NeurobiologyPurification and primary structure of the neuropeptide egg-laying hormone of Aplysia californica ( Communicated by Theodore H. Bullock, August 15,1979 ABSTRACT Egg-laying hormone (ELH), a neuropeptide synthesized by the bag cell neurons, induces egg laying and its correlated behavior in Aplysia californica. In present study, ELH has been purified to homogeneity and its primary structure has been determined. We find this molecule to have 36 amino acid residues with a M, of 4385 and a calculated isoelectric point of 9.7. Direct microsequence analysis revealed a single amino acid sequence that is in agreement with the amino acid composition determined after acid hydrolysis of ELH: HRIleSer-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-LeuThr-Glu-Gln-Ile-Arg-Glu-Arg-Gln-Arg-Tyr-Leu-Ala-Asp-Leu-

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.

1. Egg-laying hormone (ELH) is a polypeptide of about 4,500 mol wt synthesized in the bag cell neurons of the abdominal ganglion of Aplysia. We studied the effects of ELH on the 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. 2. Starved animals (n = 7) injected with crude extract containing ELH stopped eating algae at 17 +/- 4 min and their eggs first appeared at 29 +/- 4 min after injection at 20 degrees C. This cessation of eating is significant when compared to the seven controls (P less than 0.01). These data clearly indicate that a suppression of feeding activity occurs before the appearance of eggs. 3. ELH applied to the paired buccal ganglia in vitro activates a pair of neurons into a tonic pacemaker mode (approximately 1 spike/s). This activation also occurs in a high-magnesium, zero calcium solution that blocks chemical synapses. The time for the full appearance of this activity in vitro correlates well with the time for suppression of feeding in vivo. Each of these neurons has an ipsilateral axon in buccal nerve 3. The neuron has been identified by intracellular recording. 4. ELH increases the rate of firing of a second pair of buccal neurons, each with an ipsilateral axon in the cerebrobuccal connective. 5. 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; the relevant neurons remain to be identified. 6. These in vitro effects were produced by ELH partially purified from bag cell cluster homogenates using ammonium sulfate precipitation followed by anion exchange and gel filtration chromatography or by ELH released from activated bag cells in isolated abdominal ganglia and then purified by gel filtration. The isolated buccal ganglia effects have been confirmed with fully purified ELH. 7. The ELH effects on the in vitro nervous system support the hypothesis that ELH in vivo acts directly on the nervous system to suppress feeding activity, controlled by the buccal and cerebral ganglia. ELH may also produce characteristic movements of the head during egg laying, controlled probably by the pedal and cerebral ganglia.

1. The activity of units in the auditory system of alert, freely moving rats was studies during the acquisition and extinction of a tone-signaled, appetitive classically conditioned response. Responses of neurons in inferior colliculus (N = 28), medial geniculate (N = 32), posterior nucleus of thalamus (N = 28), pretectal region (N = 19), and cortex (N = 100) were studies in 74 rats across 10-trial blocks. 2. During behavioral acquisition, neurons in posterior nucleus of thalamus were the first to show response increments to CS+ onset. They were followed by neurons in cortex, pretectal region, medial geniculate, inferior colliculus and by movement behavior. 3. Prestimulus background rates during acquisition showed significant decrements in cortical neurons. These background decrements began to be evidenced in the trial series before the response increases in posterior nucleus. These data strengthened the suggestion of a previous study that posterior nucleus responses could be dependent on tonic modulation from cortex. 4. Extinction appeared to be largely a reverse of acquisition. Cortex and behavior showed response decrements first in the trial series. They were followed by medial geniculate, pretectal region, posterior nucleus, and inferior colliculus neurons. 5. The hypothesis was advanced that the auditory lemniscal adjunct afferent system may play a primary role in the early phases of auditory conditioned-response acquisition.

A continuously breeding laboratory colony of the giant leech Haementeria ghilianii has been established from a few specimens collected in French Guyana. The leeches feed on live rabbits or bovine blood, which they draw from an artifical feeding device. Leech growth is saltatory, in that at each of four successive feedings, spaced over about half a year, the weight of each specimen increases 3-to 6-fold. The male reproductive system of this hermaphroditic leech matures first, at a body weight of 3-5 g. The female reproductive system matures after at least one more feeding, at a body weight of at least 8 g. Fecundity (number of eggs per laying) depends on the weight of the leech at the time of oviposition, increasing from 60 for the smallest mature individuals to 500 for the largest. These data on the growth and reproduction of//, ghilianii gathered under laboratory conditions are consistent with its probable life cycle in the native habitat.