The Neurosecretion of the Polypeptide Egg-Laying Hormone (ELH) from the Bag Cells, Neuronal Sites of Action of ELH, and Circadian Release of Polypeptides from the Eye of Aplysia californica

The thesis is about the neurosecretion of Aplysia peptides and demonstrates that one of them acts directly upon the nervous system. Such neuronal effects of neurosecretory substances may prove to be a general phenomenon; this is discussed in the introduction. In chapter 1, radiolabeled peptides released from electrically active bag cell neurons in isolated bag cell clusters were compared with the polypeptide egg-laying hormone (ELH), 6,000 daltons, pl 9.0-9.3, as purified from homogenates of bag cell clusters. A labeled peptide which is selectively released from electrically active bag cell clusters comigrates with ELH from cluster homogenates on P-6 gel filtration columns and subsequent isoelectric focusing gels. When bag cells are activated, a released factor(s) induces egg-laying and comigrates with ELH from cluster homogenates on P-6 columns. At least three other presumed peptides of unknown function are also released. These experiments demonstrate that ELH (6,000 m.w., pl 9.0-9.3) as purified from bag cell cluster homogenates is the major, active form secreted from bag cells. In chapter 2, the effects of ELH on neuronal activity of the attached head ganglia (buccal, cerebral, pleural, and pedal), on the isolated buccal ganglia, as well as on feeding in intact Aplysia were studied. Starved animals (n = 7) injected at 20°C with a crude- extract containing ELH stopped eating algae at 17 ± 4 min and their eggs first appeared at 29 ± 4 min after injection. These data clearly indicate that a suppression of feeding activity occurs before the appearance of eggs. ELH applied to the paired buccal ganglia in vitro activates a pair of neurons into a tonic pacing mode (~1 spike/sec). The time for the full appearance of this activity in vitro correlates well with the time for suppression of feeding in vivo. These neurons each have an ipsilateral axon in buccal nerve 3. ELH increases the rate of firing of a second pair of buccal neurons, each with an ipsilateral axon in the cerebrobuccal connective. ELH when applied to the attached head ganglia causes large bursts of neuronal activity in pedal nerves to the foot, and increased activity in the nerve to the penis. These in vitro effects were produced by ELH partially purified from bag cell cluster homogenates using ammonium sulfate precipitation, an anion exchange column, and a gel filtration column or by ELH released from activated bag cells in isolated abdominal ganglia and then frationated by gel filtration. The ELH effects upon the in vitro nervous system support the working ~ hypothesis that ELH in vivo acts directly on the nervous system to suppress feeding activity. ELH may also activate neural circuits in the pedal and probably cerebral ganglia that produce characteristic movements of the head during egg-laying; the relevant neurons remain to be identified. In chapter 3, a circadian rhythm (CR) of release of presumed peptides from the isolated eye of Aplysia is demonstrated. This isolated eye is known to have a CR of compound action potentials (CAPs) as recorded from its optic nerve. Substances labeled with radioactive amino acids and released into the perfusate were separated on gel filtration columns and SDS polyacrylamide gels. In the CR experiments, the perfusate of a single, labeled, dark-maintained eye was collected every 3 h for two days while simultaneously recording the CR of CAPs. Each 3-h perfusate was applied to a P-2 gel filtration column. Excluded substances (m.w. ~ 2000) and material fractionated in the region of m.w. ~ 1000 showed a CR which was in phase with the CR of CAPs. Much of these labeled substances can be precipitated with trichloroacetic acid. Their release is stimulated by a high potassium solution and inhibited by a low calcium solution that also inhibits CAP activity. This and other previously published evidence suggests that the CAPs and the peptide release are directly produced by electrically coupled neurosecretory cells which may also contain the CR oscillator. One or more of these peptides may be a neurohormone and/or transmitter used for synchronizing, entraining and/or driving the rest of the animal's CRs.