No abstract

The bag cell neurons of the marine mollusk, Aplysia, are a putative multitransmitter system that utilizes two or more peptide transmitters derived from a common precursor protein. Two putative transmitters are egg-laying hormone (ELH), a 36 amino acid peptide that induces egg laying and mediates bag cell-induced excitatory effects on certain abdominal ganglion neurons, and alpha-bag cell peptide (alpha BCP), which mimics bag cell-induced inhibition of the left upper quadrant (LUQ) neurons and the depolarization of the bag cells that occurs during the bag cell burst discharge. Alpha BCP was previously purified from bag cell extracts in three neuroactive forms: alpha BCP(1-9), a nine amino acid peptide encoded on the ELH/BCP precursor protein, and two NH2-terminal fragments, alpha BCP(1-8) and alpha BCP(1-7). Analyzing bag cell-induced inhibition of LUQ neurons, we report here that alpha BCP fulfills the main criteria for transmitter identification: stimulation of individual bag cells produces inhibition of the neurons; inhibitory activity is present in releasate collected following an elicited bag cell burst discharge in the presence of protease inhibitors; alpha BCP(1-9) and alpha BCP(1-8) are detected in the releasate in the presence of protease inhibitors; alpha BCP is rapidly inactivated after release, as indicated by the lack of detectable alpha BCP or inhibitory activity in the releasate in the absence of protease inhibitors, and by the increase in potency of the arterially perfused peptide in the presence of protease inhibitors; alpha BCP and the endogenously released transmitter produce apparently identical changes in membrane conductance; bag cell-induced inhibition is reduced or abolished following desensitization of the inhibitory response by long-term application of high concentrations of alpha BCP. The results provide additional evidence that the bag cells are a multitransmitter system and also suggest that many of the physiological properties of alpha BCP-mediated neurotransmission differ from those of ELH. First, unlike ELH, alpha BCP is rapidly inactivated after release. Second, alpha BCP(1-9) may be activated by carboxypeptidase cleavage since alpha BCP(1-8) and alpha BCP(1-7) are 30 and 10X as potent, respectively, as alpha BCP(1-9). Third, the inhibitory action of alpha BCP on its targets has a more rapid onset and a shorter time course than the excitatory actions of ELH. Thus, alpha BCP may diffuse to less distant targets than ELH and serve to regulate the more rapidly occurring neural events underlying egg-laying behavior.

A discharge of impulse activity in a group of neuroendocrine cells, the bag cells, produces several types of prolonged responses in various identified neurons of the abdominal ganglion of Aplysia. Two excitatory responses are almost certainly mediated by egg-laying hormone, but this peptide cannot account for other responses, such as inhibition of left upper quadrant neurons. We report here the isolation from bag cell clusters of three structurally similar peptides, seven, eight, and nine residues long, that are candidate transmitters for mediating bag cell-induced inhibition. They may also serve as autoexcitatory transmitters since the seven-residue peptide produces a slow depolarization of the bag cells similar to that which occurs during bag cell discharge. The amino acid sequence of the largest peptide, termed a-bag cell peptide [1][2][3][4][5][6][7][8][9], is H-Ala-Pro-Arg-Leu-Arg-Phe-Tyr-Ser-Leu-OH. The other two peptides are identical to a-BCP[I-9] except that they lack the COOH-terminal Ser-Leu or leucine residues. The three peptides inhibit left upper quadrant neurons at relative potencies of 10:30:1 (seven-, eight-, and nine-residue peptides, respectively). Recent molecular genetic analysis shows that both a-BCP [1][2][3][4][5][6][7][8][9] and egg-laying hormone are encoded by the same bag cell-specific gene. The multiple neuronal effects of bag cells are therefore likely to be mediated by at least two transmitters that are cleaved from a common precursor molecule.The marine mollusk Aplysia is a convenient experimental system for investigating at the cellular and molecular levels the roles of neuropeptides as neurotransmitters and in the regulation of behavior. Egg laying in Aplysia is accompanied by a stereotyped behavioral pattern that lasts for several hours. The behavior pattern is thought to be initiated and controlled by the bag cells, a group of neuroendocrine cells located in the abdominal ganglion. In intact animals, egg laying is invariably preceded by a repetitive impulse discharge of the bag cells (1). In the isolated abdominal ganglion, where the central actions of the bag cells have been most extensively studied, a bag cell discharge produces at least four types of long-lasting excitatory and inhibitory effects on various identified neurons (2-4).To identify the neurotransmitters mediating these effects, extracts of bag cells have been purified and assayed for activity on various neurons. It has been determined that egg-laying hormone (ELH), a 4,400-dalton peptide that is synthesized and released by bag cells (5-8), is probably the neurotransmitter mediating two types of excitatory neuronal responses, burst augmentation of cell R15 and prolonged excitation of left lower quadrant (LLQ) neurons (9-11). However, when perfused into the abdominal ganglion at physiological concentrations, purified ELH has little or no effect on left upper quadrant (LUQ) neurons, cells L2-L4, L6, which are normally inhibited by bag cell discharge (10, 11). This indicates that ELH does not directly or in...

No abstract

The bag cell neurons of the marine mollusk Aplysia are a putative multitransmitter system which utilizes two or more neuropeptides that are enzymatically cleaved from a common precursor protein. It has been proposed that one of the neuropeptides, egg-laying hormone (ELH), acts nonsynaptically as a neurotransmitter in the abdominal ganglion by diffusing long distances to target neurons compared to conventional transmitters acting at synapses. To test this idea further, we investigated the physiological properties of neurotransmission mediated by ELH. We found that ELH acts directly to duplicate two types of responses produced by a burst discharge of the bag cells: prolonged excitation of LB and LC cells, and the previously described effect of ELH, burst augmentation of cell R15. Analysis of perfusate collected after electrical stimulation of the bag cells showed that the peptide is released in sufficient quantity to diffuse long distances within the ganglion without being completely inactivated. To mimic the way the peptide is thought to be released physiologically, ELH was arterially perfused into the ganglion. The response normally produced by bag cell activity was duplicated by 0.5 to 1.0 microM concentrations of ELH and showed no rapid desensitization. ELH had no effect on cells that are unaffected by bag cell activity and no effect on cells that are inhibited (LUQ cells) or transiently excited (cells L1 and R1) by bag cell activity. Acidic peptide, another peptide encoded on the ELH precursor protein, was found to be synthesized and released by the bag cells, but it had no effect on the cells we tested. We conclude that the combined properties of ELH neurotransmission resemble the properties of transmission at autonomic nerve endings on cardiac and smooth muscle rather than those of conventional synaptic transmission. ELH released from bag cells is dispersed throughout the interstitial and vascular spaces of the ganglion to produce responses in the cells that have receptors for the peptide. The results also suggest that ELH mediates only a subset of the responses induced by bag cell activity; they are consistent with data indicating that the other responses are mediated by other bag cell peptides derived from the same precursor protein as ELH.