Article Figures & Data
Figures
- Fig. 1.
A, Myomodulin immunostaining in the ARC muscle using lissamine–rhodamine-conjugated donkey anti-rat secondary antibody. B, Buccalin immunostaining of the same field in the ARC muscle using fluorescein-labeled donkey anti-rabbit secondary antibody. Short arrows point to processes that stain for buccalin alone and correspond to B15 processes. Longer arrows point to processes that stain for both myomodulin and buccalin, corresponding to B16 processes. Scale bar, 100 μm (applies to A and B).C, Post-embedding immunogold dual labeling of BUC and MM in a B16 neuromuscular junction of the ARC. BUC is labeled with a rabbit antibody and a 5 nm gold-labeled secondary antibody. MM is labeled with a rat antibody and a 10 nm gold-labeled secondary antibody. Most of the DCVs are labeled with both small (5 nm) and large (10 nm) gold particles, indicating the presence of both BUCs and MMs in the same DCVs. Scale bar, 100 nm.
- Fig. 2.
Effect of extracellular calcium and hexamethonium on peptide release from B16 in the ARC. A1, Results from a single experiment in which MM release is measured with and without normal calcium in the perfusate. Motorneuron B16 was stimulated during four 5 min periods (indicated by the black bars) with a physiologically relevant frequency and pattern (20 Hz for 3.5 sec every 7 sec). A2, The average of four such experiments for each peptide. For each peptide, the mean ± SE of four separate experiments is plotted at each calcium concentration. The results show that release of both peptides is dependent on extracellular calcium (p < 0.0001). B1, Results from a single experiment in which BUC release is measured without and with 0.1 mm hexamethonium (which completely abolished the contraction of the muscle) in the perfusate. Motorneuron B16 was stimulated during four 5 min periods (indicated by the black bars) with a physiologically relevant frequency and pattern (20 Hz for 3.5 sec every 7 sec). B2, The mean ± SE of the normalized release from four such experiments for each peptide. The results show that release of both peptides is not dependent on contraction of the muscle (p > 0.5).
- Fig. 3.
Corelease of MMs and BUCs within the ARC in response to stimulation of motorneuron B16. During the 1 hr period indicated by the bar, the neuron was fired at 20 Hz for 3.5 sec every 7 sec, which is within the physiological range of B16 firing. Samples of ARC perfusate were collected every 2.5 min and analyzed by radioimmunoassay for their peptide content. Alternate samples were analyzed for their MM or BUC content. A1, MM and BUC release in a single experiment. A2, Same asA1 except that BUC was scaled so those total amounts released were equal for both MM and BUC, enabling a more direct comparison of their profiles. B1, MM and BUC release from four experiments was expressed as a percentage of the total amount of peptide (MM + BUC) released in the experiment, and the percentages were averaged for each 5 min (2 sample) period. B2, Same as B1, except that BUC was scaled so that total percentages were equal for both MM and BUC, enabling a more direct comparison of their profiles. The ratio of MM and BUC remains constant even though the absolute peptide content of the samples varies considerably over the course of B16 stimulation.
- Fig. 4.
Effect of increasing either contraction frequency or amplitude on peptide release from motorneuron B16.A1, Hypothetical consequences of increased rate of feeding with no concomitant alteration of muscle properties. The closer is not sufficiently relaxed and opposes the contractions of opener muscles. Consequently, the radula does not open, and functional feeding is compromised. When the relaxation rate of the closer muscle is increased, functional feeding can be restored. A2,Results from a single experiment in which BUC release is measured at different interburst intervals, simulating changes in the rate of feeding, whereas the intraburst frequency (20 Hz) and the duration of bursts (3.5 sec) are kept constant. The duration of stimulation in this type of experiment was 5 min. The physiological range of interburst intervals for B16 varies from 3.5 to 10 sec (or more) (Cropper et al., 1990b). BUC release decreases as the interval between bursts increases.A3, Peptide release is normalized to the total release during an experiment and for the total number of action potentials delivered in each condition. The results from four separate experiments for each peptide were averaged and plotted (± SE) for each of the three interburst intervals. There is a statistically significant (BUC:F = 23.13; df = 2,6; p < 0.002 MM: F = 25.1; df = 2,6;p < 0.002) inverse relationship between peptide released per action potential and the duration between bursts. The fact that this relationship is nearly identical for the two peptides indicates that they are released at a fixed ratio. B1, Hypothetical consequences of increased contraction amplitude of the radula closer with no concomitant alteration of muscle properties. The closer is not sufficiently relaxed and opposes the contractions of opener muscles. Consequently, the radula does not open, and functional feeding is compromised. As in A1, when the relaxation rate of the closer muscle is increased, functional feeding can be restored. B2, Results from a single experiment in which MM release is measured at three different intraburst frequencies, which produce contractions of differing amplitudes, while the duration of bursts (3.5 sec) and the interburst interval (3.5 sec) are kept constant. The physiological range of firing frequencies for B16 varies between 10–20 Hz (Cropper et al., 1990b). The duration of stimulation in this type of experiment was 5 min. MM release is lower at the lower intraburst frequencies. B3, Normalized release per action potential at each of the three intraburst frequencies corrected to give the release per action potential. The results from four separate experiments for each peptide were averaged and plotted (± SE) for each of the three intraburst frequencies of stimulation. There is a statistically significant (BUC: F = 47.36; df = 2,6; p < 0.0005; MM: F = 126.78; df = 2,6; p < 0.0001) increase of peptide released per action potential and as the frequency of action potentials increases. The fact that this relationship is nearly identical for the two peptides also indicates that they are released at a fixed ratio.
- Fig. 5.
Comparison of the peptide release per action potential from motorneurons B15 and B16 as a function of intraburst frequency and interburst interval. A, Normalized release per action potential at each of the three intraburst frequencies corrected to give the release per action potential. Burst duration (3.5 sec) and interburst interval (3.5 sec) were identical in all experiments. The results from eight separate experiments for each motorneuron were averaged and plotted (± SE) at three different intraburst frequencies of stimulation. The physiological range of firing frequencies for B15 varies between 7.5 and 12 Hz, and B16 varies between 10 and 20 Hz. Peptide release appears to be coordinated with the normal operating range of frequencies of each motorneuron.B, Normalized release per action potential at each of the three interburst intervals corrected to give the release per action potential. Burst duration (3.5 sec) and intraburst frequencies (12 Hz for B15, 20 Hz for B16) were identical in all experiments. The results from eight separate experiments for each motorneuron was averaged and plotted (± SE) at three interburst intervals of stimulation (3.5, 5, and 7 sec). The data for B15 have been described previously (Vilim et al., 1996b). The data for B16 were recalculated from Figure 4.
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