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First published online August 30, 2006
Journal of Experimental Biology 209, 3664-3676 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02415

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Actions of kinin peptides in the stomatogastric ganglion of the crab Cancer borealis

Shari R. Saideman¹, Andrew E. Christie², Pieter Torfs³, Jurgen Huybrechts³, Liliane Schoofs³ and Michael P. Nusbaum^1,*

¹ Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6074, USA,
² Department of Biology, University of Washington, Box 351800, Seattle, WA 98195-1800, USA and
³ Laboratory of Developmental Physiology, Genomics and Proteomics, Katholieke Universiteit Leuven, Naamsestraat 59, B-3000 Leuven, Belgium

View larger version (30K): [in a new window]   Fig. 1. Schematic of the stomatogastric nervous system of Cancer borealis and recordings of the spontaneously active pyloric rhythm. (Top) A schematic of the stomatogastric nervous system, which is composed of the paired commissural ganglia (CoGs) and unpaired esophageal (OG) and stomatogastric (STG) ganglia, plus their connecting nerves and a subset of peripheral nerves. Projection neurons from the CoGs innervate the STG and influence the pyloric and gastric mill rhythms. The axons of a subset of these projection neurons are illustrated projecting through the nerves (ions, sons, stn) that connect these ganglia. The axons projecting through the dorsal ventricular nerve (dvn) originate from STG motor neurons and innervate identified pyloric and/or gastric mill muscles. (Bottom) Extracellular recordings of the pyloric motor neurons during a spontaneously active pyloric rhythm in the completely isolated stomatogastric nervous system. Each pyloric motor neuron was identified on the basis of the identity of the recorded nerves, the relative timing of the action potential bursts and the relative amplitude of the recorded action potentials. In C. borealis, there are single copies of the LP, IC and VD neurons, two copies of the PD neuron and five copies of the PY neuron (Kilman and Marder, 1996).

View larger version (62K): [in a new window]   Fig. 2. Kinin-like immunolabeling (KLI) in the pericardial organ (PO) of Cancer borealis. (A) KLI in the ventral-most nerve trunk of the PO. This image, a brightest pixel projection of 11 optical sections taken at 1.95 µm intervals, shows that KLI axons, fine neurites and varicosities are present in the PO. Boxed region is shown at a larger scale in B. Scale bar, 100 µm. (B) A higher magnification image of the central core of the nerve trunk. Note that the large diameter immunopositive axons (arrowheads) are present in the center of the trunk, with labeled fine neurites radiating from them toward the perimeter of the nerve where they terminate in a dense network of immunopositive varicosities. Several of these labeled varicosities that appear to be in contact with the hemolymph space are indicated by arrows. This image is a brightest pixel projection of 12 optical sections taken at 0.2 µm intervals. Scale bar, 100 µm. Abbreviations: AB, anterior bar; PB, posterior bar. (C) Schematic representation of KLI in the PO. In brachyuran crabs, including C. borealis, each of the bilaterally symmetric POs consists of two or more longitudinal nerve trunks that are connected by vertical nerve bars. The trunks and bars that form each PO are elaborations of the segmental nerves (sn) that originate from the thoracic ganglion. In C. borealis the distribution of KLI neuropil in each PO, represented in this schematic by the local branching structures, was patchy, variable in location and often restricted to one or more small regions in the nerve trunks that form the PO.

View larger version (29K): [in a new window]   Fig. 3. Pevkinin-2 excites the pyloric rhythm. (Left) During saline superfusion in a preparation where the STG was isolated from the CoGs, there was an ongoing pyloric rhythm. Note that, without the influence of spontaneously active CoG projection neurons, the pyloric rhythm was relatively slow and weak (see Fig. 1). (Right) During superfusion of pevkinin-2 (PevK-2), there was an increased pyloric rhythm speed as well as increased activity in the PD and LP neurons. Note also the increased number and amplitude of the LP-mediated IPSPs in the PD neuron during PevK-2 superfusion. The IC and VD neurons (mvn) were not activated by PevK-2 application.

View larger version (33K): [in a new window]   Fig. 4. Pevkinin-2 initiates the pyloric rhythm and elicits slow, rhythmic bursting in a gastric mill motor neuron. (Left) During saline superfusion in the isolated STG, there was no pyloric rhythm (dvn) and the dorsal gastric (DG) neuron was inactive (dgn). Note that the there was ongoing tonic activity in the PY neurons and occasional action potentials in the LP neuron, while the PD neurons were silent. (Right) Superfusion of PevK-2 initiated the pyloric rhythm and elicited rhythmic bursting in the DG neuron. There was no temporal relationship evident between the bursting patterns of the pyloric rhythm and the DG neuron.

View larger version (19K): [in a new window]   Fig. 5. Pevkinin-2 increases the speed of the pyloric rhythm. (Left) Scatter plot of the pyloric cycle frequency during superfusion of PevK-2 (10-6 mol l-1) as a function of the pyloric cycle frequency during saline superfusion. Each data point represents the mean (±s.d.) cycle frequency from a single experiment during PevK-2 superfusion and its pre-application control (N=39). Data points located along the diagonal, unity line indicate experiments in which PevK-2 application did not change the ongoing cycle frequency. (Right) PevK-2 (10-6 mol l-1) application increased the mean pyloric cycle frequency across preparations (N=39; *P<0.0001).

View larger version (19K): [in a new window]   Fig. 6. Pevkinin-2 increases the pyloric rhythm-timed activity level of the LP neuron. (Left) Scatter plot of the number of LP neuron spikes/burst in PevK-2 (10-6 mol l-1; N=40) as a function of LP neuron activity during saline superfusion. Each data point represents the mean (±s.d.) number of LP spikes/burst from a single experiment during PevK-2 superfusion and its pre-application control. The diagonal, unity line indicates the points at which PevK-2 application and saline superfusion resulted in the same level of LP neuron activity. (Right) PevK-2 (N=32) increased the mean number of LP spikes/burst, relative to saline controls, during the ongoing pyloric rhythm (*P<0.0001).

View larger version (13K): [in a new window]   Fig. 7. Pevkinin-2 alters the phase relationships among the pyloric neurons. The phase relationships of the pyloric neurons PD, LP and PY are plotted as a function of the normalized pyloric cycle period during saline (filled boxes) and PevK-2 (open boxes) superfusion. A single pyloric cycle extends from the onset of the PD neuron burst to the start of the next PD neuron burst. All five analyzed parameters (mean phase offset of the PD neuron burst, mean phase onset and offset of the LP and PY neuron bursts) were changed during PevK-2 (10-6 mol l-1) superfusion (N=22; *P<0.05, **P<0.01, ***P<0.001).

View larger version (11K): [in a new window]   Fig. 8. The actions of pevkinin-2 on the pyloric rhythm are dose dependent. (Left) PevK-2 application had dose-dependent actions on the pyloric cycle frequency. As shown in Fig. 5, PevK-2 (10-6 mol l-1) increased the pyloric cycle frequency relative to saline controls. However, the pyloric cycle frequency was unchanged during application of lower peptide concentrations. (Right) PevK-2 superfusion increased the number of LP neuron spikes/burst in a dose-dependent manner. The threshold concentration for PevK-2 enhancement of LP neuron activity (10-7 mol l-1), relative to levels during saline superfusion, was lower than for pyloric cycle frequency (10-6 mol l-1). The mean number of LP neuron spikes/burst was also larger when PevK-2 was applied at 10-6 mol l-1 than at 10-7 mol l-1 (N=6 or 7 for each concentration; *P<0.005, P<0.05).

View larger version (42K): [in a new window]   Fig. 9. The LP neuron regulates the speed of the pevkinin-modulated pyloric rhythm. (A) Suppressing LP neuron activity during an ongoing pyloric rhythm in saline did not alter the pyloric cycle frequency. Note that there were five pyloric cycles (six PD neuron bursts) in the same duration regardless of whether the LP neuron was active (pre- and post-LP hyperpolarization) or was silenced (middle) by constant amplitude hyperpolarizing current injection. The pyloric rhythm was unchanged despite the fact that, when LP was silenced, the LP-mediated IPSPs were eliminated in the PD neuron and the trough of the PD neuron slow wave oscillation was depolarized. (B) Suppressing LP neuron activity during an ongoing pyloric rhythm in the presence of PevK-2 increased the pyloric cycle frequency. During the same duration, there were seven pyloric cycles when the LP neuron was active both pre- and post-LP hyperpolarization whereas there were ten pyloric cycles when LP activity was suppressed by hyperpolarizing current injection. Traces in A and B are from the same experiment.

View larger version (15K): [in a new window]   Fig. 10. The LP neuron regulates the speed of the pevkinin-modulated pyloric rhythm. (Left) Scatter plot comparing the pyloric cycle frequency when the LP neuron was active and silent, during both saline superfusion (pre-PevK-2, open squares; post-PevK-2, open triangles) and PevK-2 (10-6 mol l-1) application (filled circles). The LP neuron was silenced via hyperpolarizing current injection (see Fig. 9). During saline superfusion, suppressing LP neuron activity generally did not alter the mean pyloric cycle frequency. Note that these data points lie along the diagonal, unity line. By contrast, during PevK-2 superfusion, suppressing LP neuron activity consistently increased the pyloric cycle frequency relative to times during the same preparation when the LP neuron was active. (Right) Across preparations, the mean pyloric cycle frequency during saline superfusion was unchanged by suppressing LP neuron activity. However, during PevK-2 application, suppressing LP neuron activity did increase the pyloric cycle frequency. Both examined conditions during PevK-2 application resulted in faster pyloric rhythms than either condition examined during saline superfusion (N=9; *P<0.05, **P<0.001).

View larger version (25K): [in a new window]   Fig. 11. The VCN-elicited gastric mill rhythm was not altered by pevkinin-2 application. There was no evident alteration in the VCN-elicited gastric mill rhythm during superfusion of PevK-2 (10-6 mol l-1). These three gastric mill rhythms were elicited, in the same preparation, by three separate VCN stimulations, performed in the order shown.

View larger version (12K): [in a new window]   Fig. 12. The VCN-elicited gastric mill rhythm is largely unchanged by the presence of pevkinin-2. Superfusion of PevK-2 (10-6 mol l-1) did not alter most of the analyzed parameters of the VCN-elicited gastric mill rhythm. The only change occurred in the DG neuron mean burst offset phase, which was delayed by peptide application (N=7; P<0.05).