Monoamine control of the pacemaker kernel and cycle frequency in the lobster pyloric network

Abstract

The monoamines dopamine (DA), serotonin (5HT), and octopamine (Oct) can each sculpt a unique motor pattern from the pyloric network in the stomatogastric ganglion (STG) of the spiny lobster Panulirus interruptus. In this paper we investigate the contribution of individual network components in determining the specific amine-induced cycle frequency. We used photoinactivation of identified neurons and pharmacological blockade of synapses to isolate the anterior burster (AB) and pyloric dilator (PD) neurons. Bath application of DA, 5HT, or Oct enhanced cycle frequency in an isolated AB neuron, with DA generating the most rapid oscillations and Oct the slowest. When an AB-PD or AB-2xPD subnetworks were tested, DA often reduced the ongoing cycle frequency, whereas 5HT and Oct both evoked similar accelerations in cycle frequency. However, in the intact pyloric network, both DA and Oct either reduced or did not alter the cycle frequency, whereas 5HT continued to enhance the cycle frequency as before. Our results show that the major target of 5HT in altering the pyloric cycle frequency is the AB neuron, whereas DA's effects on the AB-2xPD subnetwork are critical in understanding its modulation of the cycle frequency. Octopamine's effects on cycle frequency require an understanding of its modulation of the feedback inhibition to the AB-PD group from the lateral pyloric neuron, which constrains the pacemaker group to oscillate more slowly than it would alone. We have thus demonstrated that the relative importance of the different network components in determining the final cycle frequency is not fixed but can vary under different modulatory conditions.

Fig. 1.

Schematic diagram of the different preparations used in this study. A, Isolated AB or PD neurons (descending modulatory inputs were either blocked by a tetrodotoxin block on the stomatogastric nerve or kept intact). B, An AB–PD (1) or AB–2xPD (2) pacemaker subnetwork (with or without descending inputs).C, The intact pyloric circuit (modulatory inputs were kept intact). An example of the method of isolating neurons and subnetworks is also shown in C. To isolate the AB–PD subnetwork (shown in B1), the VD neuron and one PD neuron were photoinactivated, and the LP → PD synapse was pharmacologically blocked with picrotoxin. All synaptic connections are either electrical (nonrectifying synapses, resistors; rectifying synapses, diodes) or chemical inhibitory (small circles).

Fig. 2.

Effects of bath-applied amines on the AB and PD neurons. Each neuron was isolated from all pyloric synaptic inputs as described in Materials and Methods. Descending modulatory inputs via the stomatogastric nerve were either blocked (A) or kept intact (B). Both A1 and B1 for the AB neuron, as well as A2 and B2 for the PD neuron, show recordings from a single neuron. Dopamine (DA), serotonin (5HT), and octopamine (OCT) induced fully reversible changes in the cellular activity. The control resting membrane potential or potential at the lowest voltage of oscillations is marked byarrows.

Fig. 3.

Effects of bath-applied dopamine (DA), serotonin (5HT), and octopamine (Oct) on the AB neuron, when isolated from all pyloric synaptic inputs as described in Materials and Methods, with descending modulatory inputs in the stomatogastric nerve blocked (A) or left intact (B).Filled bars, AB cycle frequency; open bars, mean spike frequency within the AB burst. The data show the mean of three (A) or five (B) different preparations ± SD. InA, both parameters measured in Oct (*) are significantly different from DA and 5HT frequencies. Both parameters measured in all three amines in B (*) are significantly different from control. In addition, the measurements in Oct are significantly different from those in DA (**).

Fig. 4.

A, Effects of bath-applied amines on an AB–PD subnetwork isolated from all pyloric synaptic inputs. Descending modulatory inputs were kept intact. A1, Simultaneous recordings from the AB and PD neurons. The recordings shown in all panels are from a single preparation. Dopamine (DA), octopamine (Oct), and serotonin (5HT) induced fully reversible and reproducible changes in the subnetwork activity. The control oscillation trough membrane potential of both neurons is marked by arrowsin all panels. A2, Effects of the bath-applied amines on cycle frequency in the AB–PD subnetwork. Open bars, Control; filled bars, amine bath application.B, Effects of bath-applied amines on cycle frequency in an AB–2xPD subnetwork, with descending modulatory inputs intact. InA2 and B, data show the mean of five to eight different preparations ± SD. Cycle frequencies in 5HT and Oct (*) are significantly different from control and DA but not from each other.

Fig. 5.

Effects of bath-applied dopamine (DA), serotonin (5HT), and octopamine (Oct) on the intact pyloric network with descending modulatory inputs. A, Simultaneous recordings from four of the six pyloric neuron classes. All of the recordings are from a single preparation. All amine effects were reversible after wash with normal saline. B, Effects of bath-applied amines on the pyloric cycle frequency in the intact pyloric network with descending modulatory inputs. Open bars, Control;filled bars, amine bath application. Data show the mean of six different preparations ± SD. Cycle frequency in 5HT (*) is significantly different from control, Oct, and DA frequencies.

Fig. 6.

Summary of amine effects on the cycle frequency of an isolated AB neuron, an AB–PD subnetwork, an AB–2xPD subnetwork, and an intact pyloric circuit, all with intact descending modulatory inputs. Data shown as percentage relative to control frequencies in each condition. Means of five to eight different preparations ± SEM are shown. An asterisk signifies a significant difference between amines within the same type of preparation. For each amine, bars marked by adifferent letter show statistically significant difference between a single amine-induced change in the different types of preparations; the same letter indicates no significant difference between the different types of preparations.

Fig. 7.

The effect of the LP neuron on octopamine (Oct) and dopamine (DA) induced rhythms in an intact pyloric network with descending modulatory inputs. Simultaneous recordings from the LP and a PD neuron in control conditions (A), during Oct bath application (B), and during DA bath application (C) are shown. In both Oct and DA, the traces shown in 1 demonstrate the final and stable amine-induced rhythm (∼5 min of bath application). The traces in2 show the effect of strongly hyperpolarizing the LP neuron by current injection on the amine-induced rhythm. The traces shown in 2 were recorded a few seconds after the recordings shown in 1. The amine effects and effects of hyperpolarizing the LP neuron were fully reversible.

Fig. 8.

The effect of hyperpolarizing current injections to the PD neurons in an intact pyloric network with the LP → PD synapse pharmacologically blocked. In all the experiments shown, hyperpolarizing current pulses were intracellularly injected to a PD neuron while the neuron’s membrane potential was recorded with a second electrode. A, Simultaneous recording from PD and AB neurons. Two sweeps (from the same preparation) are overlaid. In both, a cycle with no current injection is followed by one with an inhibitory stimulus mimicking the LP inhibition of PD during DA (black) or Oct (gray) (for details, see Results). The time of current injection is shown by theblack and gray bars, and the phase of two cycles is shown based on the prestimulation cycle (the first spike in the AB neuron is defined as phase 0). B, The effect of inhibitory stimulation of PD on the pyloric cycle period. Single stimuli (three different stimulus protocols as shown in the graph’s legend) were given at different phases along the PD bursting cycle. The stimuli duration and intensity correspond to those generated by the LP neuron in a fully intact network in control (open squares), DA (black squares), or Oct (gray squares) conditions. The phase of the end of the inhibitory stimulus (calculated relative to the preceding cycle with no stimulus as shown in A) is plotted against the change in cycle period generated by the inhibition (period with inhibition/period in previous cycle with no inhibition × 100). The end phase could exceed 1.0, as for example in the sweep marked Oct in A. Data points from four different preparations are shown. The dashed line is a linear fit calculated for all the data points shown (pooled together). Theoval marked Control refers to the open data points, corresponding to LP inhibition of PD in a fully intact network in control conditions. Similarly, the areas markedDA and Oct refer to the filled black and filled gray data points, respectively, and represent LP inhibition of PD during DA or Oct bath application.C, The effect of repetitive inhibitory inputs to the PD neuron on the pyloric rhythm. The three PD traces (from the same neuron) show the effect of control- (cont.), DA-, and Oct-like repetitive inhibitory stimulation using stimulus parameters and phasing indicated by the ovals in B. The time of current injection is shown by thebars.

Fig. 9.

The onset of the effect of 10⁻⁴m (A) or 5 × 10⁻⁶m(B) dopamine bath application on an AB–PD subnetwork isolated from all pyloric and descending inputs. In bothA and B, panel 1 shows a 1 min period around the onset of dopamine’s effect on the two neurons (the first AB spike is marked by an arrow). Panel 2 shows the final and stable dopamine-induced rhythm (∼5 min of bath application).

Fig. 10.

A, The biphasic effect of dopamine bath application (10⁻⁴m) on the pyloric pacemaker subnetwork isolated from all pyloric synaptic inputs and with intact descending inputs. All the data were collected from a single preparation. Dopamine was bath-applied and washed before and after photoinactivation to sequentially remove the first and then the second PD neuron (AB–2xPD, AB–PD, and isolated AB). The three panels in B show simultaneous recording from the AB (top traces) and PD (bottom traces) neurons in the AB–PD subnetwork, at the time data points marked in Aby corresponding letters.

Fig. 11.

The pyloric network components that are the major targets of monoamine modulation of cycle frequency. Schematic diagrams of the intact pyloric network are shown. In each panel (5HT, DA, and Oct) the circuit components that are instrumental in determining the specific amine-induced cycle frequency are highlighted.