Aplysia Bag Cells Function as a Distributed Neurosecretory Network

1. Radiolabeled peptides released from an isolated cluster of bag cell neurons, during an after discharge, were compared with the polypeptide egg-laying hormone (ELH), 4,500 daltons, pI 9.0-9.3, as purified from homogenates of bag cell clusters. A peptide, labeled with methionine, leucine, and arginine, which is selectively released from after discharging bag cell clusters comigrates with marker ELH, purified from cluster homogenates, on P-6 gel filtration columns and subsequent isoelectric focusing gels. At least three other presumed peptides of unknown function are also released, including one of 5,000-6,000 mol wt, pI 4.5-5.0. 2. When bag cells afterdischarge in vitro, bioactive material is released that will induce egg laying when injected into an Aplysia. This released bioactive material also comigrates with bioactive material from cluster homogenates on P-6 columns. 3. These experiments demonstrate that ELH (4,500 mol wt, pI 9.0-9.3), as purified from bag cell cluster homogenates, is also the major form secreted from bag cells that induces egg laying. This purified ELH can now be used to study the physiological effects of a secreted neurohormone and their relationship to behavior.

Regulated release of multiple peptides from the bag cell neurons ofAplysia californica

Control of potassium currents and cyclic AMP levels by autoactive neuropeptides in Aplysia neurons

A variety of chemical and electrophysiological evidence indicates that the onset of afterdischarge and the subsequent profound enhancement of spike broadening that occur in the bag cell neurons of Aplysia are related to an increase in adenosine 3',5'-monophosphate-(cAMP) dependent protein phosphorylation. We have now used a two-electrode voltage clamp to study the properties of isolated bag cell neurons in cell culture and their response to 8 benzylthio-cAMP (8BTcAMP) and N6-n-butyl 8BTcAMP. These membrane-permeant and phosphodiesterase-resistant cAMP analogs induce spontaneous discharge and spike broadening in both the intact bag cell cluster and isolated bag cell neurons in cell culture. The dominant inward current in these cultured cells was found to be the calcium current, Ica, which was abolished by Co2+ (20 mM) or Ni2+ (10 mM) and could be observed in Na+-free media. In a minority of cells (2 of 12), in normal ionic media, a transient inward current was observed that was unaffected by Co2+ and Ni2+ and probably represents a sodium current. The three characterized potassium currents, the delayed rectifying current IK, the calcium-dependent current IC, and the early transient current IA, distinguished by their differing pharmacological and voltage-activation properties, were present in all healthy cells. Three effects of the cyclic AMP analogs (0.5 mM) on the electrical properties of these cells were 1) the emergence of a region of negative slope resistance in the steady-state I-V relations, 2) a depression of the net sustained outward currents due to depolarizing commands, and 3) a marked reduction in IA. When outward currents had been largely suppressed using high concentrations of tetraethylammonium (TEA) ions (100-460 mM) no effects of the cyclic AMP analogs could be observed on peak inward currents using NA+ and Ca2+ or Ba2+ as carriers of inward current. At least part of these electrical effects of the cyclic AMP analogs could be accounted for by a depression of a delayed potassium current and the A current.

The bag cell neurons of Aplysia synthesize and secrete several peptides. Some of these, in addition to the egg-laying hormone (ELH), are strongly implicated in the various alterations of central neuronal activity that accompany an electrical discharge of the bag cells. Thus, the secreted peptides appear to play a variety of roles in the animal's physiology. We have been interested in the intracellular mechanisms that precede peptide secretion from the bag cells because of the evidence that most, if not all, of these peptides are derived from a common precursor. Our objective has been to determine if presumed products of this precursor are processed coordinately following their synthesis. We have concentrated on two peptides (ELH and the acidic peptide, AP) because they are most easily identified in our analytical systems. On pulse-chase radiolabeling of the cells in vitro, we found that labeled AP appears before labeled ELH in axonal transport. This observation is not easily accounted for by the assumption, taken from studies of other peptide-secreting cells, that a precursor to both peptides is loaded into secretory granules before further processing ensues. Since the initial disproportion in the representation of the peptides in transport is no longer detectable at long chase times (18 and 24 hr), we examined the possibilities that ELH production is delayed relative to that of AP or that AP is degraded more rapidly than ELH. No evidence was found for either process. The disproportion between the newly labeled peptides in transport was evident on analysis of the medium bathing bag cells depolarized after 24 hr of chase.(ABSTRACT TRUNCATED AT 250 WORDS)

In the opisthobranch mollusc Aplysia one aspect of reproduction, egg-laying, is controlled by a neuropeptide hormone named egg-laying hormone (ELH). The primary structure of ELH from A. californica has been determined; it has 36 amino acid residues and the C-terminal lysine is amidated (Chiu et al. 1979). ELH is synthesized by the bag cell neurons, which are located in two clusters of about 400 neurons each at the anterior pole of the abdominal ganglion. There are at least three genes coding for ELH or ELH- like peptides in Aplysia (Scheller et al. 1983 b). Two of these genes are expressed in the atrial gland, an exocrine gland (Beard et al. 1982) at the end of the reproductive tract (large hermaphroditic duct) which leads to the external genital aperture. The function of these ELH-like peptides, called califms, in the atrial gland are not known, but it has been speculated that they may be pheromones to induce egg-laying in nearby Aplysia (Rothman et al. 1986). The third gene is expressed in the bag cells and codes for ELH. This chapter deals primarily with the bag cell system in Aplysia but some comparative information is included. In the freshwater snail Lymnaea stagnalis the caudodorsal neurons synthesize an ELH and are located in the cerebral ganglion (Joosse and Geraerts 1983).KeywordsAcidic PeptideFreshwater SnailAbdominal GanglionPeptidergic NeuronCell PeptideThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The bag cell neurons of the marine mollusk Aplysia have been used extensively for investigating the electrophysiology and molecular biology of neurosecretory cells. However, there has been little attempt to carefully describe the pattern of secretion of the 36-amino acid peptide hormone that controls egg-laying behavior. This egg-laying hormone (ELH) is secreted in response to a highly characteristic pattern of action potential firing called the afterdischarge. The purpose of this study was to document the pattern of ELH secretion in response to the bag cell afterdischarge. To accomplish this goal, we developed the first RIA for measurement of ELH. The study was conducted in vitro in excised neural preparations that included the bilateral bag cell clusters; preparations were maintained in static culture of filtered artificial sea water (ASW) at 21-22 C. Afterdischarges were electrically stimulated, and action potentials were monitored via extracellular electrodes. The entire volume of ASW was collected every 5 min and immediately replaced with fresh ASW. In all 20 preparations, ELH levels were low to undetectable before stimulation of the afterdischarge; ELH rose above baseline within 5-10 min of the start of the afterdischarge, reached peak levels near the end or after the end of the afterdischarge, and then remained elevated long after action potentials had ceased firing. These results indicate a temporal dissociation between action potential firing and secretion. We suggest that the afterdischarge is important for triggering intracellular events that culminate in peptide release. Importantly, once these secretion-inducing events have been initiated, they can continue in the absence of further action potential stimulation.

SUMMARY Neurosecretory phenomena are apparently ubiquitous among Metazoa. In Hydra, neurosecretory products are probably involved in the control of growth and reproduction. Secretory elements in the central nervous system of turbellarians probably promote fission, regeneration and reproduction. The cerebral ganglia of nemertines are the source of a hormone which exercises an inhibitory influence on maturation of the gonads and the development of somatic sexual characteristics. A principle secreted by the ganglia and/or the associated cerebral organs controls weight regulation. A sex hormone controls sexual differentiation. Secretory neurons may influence the production of exsheathing fluid and ecdysis in nematodes. In nereid polychaetes, a single hormone which originates from the cerebral neuroendocrine system apparently promotes segment proliferation and inhibits maturation. However, in certain other polychaetes, contrasting endocrine mechanisms seem to operate. In lumbricid oligochaetes, a hormone secreted by the cerebral ganglion (and possibly by other nervous centres also) promotes gonadal activity and the differentiation of somatic sexual characters. In some species the ganglion exerts an inhibitory influence on the associated processes of ‘diapause’ and posterior regeneration. The cerebral and suboesophageal ganglia are implicated in the control of osmoregulation. Cerebral neurosecretory cells in limicolous oligochaetes may influence regenerative growth and osmoregulation. The cerebral ganglia of leeches secrete a gonadotrophic hormone. In gastropod molluscs, hormones secreted by the cerebral ganglia, glandular ‘dorsal bodies’ and/or optic tentacles are responsible for the control of the development of the reproductive tract, and the activity of the gonads. Contrasting mechanisms are thought to operate in the different gastropod groups and there is conflicting evidence particularly with respect to the endocrine functions of the optic tentacles and the gonads. The ‘bag cells’ of the abdominal ganglion of ApZysiu secrete a hormone which induces egg‐laying. Cells in the pleural and parietal ganglia probably control osmoregulation in Lymnaea. The optic glands of cephalopods secrete a gonadotrophic hormone, but sex hormones are apparently absent. The functional significance of the neurovenous tissues (presumptive neurosecretory complexes of unusual character) is obscure in most cases. The radial nerves of starfish are the source of a hormone which induces the production of I‐methyl adenine by the follicle cells of the gonad. This second principle stimulates oocyte maturation and the shedding of male and female gametes. The relevance of such information to the development of certain biological concepts and to various aspects of comparative physiology is briefly discussed.

Two neuronal populations in the head ganglia of Aplysia californiaa with egg-laying hormone-like immunoreactivity

Two neuroactive peptides, A and B, have been isolated from the atrial gland in the reproductive tract of Aplysia. Each of the two peptides is able to induce egg-laying behavior in recipient animals. In vitro recordings from the abdominal ganglion show that both peptides also trigger longlasting discharges in the bag cell neurons at concentrations around 0.1 muM. The peptides were purified by a combination of ammonium sulfate precipitation, agarose gel filtration, and cation exchange chromatography. Each peptide has 34 amino acid residues. Microsequencing together with carboxypeptidase Y degradation and analysis of tryptic peptides revealed the following sequence for peptide A: H-Ala-Val-Lys-Leu-Ser-Ser-Asp-Gly-Asn-Tyr-Pro-Phe-Asp-Leu-Ser-Lys-Glu-Asp-Gly -Ala-Gln-Pro-Tyr-Phe-Met-Thr-Pro-Arg-Leu-Arg-Phe-Tyr-Pro-Ile. Peptide B differs from A in only four positions. The first nine residues of B are: Ala-Val-Lys-Ser-Ser-Ser-Tyr-Glu-Lys-, whereas residues 10-34 of B are identical to those of A. The calculated M(r) of A is 3924 and that of B is 4032. The pI of peptide A as determined by isoelectric focusing in polyacrylamide gels is 7.9-8.1 and that of peptide B is 9.0-9.2. It is estimated that each atrial gland contains at least 150 mug of peptide A and 50 mug of B. Neither peptide resembles the egg-laying hormone isolated from bag cell neurons. It is postulated that the atrial gland peptides are released during copulation, and then by interacting with neuronal receptors in the head ganglia and pleuroabdominal connectives they cause the bag cells to afterdischarge, thereby releasing egg-laying hormone.

The modulation of voltage-activated calcium currents by protein kinases provides excitable cells with a mechanism for regulating their electrical behaviour. At the single channel level, modulation of calcium current has, to date, been characterized only in cardiac muscle, where beta-adrenergic agonists, acting through cyclic AMP-dependent protein kinase, enhance the calcium current by increasing channel availability and opening. We now report that enhancement of calcium current in the peptidergic bag cell neurons of Aplysia by protein kinase C occurs through a different mechanism, the recruitment of a previously covert class of calcium channel. Under control conditions, bag cell neurons contain only one class of voltage-activated calcium channel with a conductance of approximately 12 pS. After exposure to agents that activate protein kinase C, these neurons also express a second class of calcium channel with a different unitary conductance (approximately 24 pS) that is never seen in untreated cells.

A brief synaptic input to the bag cell neurons of Aplysia evokes a lengthy afterdischarge and the secretion of peptide hormones that trigger ovulation. The input transmitter is unknown, although prior work has shown that afterdischarges are prevented by strychnine. Because molluscan excitatory cholinergic synapses are blocked by strychnine, we tested the hypothesis that acetylcholine acts on an ionotropic receptor to initiate the afterdischarge. In cultured bag cell neurons, acetylcholine induced a short burst of action potentials followed by either return to near baseline or, like a true afterdischarge, transition to continuous firing. The current underlying the acetylcholine-induced depolarization was dose dependent, associated with increased membrane conductance, and sensitive to the nicotinic antagonists hexamethonium, mecamylamine, and α-conotoxin ImI. Whereas nicotine, choline, carbachol, and glycine did not mimic acetylcholine, tetramethylammonium did produce a similar current. Consistent with an ionotropic receptor, the response was not altered by intracellular dialysis with the G protein blocker guanosine 5'-(β-thio)diphosphate. Recording from the intact bag cell neuron cluster showed acetylcholine to evoke prominent depolarization, which often led to extended bursting, but only in the presence of the acetylcholinesterase inhibitor neostigmine. Extracellular recording confirmed that exogenous acetylcholine caused genuine afterdischarges, which, as per those generated synaptically, rendered the cluster refractory to further stimulation. Finally, treatment with a combination of mecamylamine and α-conotoxin ImI blocked synaptically induced afterdischarges in the intact bag cell neuron cluster. Acetylcholine appears to elicit the afterdischarge through an ionotropic receptor. This represents an expedient means for transient stimulation to elicit prolonged firing in the absence of ongoing synaptic input.

Flufenamic acid (FFA) is a nonsteroidal antiinflammatory agent, commonly used to block nonselective cation channels. We previously reported that FFA potentiated, rather than inhibited, a cation current in Aplysia bag cell neurons. Prompted by this paradoxical result, the present study examined the effects of FFA on membrane currents and intracellular Ca2+ in cultured bag cell neurons. Under whole cell voltage clamp, FFA evoked either outward (I out) or inward (I in) currents. I out had a rapid onset, was inhibited by the K+ channel blocker, tetraethylammonium, and was associated with both an increase in membrane conductance and a negative shift in the whole cell current reversal potential. I in developed more slowly, was inhibited by the cation channel blocker, Gd3+, and was concomitant with both an increased conductance and positive shift in reversal potential. FFA also enhanced the use-dependent inactivation and caused a positive-shift in the activation curve of the voltage-dependent Ca2+ current. Furthermore, as measured by ratiometric imaging, FFA produced a rise in intracellular Ca2+ that persisted in the absence of extracellular Ca2+ and was reduced by depleting either the endoplasmic reticulum and/or mitochondrial stores. Ca2+ appeared to be involved in the activation of I in, as strong intracellular Ca2+ buffering effectively eliminated I in but did not alter I out. Finally, the effects of FFA were likely not due to block of cyclooxygenase given that the general cyclooxygenase inhibitor, indomethacin, failed to evoke either current. That FFA influences a number of neuronal properties needs to be taken into consideration when employing it as a cation channel antagonist.

Calcium changes in isolated peptidergic neurons during activation by a cAMP analog

Persistent activation of calcium-activated and calcium-independent protein kinase C in response to electrical afterdischarge from peptidergic neurons of Aplysia