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First published online October 5, 2006
Journal of Experimental Biology 209, 4000-4010 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02479

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The control of anterior foregut motility during a larval molt of the moth Manduca sexta involves the modulation of presynaptic activity

Jennifer E. Bestman^* and Ronald Booker

Department of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853, USA

View larger version (7K): [in a new window]   Fig. 1. Exposure to the active factor present in the hemolymph of early-molt stage larvae triggers a dramatic decline in anterior foregut contraction amplitude. (A) In saline, anterior foreguts isolated from nonmolting 5th instar larvae respond to a 10 ms stimulation of the severed recurrent nerve with a large amplitude contraction. Within a few minutes of exposure to the hemolymph fraction from early-molt stage larvae, however, the amplitude of the elicited contractions was markedly decreased. The response rebounded following a saline wash. The anterior foregut contractions were recorded using a movement transducer attached to the anterior esophageal constrictor muscles. (B) Summary of the results (N=6; P<0.001, paired t-test). In saline rinse we found that the average contraction amplitude recovered and surpassed the initial control levels (P<0.05, paired t-test). *Significant difference from saline value.

View larger version (7K): [in a new window]   Fig. 2. (A) Example of excitatory junctional potentials (EJPs) recorded from musculature of active anterior foreguts in response to a 10 ms stimulation of the severed recurrent nerve. Following exposure to the active hemolymph factor the amplitude of the EJP dropped significantly but recovered after a saline rinse. (B) We found a significant decrease in the average amplitude of the EJPs recorded from anterior esophageal constrictor muscles of the anterior foreguts (N=10; paired t-test, P<0.001). The average EJP amplitude returned to the initial saline level in the rinse. *Significant difference from both saline and rinse levels.

View larger version (50K): [in a new window]   Fig. 3. The activity-dependent dye FM1-43 reveals that the active hemolymph fraction triggers a decline in the efficacy of the synaptic terminals on the anterior foregut musculature. Presynaptic terminals of intermolt 5th instar larvae were first preloaded with FM1-43 (A,C), and then allowed to unload under one of two conditions: (B) after a continued 20 min incubation in normal physiological saline or, (D) after a 20 min incubation in the hemolymph fraction. (E) In the preparations incubated in the hemolymph fraction, the average percentage change in the fluorescence of the terminals was significantly lower than that of the preparations that had been incubated in saline (unpaired t-test, P<0.01) *Significant difference from the normal saline levels. Scale bar, 50 µm.

View larger version (118K): [in a new window]   Fig. 4. Crustacean cardioactive peptide (CCAP) immunostaining of the esophageal dilator muscles of an early-molt larva. The diffuse, punctate staining is indicative of neurohemal release sites. Inset: diagram of the anterior foregut musculature, brain and frontal ganglion. The box indicates approximate position of the esophageal dilator muscles shown in the figure. Scale bar, 50 µm.

View larger version (10K): [in a new window]   Fig. 5. Exposing the inactive anterior foreguts isolated from early-molt stage larvae to crustacean cardioactive peptide (CCAP) triggers a dramatic increase in anterior foregut motility. (A,B) Examples of anterior foregut activity in saline conditions (A) and of the same preparation after 3 min in 10–8 mol l–1 CCAP (B). The top trace is the endogenous activity recorded by a movement transducer attached to the anterior esophageal constrictor muscle group. The bottom trace is a concurrent extracellular recording of excitatory junctional potentials (EJPs) from the esophageal constrictor muscles. (C) Within 30 min of the CCAP application to the anterior foreguts isolated from larvae early in the molt cycle, the average contraction amplitude increased significantly (P<0.05, paired t-test) to levels similar to that observed around the time of molting fluid ingestion. *Significant difference from the initial saline contraction levels.

View larger version (8K): [in a new window]   Fig. 6. Crustacean cardioactive peptide (CCAP) dose response. Anterior foregut contractions were elicited by delivering 5 ms pulses at 40 pulses s–1 for 500 ms duration to the severed recurrent nerve. (A) An example of a movement transducer record from an early-molt larva initially in saline and then 10 min after exposure to 10–8 mol l–1 CCAP. (B) There is a strong dose–response relationship between the amount of contraction and increasing concentrations of CCAP. Plotted are the percentage increases in contraction amplitude elicited from stimulation of the recurrent nerve relative to the maximum response elicited in the initial saline conditions. Each point is an average of the responses of between five and 22 animals.

View larger version (9K): [in a new window]   Fig. 7. Excitatory junctional potentials (EJPs) elicited in the presence of 10–8 mol l–1 crustacean cardioactive peptide (CCAP) produced significantly greater responses than those in saline. EJP responses recorded intracellularly from the anterior foregut muscles of early-molt larvae (A), and intermolt larvae (B), in control saline and after a 10 min exposure to saline plus 10–8 mol l–1 CCAP. The early-molt larvae produced significantly greater EJP responses after incubation in CCAP (paired t-test, P<0.0001), reaching values that were similar to those of the intermolt larvae in saline (unpaired t-test, P>0.5). After treatment with 10–8 mol l–1 CCAP, the average EJP amplitude recorded from the intermolt larvae increased, but was not significantly different from the saline values (paired t-test, P>0.05). *Significant increase over initial saline levels (paired t-test, P<0.0001) but is not significantly different from the intermolt saline values (unpaired t-test, P>0.05).

View larger version (53K): [in a new window]   Fig. 8. Application of crustacean cardioactive peptide (CCAP) results in a significant increase in the activity of the synaptic endings on the anterior foreguts of early-molt stage larvae. For these experiments the density of FM1-43 loaded puncta was used to estimate the endogenous activity of the presynaptic terminals found on the anterior foregut musculature. (A) Under control conditions there were relatively few FM1-43-labeled puncta found on the esophageal dilator muscles of the anterior foregut of an early-molt larva. (B) However, following a 15 min incubation in 10–8 mol l–1 CCAP, there is a dramatic increase in the density of FM1-43-labeled puncta on the muscle. (C) A plot summarizing the FM1-43 results. When loading occurred in control saline the density of FM1-43 puncta on the early-molt stage anterior foreguts was only about a quarter of the value calculated for intermolt larvae. Following exposure to 10–8 mol l–1 CCAP for 15 min, the density of labeled puncta on the anterior foregut muscles of early-molt stage larvae was similar to that observed in anterior foreguts isolated from intermolt larvae (P>0.05, unpaired t-test). *Significant difference from both intermolt saline level (P<0.005, unpaired t-test) and from the 10–8 mol l–1 CCAP level (P<0.005, paired t-test). Scale bar, 20 µm.