Frequency-dependent release of peptide cotransmitters from identified cholinergic motor neurons in Aplysia

We have investigated the release of two peptide cotransmitters from the terminals of a cholinergic motor neuron in Aplysia. Identified motor neuron B15 synthesizes the two small cardioactive peptides (SCP) A and B in addition to acetylcholine. A symmetrical pair of B15 neurons innervate symmetrical buccal muscles, termed I5, which are involved in generating biting movements. The amplitude of I5 contractions is enhanced by the SCPs. Intracellular stimulation of one B15 produces depletion of the SCPs from the stimulated muscle as compared to the unstimulated control muscle. Significant depletion requires either high-frequency stimulation or prolonged bursts at lower frequencies. A second cholinergic motor neuron, B16, also innervates I5 but does not synthesize the SCPs. Stimulation of B16 produced no depletion of the SCPs. Exogenous SCPs potently increase cAMP levels in the muscle. If depletion is a reflection of release, it should be possible to demonstrate an effect of B15 stimulation on muscle cAMP levels. Indeed, stimulation of B15 did elevate cAMP levels in I5. Stimulation of B16 had no effect on cAMP levels. Increases in cAMP were observed only when B15 was stimulated in a manner that would produce significantly facilitated acetylcholine release. This facilitation could be produced by increased stimulation frequency, longer burst durations, or shorter interburst intervals. However, B15 is capable of producing cholinergically mediated contractions with stimulation parameters that would not cause release of the SCPs. Thus, B15 appears to function as a purely cholinergic motor neuron when firing slowly, and as a cholinergic/peptidergic neuron when firing rapidly.