Neuron, Vol. 12, 1223-1233, June, 1994, Copyright 0 1994 by Cell Press Cyclic AMP Modulates Fast Axonal Transport in Aplysia Bag Cell Neurons by Increasing the Probability of Single Organelle Movement E. M. Azhderian,*+ D. Hefner,+* Chi-Hung Lin,+* 1. K. Kaczmarek,* and P. Forscher* *Department of Pharmacology *Department of Biology Yale University New Haven, Connecticut 06510 Summary Stimulation of Aplysia bag cell neurons triggers elevation of CAMP and prolonged secretion of ELH neuropeptide. Using video-enhanced microscopy to track individual organelle movements in bag cell neurons, we find that organelle translocation consists of periods of movement interrupted by stationary episodes. CAMP elevation leads to a 2- to 3-fold enhancement of the average rate of organelle transport in both anterograde and retrograde directions. This effect does not result from alteration of the instantaneous velocity of organelle transport along microtubules, but rather from an increase in the propor- tion of time individual organelles spend in motion. Bio- chemical measurements also provided evidence that CAMP elevation promotes ELH peptide translocation from the somata into axons. Enhanced transport of ELH as a result of these effects may contribute to the replen- ishment of neuropeptide-containing vesicles at release sites during prolonged periods of secretion. Introduction Neurons in pathways that control episodic behaviors such as feeding and mating typically undergo periods of intense activity and neurotransmitter secretion, fol- lowed by prolonged periods of relative inactivity. An unresolved question in such neurons is how the pro- cesses of neurotransmitter synthesis, transport, and mobilization are adjusted to adapt to rapid changes in demand. One example of such cells is the bag cell neurons of Aplysia californica, which act as a biologi- cal switch to control a sequence of reproductive be- haviors leading to egg laying in this animal (Conn and Kaczmarek, 1989). Although these neurons may re- main inactive for periods of days or weeks, they are induced to depolarize and generate a long lasting af- terdischarge of action potentials in response to tran- sient stimulation of an afferent input. This discharge leads to the secretion of several neuroactive peptides including the 36 amino acid egg-laying hormone (ELH). Because of the anatomical simplicity and apparent homogeneity of the clusters of bag cell neurons, which are located in the abdominal ganglion of Aplysia, these cells have served as a model system for investi- gations of the synthesis, processing, and release of “The first three authors made equal contributions to this study. neuroactive peptides (Arch and Berry, 1989). ELH is first synthesized as part of a 32 kDa precursor protein, which begins proteolytic processing in the Golgi ap- paratus to yield secretory vesicles containing ELH, as well as several other neuropeptides (Arch, 1972; Berry, 1981; Scheller et al., 1983; Fisher et al., 1988; Newcomb et al., 1988; Nagle et al., 1990; Rajpara et al., 1992). The onset of the afterdischarge is associated with an elevation of CAMP levels (Kaczmarek et al., 1978; Kauer and Kaczmarek, 1985). This second messenger regulates both the electrical and biochemical proper- tiesofthe bagcell neurons(KaczmarekandStrumwas- ser, 1984; Bruehl and Berry, 1985; Loechner and Kacz- marek, 1990; Azhderian and Kaczmarek, 1990; Knox et al., 1992) and modulates the spatial distribution of secretory organelles in growth cones (Forscher et al., 1987). Changes in CAMP levels may therefore contrib- ute to the progressive potentiation in the rate of secre- tion of ELH that occurs over the first several minutes of the afterdischarge (Loechner et al., 1990). We have now found that elevation of CAMP levels in the bag cell neurons rapidly promotes the translo- cation of a set of ELH-containing proteins from the somata into and along the axons of these neurons toward the sites of release. Experiments presented here using high resolution video-enhanced micros- copy of isolated neurons show that transport of secre- tory organelles along microtubules occurs by a salta- tory process: periods of movement are interrupted byperiodsduringwhichorganelles remain stationary. We provide evidence that the net enhancement of organelle translocation after CAMP treatment results from an increase in the proportion of time which the secretory organelles are in motion, not from an in- crease in instantaneous translocation velocity. The re- sults suggest that CAMP may modulate the activity of a “clutch-like” protein, capable of controlling the linkage between organelles and molecular motors or microtubules. The results also show that the process of axoplasmic transport of neuropeptides in bag cell neuronsissubjecttomodulationwhich isfunctionally coupled to the state of excitability of these cells. Results ELH Peptide Localization in Bag Cell Axons The bag cell neurons comprise two clusters of 200-400 cells, each situated at the rostra1 end of the abdominal ganglion of Aplysia. They are anatomically isolated from other neurons and send their axonal branches into the surrounding connective tissue sheath (Frazier et al., 1967; Haskins et al., 1981) (see Figure 8A). After isolation from the abdominal ganglion, bag cell neu- rons continue to synthesize and transport ELH in se- cretory granules. As reported previously (Chiu and