Modulators with convergent cellular actions elicit distinct circuit outputs

Abstract

Six neuromodulators [proctolin, Cancer borealis tachykinin-related peptide Ia, crustacean cardioactive peptide (CCAP), red pigment-concentrating hormone, TNRNFLRFamide, and pilocarpine] converge onto the same voltage-dependent inward current in stomatogastric ganglion (STG) neurons of the crab C. borealis. We show here that each of these modulators acts on a distinct subset of pyloric network neurons in the STG. To ask whether the differences in cell targets could account for their differential effects on the pyloric rhythm, we systematically compared the motor patterns produced by proctolin and CCAP. The motor patterns produced in proctolin and CCAP differed quantitatively in a number of ways. Proctolin and CCAP both act on the lateral pyloric neuron and the inferior cardiac neuron. Proctolin additionally acts on the pyloric dilator (PD) neurons, the pyloric (PY) neurons, and the ventricular dilator neuron. Using the dynamic clamp, we introduced an artificial peptide-elicited current into the PD and PY neurons, in the presence of CCAP, and converted the CCAP rhythm into a rhythm that was statistically similar to that seen in proctolin. This suggests that the differences in the network effects of these two modulators can primarily be attributed to the known differential distributions of their receptors onto distinct subsets of neurons, despite the fact that they activate the same current.

Fig. 1.

Modulators acting on the PD and PY neurons.A, Convergence of proctolin, CabTRP, and pilocarpine onto the PD neuron. All three modulators yield similar current–voltage curves with V_peak values of approximately −35 mV. Recordings were made from the same PD neuron. The PD neuron was isolated from the electrically coupled AB and LPG neurons by photoinactivation. B, Two of the response types exhibited by PY neurons. The responses of the PY neuron fell into three categories: those that responded to only TNRNFLRFamide (type I;top), those that responded to TNRNFLRFamide, proctolin, and pilocarpine (type II; bottom), and those that responded to TNRNFLRFamide, proctolin, pilocarpine, and CabTRP (type III; data not shown). Note that all three modulators converging onto the type-II neurons yield similar current–voltage curves with V_peak values at approximately −20 mV. Recordings were all made in TEVC. Modulators were pressure applied for 100 msec at 500 μm (proctolin, CabTRP, or TNRNFLRFamide) or 50 mm (pilocarpine). The bath contained 10 μmPTX to block the inhibitory glutamatergic synapses, 0.1 μm TTX to block action potential generation, and 10 mm TEA to block some of the K⁺currents.

Fig. 2.

Pyloric circuits summarizing the target neurons (shaded) for each of the convergent modulators. Each modulator targets a different subset of cells. Neurons that exhibited multiple responses are shaded according to the percentage of the cells that did respond to a given modulator.

Fig. 3.

Proctolin- and CCAP-elicited pyloric rhythms at two different concentrations (10⁻⁶ and 10⁻⁷m). Extracellular recordings are from STG motor nerves. These recordings show the activity of the pyloric neurons as labeled. Both sets of recordings are from the same preparation.

Fig. 4.

Dose–response curves for proctolin and CCAP in the LP neuron. The proctolin dose–response curve is adapted fromSwensen and Marder (2000). For proctolin, 90% of the maximal current was reached at 1.8 × 10⁻⁶m, and for CCAP, 90% maximal was reached at 6 × 10⁻⁷m.Plotted points represent the normalized peak inward current elicited (±SE) in the LP neuron while voltage clamped to −40 mV. The bath contained 10 μm PTX and 0.1 μmTTX. Points were fit to the equation:y = (I_max)x/(K_d+ x), where x is the concentration of the applied peptide, I_max is the normalized maximal current, and K_d is the dissociation constant. For proctolin, I_max = 1.06 ± 0.01, and K_d = 2.02 (± 0.01) × 10⁻⁷m. For CCAP, I_max = 0.99 ± 0.02, and K_d = 6.7 (± 1.1) × 10⁻⁸m.

Fig. 5.

The artificial peptide-elicited currents for the PD and PY neurons. A, Proctolin currents recorded from a PD and a PY neuron and the fits that were used as the artificial peptide currents. Recordings were made using TEVC in the presence of 10 μm PTX and 0.1 μm TTX. Values obtained from the fits were used in the dynamic-clamp program (see Materials and Methods). B, Left, The control activity of a PD and a PY neuron. Right, How the activity of the PD and the PY neurons change with the introduction of the artificial current.Below each intracellular trace is a current trace showing the amplitude of the artificial current being injected at any given time. The horizontal line indicates −50 mV. In these two examples the peak current values were set at −1.8 nA (PD neuron) and −1.6 nA (PY neuron).

Fig. 6.

The proctolin-elicited, CCAP-elicited, and artificial pyloric rhythms. The artificial rhythm is the rhythm produced in the presence of CCAP while the artificial “peptide” currents were being applied to the PD and PY neurons simultaneously.A, Extracellular recordings from STG motor nerves. Spike units for the different pyloric neurons are labeled. Some of the measured values from this experiment are shown to theright. These values are averages over longer portions of the traces shown. In this experiment, the number of spikes per burst of the PY neuron was measured directly from a PY neuron. All three sets of recordings are from the same preparation.lvn, Lateral ventricular nerve; pdn, pyloric dilator nerve; mvn, medial ventricular nerve.B, Phase relationships of the PD, LP, PY, and IC neurons derived from the traces in A. Measurements are the means ± SD. For this experiment, the peak current values for the artificial peptide currents were set at −1.8 nA for the PD neuron and −1.6 nA for the PY neuron. The CCAP concentration was 2 × 10⁻⁷m.

Fig. 7.

Comparison of proctolin-elicited, CCAP-elicited, and the artificial rhythms. A, Comparison of the activity-related measurements across all experiments. Only the values found to be significantly different for the CCAP rhythm, as compared with the proctolin rhythm, are shown. B, The phase relationships averaged across all experiments. The on-time of the PD neuron was defined as phase zero. Measurements are the means ± SD (n = 6). All comparisons were made using a pairedt test. * denotes p < 0.05. The concentrations of CCAP used in the experiments were the following: 1.5 × 10⁻⁷, 2 × 10⁻⁷, 3 × 10⁻⁷, 3.5 × 10⁻⁷, 5 × 10⁻⁷, and 5 × 10⁻⁷m.