Typically, the marine mollusk, Clione limacina, exhibits a slow, hovering locomotor gait to maintain its position in the water column.  However, the animal exhibits behaviorally relevant locomotor swim acceleration during escape response and feeding behavior.  Both nitric oxide and serotonin mediate this behavioral swim acceleration.  In this study, we examine the role that the second messenger, cGMP, plays in mediating nitric oxide and serotonin induced swim acceleration.  We observed that application of an analogue of cGMP or an activator of soluble guanylyl cyclase, increased fictive locomotor speed recorded from Pd-7 interneurons of the animal’s locomotor central pattern generator. Moreover, inhibition of soluble guanylyl cyclase decreased fictive locomotor speed.  These results suggest that basal levels of cGMP are important for slow swimming and that increased production of cGMP mediates swim acceleration in Clione.  Because nitric oxide has its effect through cGMP signaling and because we show herein that cGMP produces cellular changes in Clione swim interneurons that are consistent to cellular changes produced by serotonin application, we hypothesize that both nitric oxide and serotonin function via a common signal transduction pathway that involves cGMP.  Our results show that cGMP mediates nitric oxide-induced but not serotonin-induced swim acceleration in Clione.

Data collected represent intracellular recordings from Pd-7 interneurons of the Clione limacina locomotor central pattern generator using Axon Instruments Axoclamp 2B or A-M Systems amplifiers with thin-walled borosilicate glass microelectrodes filled with 2 M potassium acetate.  Data are representative of recordings made using Axon Instruments, Inc. Axoscope software or Pclamp software and A/D converter connected to a PC computer.  Action potential frequency (an indication of fictive swim frequency), action potential duration, and sag potential amplitude were measured in control conditions and after application of pharmacological agents (drugs that affect nitric oxide signaling and cGMP signaling). Statistical analysis of the mean control values to mean experimental values are compared and was done using GraphPad Instat (GraphPad Software, La Jolla, CA, USA).

The following is a summary of the data set components.

Supplement 1 Fig. 1 Effect of 8-Br-cGMP on Fictive Swim Frequency

This Excel file summarizes the data showing how 8-Br-cGMP changes fictive swim frequency recorded from Pd-7 swim interneurons of the Clione swim central pattern generator.  The first sheet summarizes all of the data used in this figure, including statistical results.  Subsequent sheets show the individual data for these experiments.

Supplement 2 Fig. 2 Effect of ODQ + NO on Fictive Swim Frequency

This Excel file summarizes the data showing that ODQ, an inhibitor of soluble guanylyl cyclase, prevents nitric oxide from causing an acceleration in fictive swim frequency recorded from Pd-7 swim interneurons of the Clione swim central pattern generator.  The first sheet summarizes all of the data used in this figure, including statistical results.  Subsequent sheets show the individual data for these experiments.

Supplement 3 Fig. 3 Effect of 8-Br-cGMP on Action Potential Duration

This Excel file summarizes the data showing how 8-Br-cGMP changes the action potential duration recorded from Pd-7 swim interneurons of the Clione swim central pattern generator. 

Supplement 4 Fig. 4 Effect of 8-Br-cGMP on Baseline Depolarization

This Excel file includes the data showing how 8-Br-cGMP results in baseline depolarization recorded from Pd-7 swim interneurons of the Clione swim central pattern generator (column A is time in seconds and column B is mV for each sheet). 

Supplement 5 Fig. 5 Sag Amplitude Analysis

This Excel file summarizes the data showing how 8-Br-cGMP changes the sag potential amplitude recorded from Pd-7 swim interneurons of the Clione swim central pattern generator.  The first sheet summarizes all of the data used in this figure, including statistical results.  Subsequent sheets show the individual data for these experiments.

Supplement 6 Fig. 6 Effects of ODQ + 5HT on Fictive Swim Frequency

This Excel file summarizes the data showing that ODQ, an inhibitor of soluble guanylyl cyclase, does not prevent serotonin from causing an acceleration in fictive swim frequency recorded from Pd-7 swim interneurons of the Clione swim central pattern generator.  The first sheet summarizes all of the data used in this figure, including statistical results.  Subsequent sheets show the individual data for these experiments (column A is time in seconds and column B is mV).

Supplement 7 Fig. 7 Effect of PKG Inhibtor (Rp-8-Br-PET-cGMP) + 5HT on Fictive Swim Frequency

This Excel file summarizes the data showing that Rp-8-Br-PET-cGMP, an inhibitor of cGMP dependent protein kinase (PKG), does not prevent serotonin from causing an acceleration in fictive swim frequency recorded from Pd-7 swim interneurons of the Clione swim central pattern generator.  The first sheet summarizes all of the data used in this figure, including statistical results.  Subsequent sheets show the individual data for these experiments (column A is time in seconds and column B is mV).

Additional Data includes:

LNAME PTIO 5HT data show that serotonin increases fictive swimming speed independent of nitric oxide.

DEANO NO donor data that show that nitric oxide increases fictive swimming speed independent of serotonin.

Isoliquiritigenin