Circadian organization in Aplysia californica.

Substantial progress has been made in unraveling the organization of the circadian system of Aplysia californica. There are at least three circadian pacemakers in Aplysia. One has been localized in each eye and a third lies outside the eyes. Removal of the eyes disrupts the free-running locomotor activity rhythm; however, an extraocular oscillator can mediate a free-running rhythm in some eyeless animals. Although photoreceptors sufficient for entrainment of the ocular oscillator have been localized in the retina, photoreceptors outside the eyes are capable of "driving" a diurnal rhythm of locomotor activity and may also influence entrainment of ocular pacemakers. Finally, attention has been focused on the optic nerve as a coupling pathway between various parts of the system. The evidence suggests that information transmitted in the optic nerves is involved in entrainment of the ocular pacemaker by light, and in ocular control of the locomotor activity rhythm.     

Rhombic particle arrays in gill epithelium of a mollusc, Aplysia californica.

Gill epithelia from adult and juvenile Aplysia were examined by conventional thin section and freeze-fracture methods. Freeze-fracture replicas of adult gill epithelium revealed septate and gap junctions, which served as membrane markers for the epithelial cells. In these same cell membranes, non-junctional rhombic arrays of intramembranous particles were observed on prominent ridges on the membrane P fracture face of some epithelial cells. In thin sections of adult epithelium, nerve terminals were observed abutting the lateral plasma membranes near the basal lamina of some epithelial cells. Correlative areas of plasma membrane in freeze-fracture replicas showed a close association between rhombic particle arrays and abutting nerve terminals. In thin sections of juvenile Aplysia, nerve terminals abutting the epithelial cells were not recognizable, and rhombic arrays were not observed in freeze-fracture replicas. This suggested that a developmental association existed between the appearance of rhombic arrays in adult epithelia and their innervation. It is not known with certainty if, in invertebrates, rhombic arrays are an essential structural entity of all innervated cell membranes; however, in the cells thus far studied, there appears to be an associative condition. In the case of the gill epithelium of Aplysia, rhombic arrays are located in the same vicinity as the abutting nerve terminals. Similar arrays of intramembranous particles have been observed in myoneural postjunctional complexes of other invertebrates and have been interpreted to be the morphological expression of neurotransmitter receptors. An analogous explanation is put forth, namely that rhombic arrays may represent the structural correlates of neurotransmitter receptors and/or ionic channels in innervated membranes of invertebrates.     

Circulating free amino acids in Aplysia californica.

1. Questions regarding the availability of free amino acids and their importance as precursors and direct participants in neural functions are essential to our understanding but cannot be answered without basic data. 2. A profile of 22 circulating amino acids was developed for the often studied Aplysia californica.     

Feeding neural networks in the mollusc Aplysia

Aplysia feeding is striking in that it is executed with a great deal of plasticity. At least in part, this flexibility is a result of the organization of the feeding neural network. To illustrate this, we primarily discuss motor programs triggered via stimulation of the command-like cerebral-buccal interneuron 2 (CBI-2). CBI-2 is interesting in that it can generate motor programs that serve opposing functions, i.e., programs can be ingestive or egestive. When programs are egestive, radula-closing motor neurons are activated during the protraction phase of the motor program. When programs are ingestive, radula-closing motor neurons are activated during retraction. When motor programs change in nature, activity in the radula-closing circuitry is altered. Thus, CBI-2 stimulation stereotypically activates the protraction and retraction circuitry, with protraction being generated first, and retraction immediately thereafter. In contrast, radula-closing motor neurons can be activated during either protraction or retraction. Which will occur is determined by whether other cerebral and buccal neurons are recruited, e.g. radula-closing motor neurons tend to be activated during retraction if a second CBI, CBI-3, is recruited. Fundamentally different motor programs are, therefore, generated because CBI-2 activates some interneurons in a stereotypic manner and other interneurons in a variable manner.     

Complete DNA sequence of the mitochondrial genome of the sea-slug, Aplysia californica: conservation of the gene order in Euthyneura

We have sequenced and characterized the complete mitochondrial genome of the sea slug, Aplysia californica, an important model organism in experimental biology and a representative of Anaspidea (Opisthobranchia, Gastropoda). The mitochondrial genome of Aplysia is in the small end of the observed sizes of animal mitochondrial genomes (14,117 bp, NCBI Accession No. NC_005827). The Aplysia genome, like most other mitochondrial genomes, encodes genes for 2 ribosomal subunit RNAs (small and large rRNAs), 22 tRNAs, and 13 protein subunits (cytochrome c oxidase subunits 1-3, cytochrome b apoenzyme, ATP synthase subunits 6 and 8, and NADH dehydrogenase subunits 1-6 and 4L). The gene order is virtually identical between opisthobranchs and pulmonates, with the majority of differences arising from tRNA translocations. In contrast, the gene order from representatives of basal gastropods and other molluscan classes is significantly different from opisthobranchs and pulmonates. The Aplysia genome was compared to all other published molluscan mitochondrial genomes and phylogenetic analyses were carried out using a concatenated protein alignment. Phylogenetic analyses using maximum likelihood based analyses of the well aligned regions of the protein sequences support both monophyly of Euthyneura (a group including both the pulmonates and opisthobranchs) and Opisthobranchia (as a more derived group). The Aplysia mitochondrial genome sequenced here will serve as an important platform in both comparative and neurobiological studies using this model organism.     

Stages in the post-hatching development of Aplysia californica

In order to study the development of the nervous system of the marine mollusc, Aplysia californica, it is necessary objectively to assess the maturity of individual specimens. This can be done by defining stages in the life cycle. The post-hatching development can be divided into four phases: planktonic, metamorphic, juvenile, and adult. These phases can be further subdivided into 13 stages on the basis of behavioral and morphological characteristics visible in living specimens: Stage 1, newly hatched; Stage 2, eyes develop; Stage 3, the larval heart beats; Stage 4, maximum shell size is reached; Stage 5, the propodium develops; Stage 6, red spots appear; Stage 7, the velum is shed; Stage 8, eyebrows appear; Stage 9, pink color develops; Stage 10, white spots appear; Stage 11, rhinophores grow; Stage 12, the genital groove forms; Stage 13, egg laying begins. Reconstructions from serial sections taken from specimens fixed at each of these stages reveal the sequence of formation of the major organ systems. The nervous system develops gradually. The cerebral and pedal ganglia are present at Stage 1, the optic ganglia develop at Stage 2, the abdominal, pleural, and osphradial ganglia at Stage 3, the buccal ganglia at Stage 5, and the genital ganglion at Stage 13. Because Aplysia develops gradually, it is possible to analyze the contribution which gastropod torsion makes to the different phases of the life cycle. The Aplysia embryo undergoes 120 degrees torsion prior to Stage 1. The major visceral organs, the digestive system, heart, gill, and visceral nervous system, develop sybsequently in their post-torsional positions. After metamorphosis, there is a partial de-torsion which involves only the digestive system. Torsion of the digestive system may therefore be beneficial only to the pre-metamorphic larva, and not to the postmetamorphic juvenile.     

Ultrastructure of the osphradium of Aplysia californica

Summary The osphradium of Aplysia californica, a sensory organ, is a small yellow-brown epithelial patch located in the mantle cavity immediately anterior to the rostral attachment of the gill. Scanning electron microscopy reveals a round ellipsoid structure of 0.6–1 mm in diameter with a central, occasionally folded, sensory epithelium. The central area is covered with microvilli and surrounded by a densely ciliated epithelium. Transmission electron micrographs show that the columnar supporting cells in the sensory epithelium contain an abundance of apical pigment granules and microvilli. Between the epithelial-supporting cells, the putative sensory elements consist of thin neurites (0.4–1.5 m in diameter) that reach the sea-water side of the osphradium. The neurites contain many neurotubules, mitochondria, vesicles and cilia in their apices. The nerve endings originate from cell bodies up to 40 m below the epithelium or in the osphradial ganglion itself, as revealed by electron microscopy and retrograde labeling with Lucifer yellow. There appear to be two populations of putative sensory cells, a large population of heavily stained cell bodies 4–10 m in diameter and a few scattered cells of large diameter (25–60 m). Following lanthanum impregnation, septate junctions can be seen between all types of cells in the epithelium, 3–5 m below the sea-water surface. This study provides new information for further investigation of osmo- and mechanosensation in Aplysia californica.     

A possible sensory-motor neuron in Aplysia californica

A granule-containing cell is described in the secretory epithelia of the accessory genital mass in Aplysia californica. The ciliated apical process of this cell protrudes into the lumen of the oviduct. The granule-containing basal region of the cell is drawn out into fine processes that resemble axons. Granule-filled dilatations of these axons are found directly under large secretory cells. On this basis, it is suggested that these cells fulfill the morphological criteria for sensory-motor cells, and this data will be used as a basis for microelectrode studies to confirm or deny the above suggestion.     

Sodium-phosphate symport by Aplysia californica gut

Phosphate transport across plasma membranes has been described in a wide variety of organisms and cell types including gastrointestinal epithelia. Phosphate transport across apical membranes of vertebrate gastrointestinal epithelia requires sodium; whereas, its transport across the basolateral membrane requires antiport processes involving primarily chloride or bicarbonate. To decipher the phosphate transport mechanism in the foregut apical membrane of the mollusc, Aplysia californica, in vitro short-circuited Aplysia californica gut was used. Bidirectional transepithelial fluxes of both sodium and phosphate were measured to see whether there was interaction between the fluxes. The net mucosal-to-serosal flux of Na+ was enhanced by the presence of phosphate and it was abolished by the presence of serosal ouabain. Similarly, the net mucosal-to-serosal flux of phosphate was dependent upon the presence of Na+ and was abolished by the presence of serosal ouabain. Theophylline, DIDS and bumetande, added to either side, had no effect on transepithelial difference or short-circuit current in the Aplysia gut bathed in a Na2HPO4 seawater medium. However, mucosal arsenate inhibited the net mucosal-to-serosal fluxes of both phosphate and Na+ and the arsenate-sensitive Na+ flux to that of phosphate was 2:1. These results suggest the presence of a Na-PO4 symporter in the mucosal membrane of the Aplysia californica foregut absorptive cell.     

Sodium-Sulfate Symport by Aplysia californica Gut

Sulfate transport across plasma membranes has been described in a wide variety of organisms and cell types including gastrointestinal epithelia. Sulfate transport can be coupled to proton, sodium symport or antiport processes involving chloride or bicarbonate. It had previously been observed in Aplysia gut that sulfate was actively absorbed. To understand the mechanism for this transport, short-circuited Aplysia californica gut was used. Bidirectional transepithelial fluxes of both sodium and sulfate were measured to see whether there was interaction between the fluxes. The net mucosal-to-serosal flux of Na(+) was enhanced by the presence of sulfate and it was abolished by the presence of serosal ouabain. Similarly, the net mucosal-to-serosal flux of sulfate was dependent upon the presence of Na(+) and was abolished by the presence of serosal ouabain. Theophylline, DIDS and bumetanide, added to either side, had no effect on transepithelial potential difference or short-circuit current in the Aplysia gut bathed in a Na2SO4 seawater medium. However, mucosal thiosulfate inhibited the net mucosal-to-serosal fluxes of both sulfate and Na(+) and the thiosulfate-sensitive Na(+) flux to that of sulfate was 2:1. These results suggest the presence of a Na-SO4 symporter in the mucosal membrane of the Aplysia californica foregut absorptive cell.     

DNA sequence organization in the soybean plant

The arrangement of repetitive and nonrepetitive DNA sequences in the soybean genome was ascertained by a comparison of the reassociation kinetics of short (250 nucleotides) and long (2700 nucleotides) DNA fragments, the size distribution of S-1 nuclease resistant repetitive duplexes, and a direct assay of the spectrum of DNA sequences present on long DNA fragments enriched in repetitive DNA. These measurements reveal the following: (1) The 1N genome size of the soybean plant is 1.97 pg. (2) Approximately 40% of the soybean genome consists of nonrepetitive or single-copy DNA sequences, while 60% is repetitive DNA. (3) The repetitive DNA is partitioned into three discrete classes termed very fast, fast, and slow, containing DNA sequences repeated an average of 290,000, 2800, and 19 times each. (4) Approximately 35–50% of the soybean genome is arranged in a short-period interspersion pattern of 250 nucleotide slow sequences and single-copy DNA averaging up to 2700 nucleotides in length. (5) From 30% to 45% of the soybean genome is organized into long stretches of repetitive DNA at least 1500 nucleotides in length. (6) Minimal interspersion of repetitive sequence classes occurs in soybean DNA.These experiments were supported by NSF Grants BMS74-21461 and PCM76-24593 and were conducted while the author was in the Department of Biology, Wayne State University, Detroit, Michigan.     

Urotensin I-like immunoreactivity in the central nervous system of Aplysia californica.

In the present study the occurrence and localization of urotensin I (UI, a corticotropin releasing factor-like peptide) in the CNS of Aplysia californica were investigated by immunocytochemistry and radioimmunoassay. The RIA cross-reactivity pattern indicated that the UI antiserum used recognized an epitope in the C-terminal region of the UI, but it did not cross-react with mammalian corticotropin-releasing factor (CRF) and partially recognized sauvagine (SVG, a frog CRF-like peptide). The use of CRF-specific and sauvagine-specific antisera failed to give positive immunostaining. The application of UI antiserum (which does not cross-react with CRF in RIA) gave a positive staining, which was blocked by synthetic sucker (Catostomus commersoni) UI, but not by rat/human CRF (10 microM). On the basis of immunostaining and RIA parallel to fish UI displacement curves of cerebral ganglia extracts, the unknown UI/CRF-like substance in the Aplysia ganglia is likely to have greater homology with sucker UI than with the known CRF peptides. Urotensin I-immunoreactive (UI-ir) neurons were seen mainly in the F neuron clusters, located in the midline and rostrodorsal portion of the cerebral ganglia. Few UI-ir neurons were also found in the C and D neuron clusters of the cerebral ganglia, as well as in the left pleural and abdominal ganglia. In addition, numerous fine and coarse, and beaded UI-ir fibers were found in the cerebral commissure. UI-ir fibers were also seen in the neuropile of the buccal, pedal and pleural ganglia, and abdominal ganglion. A cuff-like arrangement of UI-ir fibers was seen in the supralabial nerves.(ABSTRACT TRUNCATED AT 250 WORDS)