Coordination of fast and slow rhythmic neuronal circuits

Abstract

Interactions among rhythmically active neuronal circuits that oscillate at different frequencies are important for generating complex behaviors, yet little is known about the underlying cellular mechanisms. We addressed this issue in the crab stomatogastric ganglion (STG), which contains two distinct but interacting circuits. These circuits generate the gastric mill rhythm (cycle period, approximately 10 sec) and the pyloric rhythm (cycle period, approximately 1 sec). When the identified modulatory projection neuron named modulatory commissural neuron 1 (MCN1) is activated, the gastric mill motor pattern is generated by interactions among MCN1 and two STG neurons [the lateral gastric (LG) neuron and interneuron 1]. We show that, during MCN1 stimulation, an identified synapse from the pyloric circuit onto the gastric mill circuit is pivotal for determining the gastric mill cycle period and the gastric-pyloric rhythm coordination. To examine the role of this intercircuit synapse, we replaced it with a computational equivalent via the dynamic-clamp technique. This enabled us to manipulate better the timing and strength of this synapse. We found this synapse to be necessary for production of the normal gastric mill cycle period. The synapse acts, during each LG neuron interburst, to boost rhythmically the influence of the modulatory input from MCN1 to LG and thereby to hasten LG neuron burst onset. The two rhythms become coordinated because LG burst onset occurs with a constant latency after the onset of the triggering pyloric input. These results indicate that intercircuit synapses can enable an oscillatory circuit to control the speed of a slower oscillatory circuit, as well as provide a mechanism for intercircuit coordination.

Fig. 1.

Activation of MCN1 elicits a gastric mill rhythm in the STG. A, Schematic illustration of the isolated stomatogastric nervous system, including the soma location and axonal projection pathway ofMCN1. B, Activation of the gastric mill rhythm in the isolated STG by stimulation ofMCN1. Before MCN1 stimulation, there was no gastric mill rhythm (LG and DG were silent; Int1 fired pyloric-timed bursts), but there was an ongoing pyloric rhythm (AB neuron recording). During tonic stimulation of both MCN1 neurons (extracellular stimulation of both ions at 10 Hz each) (Bartos and Nusbaum, 1997a), the pyloric rhythm cycled faster, and the gastric mill rhythm was activated. The pyloric-timed subthreshold oscillations inLG during MCN1 stimulation result from pyloric-timed inhibition of Int1, producing rhythmic disinhibitions in LG (arrow). The smaller amplitude, bursting unit in the dgn recording is theDG neuron (arrows), whereas the tonically firing unit is the anterior gastric receptor (AGR), an identified sensory neuron. Most hyperpolarized V_mvalues: AB, −60 mV; Int1, −56 mV; andLG, −72 mV. C, Schematic circuit diagram underlying MCN1 activation of the gastric mill rhythm. The circuit represents the two phases of the gastric mill rhythm, including retraction (left; Int1 andDG active) and protraction (right;LG active). Members of the pyloric pacemaker ensemble, the AB and PD neurons, are also included. The MCN1 synapse on PD is a functional representation. It is not determined whether MCN1directly excites PD, AB, or both neurons.Arrows represent the pathway of functioningMCN1 transmission. Active neurons and synapses are labeled black, whereas inactive neurons and synapses are labeled gray. T-barsrepresent excitatory chemical transmission; filled circles represent inhibitory chemical transmission; andresistor symbols represent electrical transmission. Nerves: dvn, dorsal ventricular nerve;dgn, dorsal gastric nerve; ion, inferior oesophageal nerve; lgn, lateral gastric nerve;lvn, lateral ventricular nerve; mgn, medial gastric nerve; mvn, medial ventricular nerve;pdn, pyloric dilator nerve; pyn, pyloric nerve; son, superior oesophageal nerve;stn, stomatogastric nerve. Ganglia: CoG, commissural ganglion; OG, oesophageal ganglion;STG, stomatogastric ganglion. Neurons:AB, anterior burster; DG, dorsal gastric;Int1, interneuron 1; LG, lateral gastric;MCN1, modulatory commissural neuron 1;PD, pyloric dilator. Subsets of these abbreviations also appear on subsequent figures.

Fig. 2.

The pyloric rhythm regulates the cycle period of the gastric mill rhythm. A, Top, In the isolated STG, MCN1 stimulation (both ions at 10 Hz each; data not shown) drives a vigorous pyloric rhythm (pdn) and gastric mill rhythm, represented by the alternating bursts inDG (dgn) and LG(lgn). Bottom, In the same preparation, after the pyloric rhythm was turned off by injecting hyperpolarizing current into the pyloric pacemaker neurons (note the absence of bursting in pdn), the same level of MCN1 stimulation elicited a slower gastric mill rhythm. B, Histograms representing the mean gastric mill cycle period during MCN1 stimulation in the presence (pyloric rhythm on, 7.1 ± 2.8 sec;n = 22) and absence (pyloric rhythm off, 19.1 ± 6.0 sec; n = 22) of the pyloric rhythm are shown. The gastric mill cycle period was longer when the pyloric rhythm was off (Mann–Whitney test, **p < 0.001).

Fig. 3.

The gastric mill cycle period is a function ofMCN1-firing frequency. A,Left, With the pyloric rhythm in progress, increasing the MCN1-firing frequency decreases the gastric mill cycle period. Most hyperpolarized V_m:LG, −66 mV. Right, When the pyloric rhythm is turned off by hyperpolarization of the pyloric pacemaker neurons, the gastric mill cycle period still decreases with increasing levels of MCN1 activity. Note, however, that at any level of MCN1 activity the gastric mill cycle period is shorter when the pyloric rhythm is present. Each indicatedMCN1-firing frequency represents its activity acrossleft and right panels. Most hyperpolarized V_m: LG, −74 mV.B, Plot of the gastric mill period (mean ± ς) as a function of MCN1 frequency in the presence and absence of the pyloric rhythm is shown. Stim.Freq., Stimulation frequency.

Fig. 4.

The artificial AB to Int1 synapse reproduces the MCN1-activated gastric mill rhythm. MCN1 was stimulated (both ions at 14 Hz each) while the natural pyloric rhythm was shut off. The resulting gastric mill rhythm exhibited a decreased cycle period when pyloric-timed dynamic-clamp IPSPs (I_dyn,Int1, g = 40 nS; cycle period = 0.75 sec) were injected into Int1during each LG neuron interburst interval. Note the resulting rhythmic disinhibitions in LG, which also occur during the natural pyloric rhythm. The thickened trace at the depolarized peak of each Int1oscillation consists of action potentials. Most hyperpolarized V_m values: DG, −59 mV;Int1, −55 mV; and LG, −70 mV.dyn, Dynamic clamp; MG, medial gastric.

Fig. 5.

The gastric mill cycle period is a function of the strength of the pyloric inhibition of Int1.A, Top, With the pyloric rhythm off, the MCN1-elicited gastric mill rhythm cycles slowly. Middle, Replacement of the AB inhibition of Int1 with dynamic-clamp injections into Int1(I_dyn,Int1, g = 5 nS; cycle period = 1 sec) is sufficient to reduce the period of the MCN1-elicited gastric mill rhythm. Bottom, Increasing the strength of the dynamic-clamp injections, by increasing the conductance of the artificial synapse (g = 10 nS), further reduces the gastric mill cycle period. Most hyperpolarized V_m values: Int1, −43 mV; and LG, −78 mV. B, The mean gastric mill cycle period is presented as a function of either the dynamic-clamp conductance (g_dyn,Int1, filled circles) or the cycle period during ongoing pyloric rhythms from the preparation shown in A (open triangle). Each data point represents the mean (± SD) gastric period from a series of cycles during a 200 sec interval.Nat., Natural.

Fig. 6.

The gastric mill cycle period is a function of the strength of the pyloric-timed disinhibition of LG.A, The same procedure is used as in Figure5A, except that dynamic-clamp pulses are injected as disinhibitions into LG. Most hyperpolarized V_m: LG, −73 mV.B, The mean gastric mill cycle period is presented as a function of the dynamic-clamp conductance (g_dyn,LG, filled circles) and the mean gastric mill cycle period during ongoing pyloric rhythms (open triangle) from the preparation shown in A. Each data point represents the mean (± SD) gastric mill period from a series of cycles during a 200 sec interval.C, Dynamic-clamp injections into LG or Int1 can reproduce the gastric mill cycle period that occurs during natural pyloric rhythms. The gastric mill cycle period (mean ± SEM; n = 10) during MCN1 stimulation is plotted in the presence or absence of a natural pyloric rhythm or during dynamic-clamp injections (reconstructed). The gastric mill cycle period was longer when the pyloric rhythm was off (paired ttest, **p < 0.001). There is no significant difference in the gastric mill period between the natural pyloric rhythm and the dynamic-clamp–reconstructed rhythm (pairedt test, p > 0.05).

Fig. 7.

The gastric mill cycle period and the timing of each gastric mill cycle are a function of the frequency of the pyloric input. A, Top, With the pyloric rhythm turned off, the MCN1-elicited gastric mill rhythm cycles slowly.Middle, Replacement of the AB inhibition of Int1 with dynamic-clamp injections into LG(I_dyn,LG) reduced the cycle period of the MCN1-elicited gastric mill rhythm. Bottom, Increasing the period of the dynamic-clamp injections caused a smaller reduction in the gastric mill cycle period. Most hyperpolarized V_m: LG, −82 mV.B, Each LG burst onset was time-locked to the preceding pyloric pacemaker neuron burst, both in the presence of the natural pyloric rhythm and when the natural pyloric rhythm is replaced with dynamic-clamp injections into LG. This relationship persists at all pyloric periods. In the case of the natural pyloric rhythm, the pyloric period was changed by injecting DC current in AB or PD. The dotted line indicates the maximum possible values for the latency, where the latency is equal to the period. C, When the dynamic-clamp injections were made into Int1 or LG, on average the gastric mill rhythm became slower as the period of the injections was increased (two-way ANOVA, p < 0.005; n = 6).D, There was not a strict monotonic relationship between the cycle period of the artificial AB synapse and the resulting gastric mill period within each experimental episode (140 sec). Gastric mill periods within one episode (vertical set of data points) are plotted versus the period of the dynamic-clamp injection for that episode. Dotted lines are lines of integer slope (y = kx, wherek ranges from 3 to 12). All experimental episodes (runs of 140 sec) come from the same preparation.

Fig. 8.

Single properly timed pyloric pulses are sufficient to recreate the natural gastric mill cycle period. In this experiment, the pyloric rhythm was turned off (top LG trace), and pyloric-like dynamic-clamp pulses were injected into LG (I_dyn,LG,g = −50 nS; cycle period = 1 sec). The meanLG interburst duration (D) during the ensuing gastric mill rhythm was determined from a sequence of cycles (second LG trace from the top). This duration was used for determining the time of injection, after the end of each LG burst, of a single pulse of the same conductance. When single pulses were delivered at durationD, each pulse elicited an LG burst, and the gastric mill rhythm was entrained to these pulses (third LG trace). When these single pulses were delivered at timesd < D, no LG burst was elicited until a subsequent pulse at a time 2d> D (bottom LG trace). Most hyperpolarized V_m: LG, −80 mV.

Fig. 9.

The gastric mill cycle period is a function of both the MCN1-firing frequency and the strength of the rhythmic input from the AB neuron. Left(top), Pyloric-like dynamic-clamp pulses (g_dyn,LG = −30 nS) were injected into LG during MCN1 stimulation (25 Hz) to elicit the gastric mill rhythm. Left(middle), As in Figure 8, replacement of the rhythmic pulses with single pulses at duration D replicated the gastric mill cycle period. Left (bottom),Top, Single pulses were also able to replicate this cycle period if either parameter were reduced while the other parameter was sufficiently increased. Right,Bottom, If either parameter were reduced while the other one was maintained constant, single pulses did not elicit anLG burst. + indicates single dynamic-clamp pulses that entrained the gastric mill rhythm to the period resulting from rhythmic pulses; + with shaded circle indicates entrainment with control parameters; − indicates no such entrainment.

Fig. 10.

The gastric mill cycle period is a function of both the MCN1-firing frequency and the strength of the pyloric-timed input. Increasing either parameter speeds up the gastric mill rhythm, and increasing both parameters is more effective than increasing either one alone. Data shown are from a single preparation.