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Modulation of foregut synaptic activity controls resorption of molting fluid during larval molts of the moth Manduca sexta

Jennifer E. Bestman^* and Ronald Booker

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

View larger version (39K): [in a new window]   Fig. 1. Diagram of the foregut preparation. BD, buccal dilator; BC, buccal constrictor; ED, esophageal dilator; aEC, anterior esophageal constrictor; pEC, posterior esophageal constrictor; FN, frontal nerve; FG, frontal ganglion; RN, recurrent nerve. Examples of suction electrode and movement transducer placements are illustrated in red. Note that the muscles have been cut from their cuticular attachments and that the branching patterns of the nerves have been omitted from the drawing. Scale bar, 500 µm.

View larger version (74K): [in a new window]   Fig. 2. FITC-inulin distribution in molting larvae. FITC-inulin was injected into the molting fluid (MF) of molting larvae. All larvae were exposed to the dye for 10 h before they were dissected. Foreguts are shown in the upper panels, and midguts are shown in the lower panels. (A) An early-molt larva [13–16 h after head capsule slippage (HCS)] with low levels of fluorescent material in its digestive tract. (B) We observed high levels of FITC-inulin in the foregut and midgut of the late-molt larva (22–25 h after HCS). The robust contractions of the foregut had resumed by the late-molt stage. (C) A late-molt stage larva (22–25 h after HCS) whose foregut was silenced by lesioning the frontal ganglion (FG) shows only low levels of fluorescent material in its gut. All larvae were photographed with the same camera exposure and microscope settings. Arrows indicate the right edge of the brain. Scale bar, 500 µm.

View larger version (16K): [in a new window]   Fig. 3. The contractions of the foregut were monitored using a movement transducer attached to anterior esophageal constrictor (aEC) muscles. (A) Output of the movement transducer recorded during the intermolt 4th and 5th instars, as well as during the molt between the two larval stages. Molting stages are given in hours after head capsule slippage (HCS). There were no significant differences in the mean period of contraction between any larval stages. Scale bars, 30 µm and 2 s. (B) A movement transducer was used to determine the mean maximum amplitude of movement (± S.E.M.) for each stage. The asterisk represents significant difference from early-molt values (P<0.05, N=9–37).

View larger version (29K): [in a new window]   Fig. 4. Activity recorded from the recurrent nerve (RN) paired with alternating contractions of the buccal constrictor (BC) and posterior esophageal constrictor (pEC) muscle groups. (A) Recordings of muscle movement and nerve activity from an intermolt 5th instar larva. (B) Muscle movement and nerve activity recorded from an early-molt larva (0–16 h after head capsule slippage). Although the foregut contractions of the early-molt larva are of low amplitude, the RN shows its characteristic bursting activity and the foregut muscle contracts in a pattern similar to the 5th instar peristalsis. Scale bars, 50 µm and 3 s.

View larger version (28K): [in a new window]   Fig. 5. Recordings from the posterior esophageal constrictor motorneurons (pEC MNs) located in the frontal ganglion and concurrent extracellular suction electrode recordings from the foregut musculature from anterior and posterior esophageal constrictor (aEC, pEC) muscles. (A) Examples of intracellular recordings from a pEC MN in an intermolt 5th instar larva. (B) Recording from an early-molt stage larva. Despite the dramatic decrease in foregut motility during the early-molt, the pattern of activity recorded appears similar to that observed in intermolt animals. Scale bars, 25 mV and 2 s.

View larger version (11K): [in a new window]   Fig. 6. Intracellular recordings from anterior esophageal constrictor (aEC) muscles reveal that early-molt larvae exhibit a sharp decline in excitatory junctional potential (EJP) amplitude compared with intermolt larvae. (A) Response of foregut muscle to endogenous frontal ganglion (FG) activity. Scale bars, 5 mV and 1 s. (B) Examples of foregut EJPs elicited by a 10 ms stimulation pulse delivered to the recurrent nerve and recorded from the aEC muscles. Scale bars, 10 ms and 5 mV for the intermolt larva and 2.5 mV for the early-molt larva. (C) The mean EJP amplitudes (± S.E.M.) recorded from intermolt larvae (open bars) and early-molt larvae (filled bars). The asterisk represents significant difference from intermolt values (P=0.05, unpaired t-test).

View larger version (11K): [in a new window]   Fig. 7. Foregut contractions elicited by stimulation delivered to the recurrent nerve (RN) are significantly lower in early-molt larvae than in both late-molt and intermolt stage larvae. RNs were stimulated with 5 ms pulses delivered at 40 pulses s–1 for 500 ms duration trains. The asterisk represents significant difference from both the molt-transition and intermolt larvae (P<0.05). Values are means ± S.E.M.

View larger version (108K): [in a new window]   Fig. 8. Molt-related changes in FM1-43 loading of nerve terminals located on foregut esophageal dilator muscles of 4th instar intermolt (A,B), early-molt (C,D) and late-molt (E,F) stage larvae. (A) Endogenous activity was sufficient to load FM1-43 into the foregut synaptic terminals of the 4th instar intermolt larvae. (B) The FM1-43 unloaded when the labeled foreguts were incubated in a high K+ solution. (C) Endogenous activity failed to load the terminals of the early-molt stage larvae. (D) Exposing early-molt larvae to high K+ with FM1-43 resulted in the loading of FM1-43 into the synaptic terminals on the foregut. (E) Endogenous frontal ganglion (FG) activity was sufficient to cause FM1-43 dye labeling of neuronal terminals of late-molt larvae (>21 h after head capsule slippage) that had begun to swallow MF. (F) The terminals of the late-molt stage larvae unload the dye when the tissue is incubated in high K+ saline. (G,H) Two examples of synaptotagmin immunoreactivity, a marker of neuronal presynaptic terminals, present on the esophageal dilator muscles. Arrows indicate equivalent positions. Scale bar, 50 µm.

View larger version (17K): [in a new window]   Fig. 9. Molt-related differences in the loading of FM1-43. (A) Endogenous activity (open bars) produces fewer FM1-43-labeled synaptic terminals on the foreguts of early-molt larvae compared with late-molt and intermolt larval stages. Equal densities of labeled synapses were found when high K+ saline (filled bars) was used to stimulate FM1-43 loading. The mean number of fluorescent puncta per 20 µm2 area (± S.E.M.) is given. The asterisk represents significant difference from the high K+ saline-loaded level (P<0.05, N=6–15). (B) FM1-43 was loaded into synapses by endogenous frontal ganglion activity (open bars), by stimulation with high K+ saline (grey bars) and unloaded with stimulation with high K+ saline (black bars). For each treatment, there were no significant differences between any of the larval stages in the luminosity of the FM1-43 labeled puncta (P>0.05). For all groups, high K+ saline resulted in a significant increase in mean fluorescent luminosity (± S.E.M.) compared with endogenous-load conditions. All of the terminals unloaded FM1-43 when incubated in high K+ saline. *Significant change from endogenous levels (P<0.05); **significant change from high K+ saline-load levels (P<0.05). N=7–20.