Graded Transmission without Action Potentials Sustains Rhythmic Activity in Some But Not All Modulators That Activate the Same Current

Abstract

Neurons in the central pattern-generating circuits in the crustacean stomatogastric ganglion (STG) release neurotransmitter both as a graded function of presynaptic membrane potential that persists in TTX and in response to action potentials. In the STG of the male crab Cancer borealis, the modulators oxotremorine, C. borealis tachykinin-related peptide Ia (CabTRP1a), red pigment concentrating hormone (RPCH), proctolin, TNRNFLRFamide, and crustacean cardioactive peptide (CCAP) produce and sustain robust pyloric rhythms by activating the same modulatory current (I_MI), albeit on different subsets of pyloric network targets. The muscarinic agonist oxotremorine, and the peptides CabTRP1a and RPCH elicited rhythmic triphasic intracellular alternating fluctuations of activity in the presence of TTX. Intracellular waveforms of pyloric neurons in oxotremorine and CabTRP1a in TTX were similar to those in the intact rhythm, and phase relationships among neurons were conserved. Although cycle frequency was conserved in oxotremorine and TTX, it was altered in CabTRP1a in the presence of TTX. Both rhythms were primarily driven by the pacemaker kernel consisting of the Anterior Burster and Pyloric Dilator neurons. In contrast, in TTX the circuit remained silent in proctolin, TNRNFLRFamide, and CCAP. These experiments show that graded synaptic transmission in the absence of voltage-gated Na⁺ current is sufficient to sustain rhythmic motor activity in some, but not other, modulatory conditions, even when each modulator activates the same ionic current. This further demonstrates that similar rhythmic motor patterns can be produced by qualitatively different mechanisms, one that depends on the activity of voltage-gated Na⁺ channels, and one that can persist in their absence.SIGNIFICANCE STATEMENT The pyloric rhythm of the crab stomatogastric ganglion depends both on spike-mediated and graded synaptic transmission. We activate the pyloric rhythm with a wide variety of different neuromodulators, all of which converge on the same voltage-dependent inward current. Interestingly, when action potentials and spike-mediated transmission are blocked using TTX, we find that the muscarinic agonist oxotremorine and the neuropeptide CabTRP1a sustain rhythmic alternations and appropriate phases of activity in the absence of action potentials. In contrast, TTX blocks rhythmic activity in the presence of other modulators. This demonstrates fundamental differences in the burst-generation mechanisms in different modulators that would not be suspected on the basis of their cellular actions at the level of the targeted current.

Keywords: Cancer borealis; Cancer borealis tachykinin-related peptide 1a; oxotremorine; proctolin; red pigment concentrating hormone; stomatogastric ganglion.

Figure 1.

The triphasic pyloric rhythm before and after decentralization. A, Intracellular recordings of the PD, LP, and PY neurons, and extracellular recording of the lvn. On the right, a schematic overview of the intact STNS. B, The pyloric rhythm slowed after decentralization. Right side, The stn was blocked to abolish modulatory descending input to the STG. C, Simplified pyloric circuit. The resistor symbol between AB and PD shows an electrical synapse. All other synapses are inhibitory. Each circle represents one neuron. aln, Anterior lateral nerve; mvn, median ventricular nerve; pdn, PD nerve; pyn, pyloric nerve. Calibration: vertical lines, 20 mV; dashed lines, −50 mV.

Figure 2.

Effects of modulators and modulators in TTX on the pyloric rhythm. A, Schematic of the STNS during different experimental conditions (from left to right), as follows: decentralized, modulator application, modulator and TTX application. B–D, Intracellular recordings of the PD neuron and LP neuron in the three different conditions. In the decentralized preparation, the frequency decreased, and phase relationships remained similar (all left panels). B, Application of the muscarinic agonist oxotremorine strongly activates the pyloric rhythm and increases burst frequency. Coapplication of oxotremorine and TTX abolishes spiking but leaves the slow-wave oscillations almost unchanged. C, CabTRP1a produces a strong pyloric rhythm, after addition of TTX the slow-wave remains, but the rhythm slows. D, RPCH produces a robust pyloric rhythm, and after the addition of TTX oscillations with two time components emerge. Calibration: vertical lines, 20 mV; dashed lines, −50 mV.

Figure 3.

Effects of modulators and modulators in TTX on the pyloric rhythm. A–C, Intracellular recordings of the PD neuron and LP neuron following decentralization (left) and modulator application (middle), and after adding TTX (right). In the decentralized preparation, the frequency decreased, and phase relationships remained similar (all left panels). Proctolin, TNRNFLRFamide, and CCAP produce a robust pyloric rhythm, but activity ceases after adding TTX. Calibration: vertical lines, 20 mV; dashed lines, −50 mV.

Figure 4.

Comparison of oscillations in oxotremorine and oxotremorine plus TTX. A, Averaged intracellular traces of PD, LP, and PY from 20 oscillations in oxotremorine (black) and oxotremorine and TTX (gray) were normalized to three PD cycles and overlaid. Spikes were digitally removed from the oxotremorine traces to facilitate direct comparison with the TTX traces (see Materials and Methods). Calibration: vertical line, 20 mV; dashed lines, −50 mV. B, Comparison of the pyloric frequency in the intact STNS, after dc, in oxotremorine, and in oxotremorine + TTX. N = 16; Box plots show the median and first and third quartiles of data, and whiskers extend to the minima/maxima. Each point reports the averaged data of one experiment. One-way ANOVA: ***p < 0.001 (intact-decentralized, p = 0.0001; intact-oxo, p = 0.9594; intact-oxo-TTX, p = 0.9974; dc-oxo, p = 0.0000; dc-oxo-TTX, p = 0.0002; oxo-oxo-TTX, p = 0.9003; F = 11.5; df = 15). C, Phase relationships of PD, LP, and PY in the different conditions (N = 5). No significant differences; two-way ANOVA PD: off control-oxo, p = 0.993; control-oxo + TTX, p = 0.2764; oxo-oxo + TTX, p = 0.3951; F = 1.53; LP on control-oxo, p = 0.9715; control-oxo + TTX, p = 0.952; oxo-oxo + TTX, p = 0.9997; F = 0.31; LP off control-oxo, p = 0.9999; control-oxo + TTX, p = 0.9639; oxo-oxo + TTX, p = 0.9739; F = 0.25; PY on control-oxo, p = 1; control-oxo + TTX, p = 0.9313; oxo-oxo +TTX, p = 0.9386; F = 0.37; PY off control-oxo, p = 0.9926; control-oxo + TTX, p = 0.9989; oxo-oxo + TTX, p = 0.9992; F = 0.87, df = 4.

Figure 5.

Comparison of oscillations in CabTRP1a (Cab) and CabTRP1a + TTX. A, Normalized and averaged intracellular traces of PD, LP, and PY neurons from 20 oscillations in CabTRP1a (black) and CabTRP1a and TTX (gray) were overlaid. Spikes were removed in CabTRP1a. Calibration: vertical bars, 20 mV; horizontal bars, −50 mV. B, Comparison of the frequency in the intact STNS, after dc, CabTRP1a, and CabTRP1a + TTX. N = 14. Box plots show the median and first and third quartiles of data, and whiskers extend to the minima/maxima. Each point correlates to the averaged data of one experiment. *p < 0.05, ***p < 0.001 (intact-dc, p = 0.0003; intact-CabTRP1a, p = 0.0182; intact-CabTRP1a + TTX, p = 0.0000; dc-CabTRP1a, p = 0.5333; dc-CabTRP1a-TTX, p = 0.6041; CabTRP1a-CabTRP1a + TTX, p = 0.05; F = 11.79; df = 13). C, Phase relationships of PD, LP, and PY neurons in the different conditions (N = 6). The only significant difference was between LP-off control and CabTRP1a + TTX (two-way ANOVA PD off control-CabTRP1a, p = 0.9957; control-CabTRP1a + TTX, p = 0.2530; CabTRP1a-CabTRP1a + TTX, p = 0.3507; F = 1.56; LP on control-CabTRP1a, p = 0.9417; control-CabTRP1a + TTX, p = 0.1868; CabTRP1a-CabTRP1a + TTX, p = 0.4222; F = 1.79; LP off control-CabTRP1a, p = 0.7701; control-CabTRP1a + TTX, p = 0.0314*; CabTRP1a-CabTRP1a + TTX, p = 0.1793; F = 3.8; PY on control-CabTRP1a, p = 0.8285; control-CabTRP1a + TTX, p = 0.295; CabTRP1a-CabTRP1a + TTX, p = 0.7601; F = 1.31; PY off control-CabTRP1a, p = 0.8694; control-CabTRP1a + TTX, p = 0.0745; CabTRP1a-CabTRP1a + TTX, p = 0.2679; F = 2.78, df = 5).

Figure 6.

Spike-mediated and graded synaptic strength in oxotremorine and CabTRP1a (Cab) measured across different conditions. A, Comparison of IPSP amplitudes in the PD neuron after decentralization and in oxotremorine and CabTRP1a (dc-oxo: p = 0.0053; df = 8; dc-CabTRP1a: p = 0.002; df = 8). B, Example traces for the measurement of graded synaptic strength in TTX (first two traces) and oxotremorine + TTX (bottom two traces). The presynaptic cell (LP) was depolarized from −60 to −20 mV, and the postsynaptic cell (PD) was held at −50 mV. C, Amplitudes of the graded IPSP in PD and LP for TTX and oxotremorine + TTX (PD TTX-TTX + oxo: p = 0.0839; df = 4; LP TTX-TTX + oxo: p = 0.0015; df = 4). D, Amplitudes of the graded IPSP in PD and LP for TTX and CabTRP1a + TTX (PD TTX-TTX + CabTRP1a: p = 0.0994; df = 3; LP TTX-TTX + CabTRP1a: p = 0.42; df = 3). Box plots show the median and first and third quartiles of data, and whiskers extend to the minima/maxima. Each point correlates to the averaged data of one experiment. *p < 0.05 (one-way ANOVA). rec, Recorded; sIPSP, spike-mediated IPSP; stim, stimulated.

Figure 7.

Current injections in TTX and oxotremorine. Intracellular recordings of PD and LP are shown. A, Hyperpolarization of the pacemaker slows down the pyloric rhythm. B, PD depolarization speeds up oscillation. C, LP hyperpolarization has little effect on the frequency of the PD neurons. D, LP depolarization leads to stronger inhibition of PD, resulting in a slower PD frequency. Calibration: dashed horizontal bars, −50 mV; vertical bars, 20 mV.

Figure 8.

Activity of the pharmacologically isolated pacemaker AB–PD in TTX and either oxotremorine or CabTRP1a. A, In the presence of oxotremorine, TTX, and PTX, the rhythm in PD persists. Note the small PD–LP inhibition in the LP traces. B, In CabTRP1a, TTX, and PTX, pacemaker oscillations persist. Calibration: dashed horizontal bars, −50 mV; vertical bars, 20 mV.

Figure 9.

Schematic overview of STG neuron receptor distribution and circuit targets of different neuromodulators. A, Receptor distribution for five substances onto four pyloric neurons. B–E, Distribution of oxotremorine (B), CabTRP1a (C), RPCH (D), and proctolin (E) receptors on neurons of the pyloric circuit. The figure is a redrawn summary of data found in the studies by Swensen and Marder (2000, 2001).