Noxious stimuli, such as electrical shocks to the animal's tail, enhance Aplysia's gill- and siphon-withdrawal reflex. Previous experimental work has indicated that this behavioral enhancement, known as dishabituation (if the reflex has been habituated) or sensitization (if it has not been habituated), might be mediated, at least in part, by the endogenous monoaminergic transmitter serotonin (5-HT). To assess 5-HT's role in dishabituation and sensitization of Aplysia withdrawal reflex, we treated Aplysia with the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). We found that 5,7-DHT treatment significantly reduced the dishabituation of the withdrawal reflex produced by tail shock. Treatment with the neurotoxin also blocked the heterosynaptic facilitation of monosynaptic connections between siphon sensory neurons and their follower cells, which contributes to the behavioral enhancement. Analysis by high-performance liquid chromatography indicated that 5,7-DHT treatment significantly reduced 5-HT levels in the Aplysia CNS. Moreover, the neurotoxic effects of 5,7-DHT appeared to be relatively specific for serotonergic pathways. Thus, 5,7-DHT treatment did not disrupt the ability of nonserotonergic facilitatory interneurons, the L29 cells, to facilitate the connections of siphon sensory neurons. Also, 5,7-DHT reduced 5-HT-dependent, but not dopamine-dependent, histofluorescence in Aplysia central ganglia. Finally, 5,7-DHT does not reduce the levels of the facilitatory peptides SCPA and SCPB within the Aplysia CNS. Our results, together with those of Mackey et al. (1989), indicate that 5-HT plays a major role in mediating dishabituation and sensitization of Aplysia's withdrawal reflex.

High pressure liquid chromatography (HPLC) followed by bioassay on isolated snail hearts were used to locate two related peptides, termed small cardioactive peptides A and B (SCPA and SCPB) in each of the central ganglia of Aplysia. The peptides are most concentrated in the buccal ganglia, the ganglia involved in the control of feeding movements. Immunocytology with antisera raised to conjugated SCPB stained three groups of neurons in the buccal ganglia. One group consisted of relatively small neurons that were tightly clustered. The second group was comprised of larger neurons that were more scattered. The third group was made up of several neurons including the two largest in the ganglia, identified cells B1 and B2. B1 and B2 and other neurons in this group innervate the gut by way of the esophageal nerve. HPLC-bioassay of single, individually dissected B1 or B2 neurons demonstrated that the two peptides are present in a single cell. For B2, but not B1, choline injected into the cell body was converted to the conventional transmitter, acetylcholine. This indicates that, in addition to the two peptides, B2 also contains choline acetyltransferase, and raises the possibility that acetylcholine and the SCPs may act as co-transmitters in B2. Strong immunocytological staining of fibers and varicosities was observed in the neuropilar region of the cerebral, pleural, pedal, and abdominal ganglia. In addition to the buccal ganglia, immunoreactive neurons were observed in all of the other central ganglia. The high concentration of the SCPs and the relatively large number of immunoreactive neurons in the buccal ganglion suggest a particularly important role of these peptides specifically in feeding behavior. However, the widespread occurrence of the SCPs in fibers and neuronal cell bodies throughout the nervous system suggests that these peptides also may have additional behavioral functions in Aplysia.

The anterior portion of intrinsic buccal muscle (I3a) is innervated by two excitatory motor neurons, B3 and B38, and the newly identified inhibitory motor neuron, B47. We show that B47 is cholinergic while B3 and B38 are not. B3 and B38 have previously been shown to express the neuropeptides FMRFamide and the small cardioactive peptides (SCPs) A and B, respectively. We present evidence here that B47 synthesizes the neuropeptide myomodulin A (Mma). When placed in culture, B3, B38, and B47 continued to synthesize their respective peptides. These peptides were released in a stimulation- and Ca(2+)-dependent manner, suggesting that they are transmitters in these neurons. By using B3-evoked excitatory junction potentials (EJPs) and muscle contractions as assays, we next examined the modulatory effects of superfusion of peptides and stimulation of motor neurons B38 and B47. Superfusing the muscle with low concentrations of the SCPs, FMRFamide, or Mma enhanced B3-evoked EJPs and contractions. Stimulation of B47 simultaneously with B3 reduced the amplitude of B3-evoked contractions. However, when either B47 or B38 was stimulated in extended bursts designed to release their peptide transmitters, subsequent B3-evoked EJPs and contractions were enhanced. We believe that this modulation is due at least in part to the release of peptides from the terminals of B38 and B47. The SCPs potently increase cAMP levels in I3a muscle fibers. Likewise, stimulation of B38 in extended bursts increased cAMP levels in the muscle. This provides independent evidence that the SCPs are released from B38 terminals in the muscle. Therefore, we have described a neuromuscular preparation amenable to the study of both excitatory and inhibitory motor neurons that utilize a variety of conventional and peptide transmitters. Our results suggest that these motor neurons can function in two states. When stimulated in single brief bursts, they primarily release conventional transmitters. When stimulated in a series of prolonged bursts, they release both conventional transmitters and peptide cotransmitters. These dual states are most pronounced in the case of B47, which, depending on the stimulation paradigm, can act selectively to inhibit or enhance the effects of a second motor neuron innervating the same muscle.

The localization of the neuropeptide FMRFamide in the buccal ganglia and buccal muscles of Aplysia was studied by immunocytology and high-pressure liquid chromatography (HPLC) combined with either a sensitive bioassay or 35S-methionine labeling. Immunocytology with an antiserum directed to FMRFamide stained a large number of fibers, varicosities, and neuronal somata. Two groups of stained neurons were of particular interest. One was the S cells, a group comprised of many small neurons, the majority of which were stained. HPLC of pooled labeled S cells confirmed that at least some of these neurons synthesize FMRFamide. The other group of stained neurons were in the ventral cluster, a group comprised of a small number of large neurons, many of which are motor neurons that innervate the buccal muscles involved in producing biting and swallowing movements. Several of the ventral neurons were previously shown to contain 2 other neuropeptides, the small cardioactive peptides SCPA and SCPB. These neurons are sufficiently large to permit HPLC analyses of the neuropeptides synthesized by individual neurons. This procedure confirmed that individual ventral neurons synthesized FMRFamide, or the SCPs, or all 3 peptides. The coexistence of FMRFamide and the SCPs in the same neuron was confirmed by simultaneous staining of sections from the buccal ganglia with a monoclonal antibody to the SCPs and an antiserum to FMRFamide. The coexistence of the 3 peptides in the same neuron was surprising in light of the observations that these peptides often have opposite biological activity. The ventral neurons are large and potentially identifiable as individuals. Thus, these neurons may be particularly useful for studying the physiological and behavioral roles of neuropeptides in generating complex behaviors.

Intrinsic buccal muscle 5 (I5) in Aplysia is innervated by 2 motor neurons (termed B15 and B16). In addition to the classical transmitter ACh, B15 also contains the 2 neuropeptides SCPA and SCPB. In a previous study, we demonstrated that the SCPs were released from the terminals of B15 in the I5 muscle and that this release was sufficient to raise cAMP levels in I5 muscle fibers. Significant peptide release occurred only when B15 was stimulated at high frequency or at lower frequencies with a relatively long burst duration (Whim and Lloyd, 1989). In the present article, we examine the possibility that the SCPs released from B15 modulate I5 muscle contractions produced by stimulation of the second motor neuron, B16. Application of exogenous SCPs to I5 muscles increased the amplitude and relaxation rate of B16-evoked contractions. Stimulation of B15 using paradigms that have been shown previously to cause release of the SCPs resulted in a long-lasting increase in the amplitude and relaxation rate of muscle contractions evoked by B16. This modulation is unlikely to be due to the B15-induced muscle contractions themselves, because modulation of B16-evoked contraction amplitude and relaxation rate was observed when the contractions were blocked transiently by a cholinergic antagonist during B15 stimulation. Conversely, stimulation of B15 at frequencies that produce no measurable release of the SCPs did not elicit significant modulation of B16-evoked contractions. The minimum B15 stimulation frequency required to elevate muscle cAMP levels or to modulate B16-evoked contractions was found to be within the physiological range at which B15 fires during feeding. Therefore, the mechanism underlying the modulation of B16-evoked contractions by B15 is likely to involve the release of the SCPs from B15 terminals in the I5 muscle. With respect to behavior, this modulation of muscle contractions would be most likely to occur during food-induced arousal when both motor neurons fire at high frequency with brief interburst intervals.