James E. Blankenship

Induction of copulatory behavior in Aplysia: Atrial gland factors mimic the excitatory effects of freshly deposited egg cordons

Egg laying in the marine mollusc Aplysia is induced and coordinated by peptide products of the egg-laying hormone (ELH) gene expressed in the neuroendocrine bag cells of the central nervous system. At least three structurally related genes, belonging to the ELH family but distinct from the ELH gene, are expressed in the atrial gland, an exocrine organ of unknown function that secretes into the oviduct of Aplysia. The experiments described in this report were designed to test the hypothesis that the atrial gland gene products serve a pheromonal function for the animal, coordinating reproductive behavior among individuals. Our studies showed that there was a significantly shorter latency to copulation when an Aplysia was paired with an animal that was actively laying eggs than when it was paired with a sexually mature but nonlaying animal. Moreover, the addition of extracts or homogenates of the atrial gland to the seawater surrounding two nonlaying animals reduced the latency to mating compared to animals exposed only to seawater or to homogenates of other regions of the reproductive tract, including oviduct. These results suggest that atrial gland products, secreted onto the egg cordon as it passes through the oviduct, may play a pheromonal role and induce mating behavior between individuals. Experiments are in progress to determine whether the active atrial gland factor(s) are products of the ELH-family genes expressed in the gland.

A light and electron microscopic investigation of the neurosecretory bag cells ofAplysia

SummaryThe two bilateral clusters of neurosecretory bag cells ofAplysia were studied with both light and electron microscopy. Autoradiography revealed that the bag cells rapidly accumulate3H-labelled amino acids and that after 1–2 h, heavy concentrations of silver grains appear over Golgi complexes and in the proximal axons. Intrasomatic injections of CoCl2 or lucifer yellow showed clear branch points and numerous varicosities along individual axons. Many of the bag cells are multipolar. Electron-microscopic observations confirmed that individual fibres branch and showed that the varicosities are packed with dense-cored vesicles similar in size (180 nm diameter) and electron density to those found in the somata. The axons of several cells are usually associated into bundles that travel (within the connective tissue sheath) either rostrally up the pleurovisceral connective or toward the contralateral bag cell cluster. Bundled in groups of tens to hundreds, a total of many thousands of axons fill the sheath around each cell cluster and around the proximal 2–5 mm of the pleurovisceral connective; the number of axon bundles in the sheath decreases rapidly with distance from the cluster. Individual axons reaching the outer edges of bundles form neurosecretory endings near blood sinuses in the sheath, creating an extensive neurohemal release area. Dense-cored vesicles are packed into the endings, often in very close apposition to the plasma membrane. Possible release profiles (omega-shaped) and smaller clear vesicles (85 nm diameter) were observed in the axon endings. A number of axons also enter and travel among the conventional (non-neurosecretory) axons in the core of the pleurovisceral connective nerve. These ‘core’ bag cell axons project for several millimetres beyond the terminations of the bundled axons of the sheath.The findings support the hypothesis proposed in physiological studies that the distribution and branching of the axonal tree are the basis for the extracellularly recorded wave forms and of the potentiation of electrical signals during bag-cell activity. Additional evidence indicates that exocytosis is the means by which bag-cell hormone is released during afterdischarges.

Aplysia behavioral biology: II. Induced burrowing in swimming A. brasiliana by burrowed conspecifics

This study demonstates that burrowing can be induced in a swimming Aplysia brasiliana by placing it into an aquarium containing a burrowed conspecific. When Burrowers (Efficient Burrowers as defined in Aspey and Blankenship, 1976) are removed from their burrows, they consistently reburrow with the latency to reburrow decreasing over 10 trials. When induced burrowers (Nonburrowers) are removed from the sand after being induced to burrow, they do not reinitiate burrowing. Although burrowing differs qualitatively and quantitatively in Burrowers and large swimming Nonburrowers induced to burrow, Burrowers and Nonburrowers are as likely to accept food when removed from the sand. However, Burrowers only partially consume the food and reburrow, whereas Nonburrowers consume the entire food ration within several minutes and do not reburrow. The occurrence of induced burrowing in Nonburrowers suggests pheromonemediated behavior since the probability of spontaneous burrowing occurring while an Aplysia is swimming approaches zero. Since Burrowers consistently reinitiate burrowing following removals from the sand, and Nonburrowers do not, it appears as if some internal physiological state may be necessary to maintain the behavior.

Modulation of arterial muscle contraction in Aplysia by glycine and neuron R14

Glycine and electrical activity in neuron R14 both enhance the contractility of the anterior aorta of the gastropod Aplysia californica. Glycine and R14 do not seem to cause contraction directly, change membrane permeabilities or alter junctional potentials occurring in the muscle fibers, yet they increase the force of contractions induced by other means. Modulation of muscle contraction is a new function for glycine.

Aplysia behavioral biology: I. A multivariate analysis of burrowing in A. brasiliana.

This study describes burrowing for the first time in the marine gastropod mollusc Aplysia brasiliana. The animals were collected along the southern coast of Texas in the Gulf of Mexico during the late summer. Ten burrowing parameters were operationally defined from burrowing observations on 32 subjects. In an effort to determine the relationship among the heterogeneous subjects to underlying pure-types along distinct dimensions of variation, the data were subjected to Linear Typal Analysis and Q-type orthogonal powered-vector Factor Analysis with a cluster-oriented solution. Three independent subject-related factors were extracted which clustered the subjects according to behavioral profiles on the burrowing parameters. Intercorrelations among the 10 burrowing parameters revealed two general behavior-related groups descriptive of burrowing; burrowing speed and burrowing responsiveness. Factor I contained nine subjects considered “Inefficient Burrowers” on the basis of scores indicating slower burrowing speed and lowered burrowing responsiveness. Factor II contained 15 subjects considered “Efficient Burrowers” due to scores indicating faster burrowing speed and greater burrowing responsiveness. Factor III contained eight subjects considered “Intermediate Burrowers” on the basis of low- to middle-range scores on the 10 burrowing parameters. Since these analytical techniques reliably identified and characterized three clusters of subjects from a naturally selected heterogeneous sample on the basis of their burrowing behaviors, testable hypotheses can now be generated to study the adaptive mechanism and functional significance of burrowing. Preliminary evidence suggests that Efficient Burrowers were probably young and/or healthy animals for whom burrowing might represent a preparatory state for subsequent reproductive activities. Inefficient Burrowers were probably old and/or unhealthy animals for whom burrowing represented an energyconserving response to deteriorating health or lowered tolerance to unfavorable environmental conditions. Intermediate Burrowers seemed to represent transitional animals, those older and less vigorous than Efficient Burrowers, but considerably younger and more vigorous than Inefficient Burrowers.

I: Aplysia californica neurons R3-R14: primary structure of the myoactive histidine-rich basic peptide and peptide I

The R3-R14 neurons of the marine mollusc Aplysia are neuroendocrine cells that express a gene encoding peptides I, II and histidine-rich basic peptide (HRBP), a myoactive peptide that excites Aplysia heart and enhances gut motility in vitro. Peptide II has been chemically characterized (35), but the complete primary structures of peptide I and HRBP have not been established by amino acid sequence analysis. HRBP, peptide I, and the prohormone (proHRBP) were therefore purified from acid extracts of Aplysia californica neural tissue using sequential gel filtration and reverse-phase high-performance liquid chromatography and chemically characterized. Amino acid sequence analysis demonstrated that HRBP was a 43-residue peptide whose sequence was: less than Glu-Val-Ala-Gln-Met-His-Val-Trp-Arg-Ala-Val-Asn-His-Asp-Arg-Asn-His-Gly- Thr-Gly - Ser-Gly-Arg-His-Gly-Arg-Phe-Leu-Ile-Arg-Asn-Arg-Tyr-Arg-Tyr-Gly-Gly-Gly- His-Leu - Ser-Asp-Ala-COOH. Compositional and sequence analyses of peptide I and proHRBP demonstrated that peptide I was a 26-residue peptide with the following sequence: NH2-Glu-Glu-Val-Phe-Asp-Asp-Thr-Asp-Val-Gly-Asp-Glu-Leu-Thr-Asn-Ala- Leu-Glu-Ser-Val-Leu-Thr-Asp-Phe-Lys-Asp-COOH. These results demonstrated that the pro-HRBP sequence predicted by nucleotide sequence analysis of a cDNA clone (24) was in fact synthesized in R3-R14 neurons. Hydrophilicity and hydrophobicity profiles of preproHRBP, combined with charge distribution profiles and predictive secondary structural analysis, showed that cleavage at dibasic sequences was strongly associated with peaks of hydrophilicity in alpha-helical regions of the preprohormone.

Atrial gland cells synthesize a family of peptides that can induce egg laying inAplysia

Summary1.Endogenous peptides induced egg laying in the marine molluscAplysia in two ways: egg-laying hormone (ELH) from the neuroendocrine bag cells acts directly, causing the release of eggs from the ovotestis; peptides A and B from the atrial gland act indirectly, activating the bag cells to release ELH. Another atrial gland peptide (egg-releasing hormone; ERH) is a structural and functional hybrid of ELH and peptides A and B; it can act both directly and indirectly to induce egg laying.2.Atrial glands were incubated in a mixture of3H-amino acids for 18 h, and the biosynthetically labelled peptides isolated using sequential Sephadex G-50 column chromatography and isoelectric focusing. Radiolabelled peaks were localized and bioassayed in intact animals. Bioactive peaks were then characterized functionally using two additional assays: egg laying in bag cell-less animals (ELH-like peptides) and in vitro induction of bag cell discharge (A- and B-like peptides). ERH-like molecules are active in both assays. Homogeneity of bioactive IEF peaks was assessed by SDS-PAGE.3.Sephadex G-50 gel filtration of biosynthetically labelled atrial gland extracts reveals two major peptide peaks. Peak D (apparentMr 6,000) is strongly radiolabelled and contains most of the egg-laying activity, but has a low absorbance at 274 nm. Peak E (apparentMr 3,500) is weakly labelled and contains a small proportion of the total egg-laying activity, but has a large absorbance at 274 nm.4.Isoelectric focusing of radiolabelled peptides in peak D reveals seven distinct ELH-like species (pI 5.5, 7.5, 8.5, 8.7, 8.9, 9.1, 9.4), and two peaks (pI 5.9, 8.1) that have both ELH-like and A-/B-like activity. The pI 8.1 peak may result from the comigration of peptide A with ERH or with an unidentified ELH-like peptide. It is not yet clear whether the pI 5.9 activity results from comigration of distinct peptides or from the presence of a previously uncharacterized ERH-like molecule.5.Isoelectric focusing of radiolabelled peptides in peak E reveals five distinct ELH-like species (pI 7.3, 8.5, 8.7, 9.1, 9.4), and one peak (pI 8.9) with both ELH-like and A-/B-like activity. The pI 8.9 peak may result from the comigration of an ELH-like peptide with peptide B. Three of the ELH-like peptides (pI 8.5, 8.9, 9.1) found in peak E are probably identical to the ELH-like peptides found at the same pIs in peak D.6.The ELH-like pI 7.5 species is a peptide complex held together by a disulfide linkage and hydrophobic interactions. The complex dissociates when treated with 2-mercaptoethanol and pyridine, resulting in a reduction in apparent molecular weight, and the appearance of one basic (pI 9.4) and two acidic (pI 3.3, 3.8) peptides. The pI 9.4 peptide presumably accounts for the ELH-like activity of the parent pI 7.5 complex but differs, based on its mobility on SDS-PAGE, from the pI 9.4 ELH-like peptide(s) described above.7.These studies, utilizing a two-step purification procedure, functional bioassays, and SDS-PAGE to assess peptide homogeneity, indicate that atrial gland cells synthesize a surprisingly large number of peptides that can induce egg laying inAplysia.

Isolation of an egg-laying hormone-binding protein from the gonad of Aplysia californica and its localization in oocytes

A protein solubilized from a membrane preparation of the gonad of Aplysia californica has been isolated by affinity chromatography, using bag cell egg-laying hormone (ELH) as the bound ligand, and partially purified and characterized by gel electrophoresis. The protein has an apparent molecular weight of 52 kDa and consists of two disulfide-linked subunits of about 30 kDa each. The protein is glycosylated and has an acidic pI. Approximately 10–15 μg of this protein can be isolated from a single ovotestis, representing less than 1% of the total protein in the gonad; but the protein could not be detected in buccal mass or body wall, tissues which do not have apparent response to ELH.Antibodies generated against this ELH-binding protein (ELHBP) were used to localize sites in the ovotestis which might contain this molecule and thus represent targets for egg-laying hormone. Immunocytochemical results indicate that the oocytes are a rich source of this protein, since their cytoplasm was the only detectable site of immunoreactivity.Whether this binding protein represents an egg-laying hormone receptor is uncertain, but its prevalence in oocytes suggests that ELH plays a signaling role on these gametes.

Functional and morphological evidence for the existence of neurites from abdominal ganglion bag cell neurons in the head-ring ganglia of Aplysia

SummaryThree lines of evidence are presented indicating that axons of the Aplysia neuroendocrine bag cells extend into the head-ring ganglia of the CNS. When the abdominal ganglion was bisected longitudinally, separating the two bag cell clusters, an afterdischarge induced in one cluster generated an afterdischarge in the other via activity through the head-ring ganglia to which each half abdominal ganglion was attached by connective nerves. This suggests that some axons of bag cells in each cluster communicate through the head-ring ganglia. Retrograde labelling of bag cells occurred when rhodamine-onjugated latex microspheres were injected into the cerebral or either pleural ganglion, a direct demonstration that bag cell axons extend into these ganglia. Finally, cell LP1 in the left pleural ganglion was inhibited during a bag cell afterdischarge, an action mimicked by application of alpha-bag cell peptide (αBCP). Since αBCP can act only close to its site of release due to susceptibility to peptidase activity, it is likely that LP1 inhibition is dependent on the local release of αBCP from bag cell neurites in the pleural ganglion. These results open new possibilities for how bag cell afterdischarges may be initiated and broaden the distribution of their effects.

The bag cell egg-laying hormones of Aplysia brasiliana and Aplysia californica are identical.

Egg laying in the marine molluscan genus Aplysia is elicited by an egg-laying hormone (ELH) which induces ovulation and acts on central neurons to effect egg-laying behavior. ELH, isolated from the A. californica bag cells, and three ELH-related peptides, isolated from the A. californica atrial gland, have been chemically characterized, yet relatively little is known about homologous peptides in other Aplysia species. In these studies, the primary structure of A. brasiliana ELH was determined. Bag cell clusters were extracted in an acidic solution, and the peptides purified by sequential gel filtration and reversed-phase HPLC; ELH was identified by bioassay. Amino acid compositional and sequence analyses demonstrated that the neurohormone was a 36-residue peptide whose sequence was identical to that of A. californica ELH: NH2-Ile-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu- Gln-Ile- Arg-Glu-Arg-Gln-Arg-Tyr-Leu-Ala-Asp-Leu-Arg-Gln-Arg-Leu-Leu-Glu-Lys-COOH .