening LPPD inhibition (Ayali et al. 1998; Johnson et al. 1995). In addition, in preliminary experiments, we have monitored the effect of LP hyperpolarization on AB-PY timing in an isolated pyloric network, with no neuromodulators present except DA. Even in this simplified preparation, LP removal does not seem to significantly advance the onset of PY firing relative to the pacemaker group.
We still do not understand why DA's enhancement of the LPPY synapse fails to increase the efficacy of this synapse in determining the PY onset time. The competing increase in PY excitability seems to counterbalance the strengthening of this synaptic inhibition; yet when the inhibition is removed (by hyperpolarizing the LP), we do not see a dramatic advance in PY onset relative to control conditions. Further work will be needed to test how this arises from complex network interactions that are not yet fully understood.
Separating the complementary contributions of intrinsic and synaptic mechanisms that shape the patterns of neuronal output in a functioning neural network is a difficult challenge in most systems. This is especially true for complex vertebrate networks, although progress has been made in studies of contrast adaptation in the cat visual cortex (Nowak et al. 2005). New models of the mechanisms underlying memory consolidation are also beginning to consider both synaptic and intrinsic mechanisms (Xu et al. 2005; Zhang and Linden 2003). As we have shown here, modulatory actions that dramatically alter network synaptic interactions may in fact not be quantitatively very important for follower cell activity compared with intrinsic changes that are happening simultaneously. This emphasizes our finding in the pyloric network that the functional importance of synaptic modulation can only be understood in the context of parallel modulation of intrinsic properties in a rhythmic network.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. R. Johnson, Dept. of Neurobiology and Behavior, S.G. Mudd Hall, Cornell Univ., Ithaca, NY 14853 (E-mail: BRJ1{at}Cornell.Edu)
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