Ultrastructural comparison of Aplysia and Dolabrifera ink glands suggests cellular sites of anti-predator protein production and algal pigment processing

Ultrastructural comparison between the ink gland of a sea harespecies that produces copious purple ink (Aplysia californica)and one that produces none (Dolabrifera dolabrifera), suggeststhat the rough endoplasmic reticulum rich cell and not the inkvesicle cell is the site for synthesis of A. californica's anti-predatorink protein, escapin. Dolabrifera dolabrifera were found tohave vestigial ink glands incapable of producing ink or itsassociated anti-predator proteins regardless of diet. This studyalso suggests that the granulate cells serve only as a storagesite for excess ink pigment acquired during periods of luxuryfeeding on red algae. Slit dimensions in sieve areas of granulatecells are also significantly different between the two species.These slit sizes are larger than those of rhogocytes, a relatedcell type commonly found in connective tissue of gastropod molluscs.Several traits of granulate cells suggest that they are distinctfrom rhogocytes and are a special cell type in the ink glandof sea hares. (Received 1 July 2005; accepted 1 April 2006)     

Postsynaptic regulation of the development and long-term plasticity of Aplysia sensorimotor synapses in cell culture

The monosynaptic component of the neuronal circuit that mediates the withdrawal reflex of Aplysia californica can be reconstituted in dissociated cell culture. Study of these in vitro monosynaptic connections has yielded insights into the basic cellular mechanisms of synaptogenesis and long-term synaptic plasticity. One such insight has been that the development of the presynaptic sensory neurons is strongly regulated by the postsynaptic motor neuron. Sensory neurons which have been cocultured with a target motor neuron have more elaborate structures—characterized by neurites with more branches and varicosities—than do sensory neurons grown alone in culture or sensory neurons that have been cocultured with an inappropriate target cell. Another way in which the motor neuron regulates the development of sensory neurons is apparent when sensorimotor cocultures with two presynaptic cells are examined. In such cocultures the outgrowth from the different presynaptic cells is obviously segregated on the processes of the postsynaptic cell. By contrast, when two sensory neurons are placed into cell culture without a motor neuron, thier processes readily grow together. In addition to regulating the in vitro development of sensory neurons, the motor neuron also regulates learning-related changes in the structure of sensory neurons. Application of the endogenous facilitatory trasmitter serotonin (5-HT) causes long-term facilitation of in vitro sensorimotor synapses due in part to growth of new presynatpic varicosities. But 5-HT applied to sensory neurons alone in cultuer does not produce structural changes in these cells. More recently it has been found that sensorimotor synapses in cell culture can exhibit long-term potentiation (LTP). Like LTP of some hippocampal synapses, LTP of in vitro Aplysia syanpses is regulated by the voltage of the postsynaptic cell. Pairing high-frequency stimulation of sensory neurons with strong hyperpolarization of the motor neuron blocks the induction of LTP. Moreover, LTP of sensorimotor synapses can be induced in Hebbian fashion by pairing weak presynaptic stimulation with strong postsynaptic depolarization. These findings implicate a Habbian mechanism in classical conditioning in Aplysia. They also indicate that Hebbian LTP is a phylogenetically ancient form of synaptic plasticity. 1994 John Wiley & Sons, Inc.     

Multi‐directional sensitivity of statocyst receptor cells of the opisthobranch gastropod Aplysia limacina

The electrical activity in the static nerves of Aplysia limacina was studied in relation to the spatial orientation of the animal. Each sense cell shows a multi‐directional sensitivity. Hence, it responds during full‐circle rotations about all horizontal axes but only within a limited angular range. The statocyst receptor cells are found to be gravity receptors only, and their electrical behaviour suggests that main information about the spatial orientation of Aplysia is contained in the pattern of responding cells, i.e. which of the 13 cells are active at any one time, and not in their absolute discharge frequencies. Furthermore, evidence is given to a similar function of the statocysts of opisthobranch and pulmonate gastropods.     

Aging in Sensory and Motor Neurons Results in Learning Failure in Aplysia californica

The physiological and molecular mechanisms of age-related memory loss are complicated by the complexity of vertebrate nervous systems. This study takes advantage of a simple neural model to investigate nervous system aging, focusing on changes in learning and memory in the form of behavioral sensitization in vivo and synaptic facilitation in vitro. The effect of aging on the tail withdrawal reflex (TWR) was studied in Aplysia californica at maturity and late in the annual lifecycle. We found that short-term sensitization in TWR was absent in aged Aplysia. This implied that the neuronal machinery governing nonassociative learning was compromised during aging. Synaptic plasticity in the form of short-term facilitation between tail sensory and motor neurons decreased during aging whether the sensitizing stimulus was tail shock or the heterosynaptic modulator serotonin (5-HT). Together, these results suggest that the cellular mechanisms governing behavioral sensitization are compromised during aging, thereby nearly eliminating sensitization in aged Aplysia.     

The transcriptome of the early life history stages of the California Sea Hare Aplysia californica

Aplysia californica is a marine opisthobranch mollusc used as a model organism in neurobiology for cellular analyses of learning and behavior because it possesses a comparatively small number of neurons of large size. The mollusca comprise the second largest animal phylum, yet detailed genetic and genomic information is only recently beginning to accrue. Thus developmental and comparative evolutionary biology as well as biomedical research would benefit from additional information on DNA sequences of Aplysia. Therefore, we have constructed a series of unidirectional cDNA libraries from different life stages of Aplysia. These include whole organisms from the egg, veliger, metamorphic, and juvenile stages as well as adult neural tissue for reference. Individual clones were randomly picked, and high-throughput, single pass sequence analysis was performed to generate 7971 sequences. Of these, there were 5507 quality-filtered ESTs that clustered into 1988 unigenes, which are annotated and deposited into GenBank. A significant number (497) of ESTs did not match existing Aplysia ESTs and are thus potentially novel sequences for Aplysia. GO and KEGG analyses of these novel sequences indicated that a large number were involved in protein binding and translation, consistent with the predominant biosynthetic role in development and the presence of stage-specific protein isoforms.     

Conduction velocity and spike duration during afterdischarge in neuroendocrine bag cells of Aplysia

Changes in conduction velocity and spike duration during electrically triggered afterdischarges were determined with extracellular recordings from bag-cell neurites of Aplysia. Spikes with high conduction velocity and short duration occurred at the onset of the afterdischarge during the period of high-frequency firing and regular interspike intervals. Later in the afterdischarge, spike frequency and conduction velocity decreased, while spike duration increased. During the short bursts within the later part of the afterdischarge, conduction velocity was highest for the first spike and decreased for successive spikes in the burst. That conduction velocity and spike frequency were both maximal during the first minute of the afterdischarge and lower during the later periods of the spike train supports the hypothesis that changes in the excitability of the bag-cell neurites occur during this firing pattern. Furthermore, the slower conduction velocity and longer duration of spikes from the bag-cell neurites late in the afterdischarge, and late in the individual bursts within the afterdischarge, suggest the hypothesis of enhanced hormone release per action potential during these periods.     

Serotonin and cAMP induce excitatory modulation of a serotonergic neuron

Serotonin (5-HT) is an excitatory neurotransmitter and neuromodulator. In the Aplysia nervous system it increases excitability and induces spike broadening in sensory neurons. It is released at the synaptic terminals of the metacerebral cells (MCCs) and modulates the feeding neural circuit and buccal muscles during the aroused feeding state. We report that MCC itself is depolarized by 5-HT and becomes excitable. 5-HT induces tonic spike activity and even spike-burst activity. Conceivably, this sensitivity to its own transmitter could provide positive feedback excitation of MCC. Voltage clamp analysis of isolated cultured MCCs shows that 5-HT reduces a calcium-dependent outward current at the resting potential (-60 mV), and enhances steady state inward currents between -55 and -30 mV and between -75 and -100 mV. 8-Br-cAMP has similar effects, suggesting that cAMP mediates the 5-HT effects, in part. A transient calcium current is enhanced at voltages more positive than -40 mV. Barium and cesium selectively block the 5-HT-induced inward current between -75 and -100 mV. Substitution of N-methyl-D-glucamine for sodium and adding cobalt block this current, also indicating that it is a hyperpolarization-activated cation current. The 5-HT-induced inward current between -55 and -30 mV is also blocked by sodium substitution and added cobalt, suggesting that 5-HT increases a depolarization-activated cation current. The outward current that remains when sodium and calcium currents are blocked is reduced by 5-HT. Thus, 5-HT enhances two different cation currents and reduces potassium currents.     

Localization and Functional Characterization of a Novel Adipokinetic Hormone in the Mollusk,Aplysia californica

Increasing evidence suggests that gonadotropin-releasing hormone (GnRH), corazonin, adipokinetic hormone (AKH), and red pigment-concentrating hormone all share common ancestry to form a GnRH superfamily. Despite the wide presence of these peptides in protostomes, their biological effects remain poorly characterized in many taxa. This study had three goals. First, we cloned the full-length sequence of a novel AKH, termed Aplysia-AKH, and examined its distribution in an opisthobranch mollusk, Aplysia californica. Second, we investigated in vivo biological effects of Aplysia-AKH. Lastly, we compared the effects of Aplysia-AKH to a related A. californica peptide, Aplysia-GnRH. Results suggest that Aplysia-AKH mRNA and peptide are localized exclusively in central tissues, with abdominal, cerebral, and pleural ganglia being the primary sites of Aplysia-AKH production. However, Aplysia-AKH-positive fibers were found in all central ganglia, suggesting diverse neuromodulatory roles. Injections of A. californica with Aplysia-AKH significantly inhibited feeding, reduced body mass, increased excretion of feces, and reduced gonadal mass and oocyte diameter. The in vivo effects of Aplysia-AKH differed substantially from Aplysia-GnRH. Overall, the distribution and biological effects of Aplysia-AKH suggest it has diverged functionally from Aplysia-GnRH over the course of evolution. Further, that both Aplysia-AKH and Aplysia-GnRH failed to activate reproduction suggest the critical role of GnRH as a reproductive activator may be a phenomenon unique to vertebrates.     

Modulation of growth of Aplysia neurons by an endogenous lectin

We have purified and characterized a galactose-binding lectin from the gonads of the mollusk Aplysia californica that modulates neurite outgrowth from cultured Aplysia neurons. Agglutination of sheep red blood cells (RBC) by this lectin, termed Aplysia gonad lectin (AGL), is inhibited strongly by galactose and to a lesser extent by fucose. On SDS-PAGE, AGL appears as a single species with a molecular weight of 34 kD under reducing conditions, and 65 kD under nonreducing conditions. This suggests that AGL is a disulfide-linked dimer in its native state. Amino terminal sequence analysis of purified AGL indicates a similarity to another galactose-binding lectin, phytohemagglutinin-E (E-PHA), found in red kidney beans. By using polyclonal antibodies prepared against AGL, we have found that the lectin is present in the gonads and eggs but not in other tissues of adult Aplysia californica. We have examined biological actions of AGL on Aplysia neurons growing in primary cell culture. AGL affects several properties of these neurons. The addition of 100 nM AGL to cultured neurons enhances neurite outgrowth from the cell soma, resulting in a greater number of primary processes. In addition, AGL acts as a neurotrophic agent, increasing neurite viability in vitro. This trophic effect is not seen with concanavalin A (con A), another lectin known to affect several properties of cultured Aplysia neurons. The results are consistent with the suggestion that AGL may play a role in neuronal differentiation and/or maintenance of viability. © 1992 John Wiley & Sons, Inc.     

Computational study of enhanced excitability in Hermissenda: membrane conductances modulated by 5-HT

Serotonin (5-HT) applied to the exposed but otherwise intact nervous system results in enhanced excitability of Hermissenda type-B photoreceptors. Several ion currents in the type-B photoreceptors are modulated by 5-HT, including the A-type K⁺ current (I_K,A), sustained Ca²⁺ current (I_Ca,S), Ca-dependent K⁺ current (I_K,Ca), and a hyperpolarization-activated inward rectifier current (I_h). In this study, we developed a computational model that reproduces physiological characteristics of type B photoreceptors, e.g. resting membrane potential, dark-adapted spike activity, spike width, and the amplitude difference between somatic and axonal spikes. We then used the model to investigate the contribution of different ion currents modulated by 5-HT to the magnitudes of enhanced excitability produced by 5-HT. Ion currents were systematically varied within limits observed experimentally, both individually and in combinations. A reduction of I_K,A or I_K,Ca, or an increase in I_h enhanced excitability by 20–50%. Decreasing I_Ca,S produced a dramatic decrease in excitability. Reductions of I_K,V produced only minimal increases in excitability, suggesting that I_K,V probably plays a minor role in 5-HT induced enhanced excitability. Combinations of changes in I_K,A, I_K,Ca, I_h and I_Ca,S produced increases in excitability comparable to experimental observations. After 5-HT application, the cell's depolarization force is shifted from the I_h–I_Ca,S combination to predominantly I_h.     

State-dependent disruption of short-term facilitation due to overexpression of the apPDE4 supershort form in <Emphasis Type="Italic">Aplysia</Emphasis>

Phosphodiesterases (PDEs) play important roles in synaptic plasticity by regulating cAMP signaling in various organisms. The supershort, short, and long forms of Aplysia PDE4 (apPDE4) have been cloned, and the long form has been shown to play a crucial role in 5- hydroxytryptamine (5-HT)-induced synaptic plasticity in Aplysia. To address the role of the supershort form in 5-HT-induced synaptic plasticity in Aplysia, we overexpressed the apPDE4 supershort form in Aplysia sensory neurons. Consequently, 5-HT-induced hyperexcitability and short-term facilitation in nondepressed synapses were blocked. However, the supershort form did not inhibit 5-HT-induced short-term facilitation in highly depressed synapses. These results show that the supershort form plays an important role in 5-HT-induced synaptic plasticity and disrupts it mainly by impairing cAMP signaling in Aplysia.     

The abdominal ganglion of Aplysia brasiliana: A comparative morphological and electrophysiological study,with notes on A. dactylomela

The ultrastructure and electrophysiological properties of neurons in the abdominal (visceral) ganglion of the marine opisthobranch gastropod Aplysia brasiliana have been investigated to determine whether this preparation compares favorably with the well studied A. californica for neurobiological research. In general, the topography, morphology and physiological characteristics, including synaptic connections, of neurons in this ganglion are quite similar to those of A. californica. There is close correspondence between the two animals in terms of each of the identified cells or neuronal clusters in the ganglion, including the presence of the cell L10 (interneuron I) in A. brasiliana which makes synaptic connections comparable with those in A. californica. New follower cells of this interneuron have been found in A. brasiliana. This species offers some advantages in that the connective tissue surrounding the ganglion is thinner and more transparent, making cell identification and penetration easier. A. brasiliana appears to exhibit the behaviors of A. californica that have been used in previous functional analyses of neural circuits. In addition, this species swims and exhibits a ?burrowing”? activity less commonly seen in A. californica. The rich repertoire of behaviors and accessibility of large identifiable and functionally interconnected neurons makes this species of Aplysia an excellent model preparation for future neurobiological studies. Similar, less thorough, investigations of the abdominal ganglion of A. dactylomela indicate that this species is also very similar to A. californica in terms of the identified cells in the abdominal ganglion.     

Amino acid sequence of myoglobin from the molluscBursatella leachii

The complete amino acid sequence of myoglobin from the triturative stomach of gastropodic molluscBursatella leachii has been determined. It is composed of 146 amino acid residues, is acetylated at the N-terminus, and contains a single histidine residue at position 95 which corresponds to the heme-binding proximal histidine. The E7 distal histidine, which is conserved widely in myoglobins and hemoglobins, is replaced by valine inBursatella myoglobin. The amino acid sequence ofBursatella myoglobin shows strong homology (73–84%) with those ofAplysia andDolabella myoglobins.     

Aplysia myoglobins with an unusual amino acid sequence

The complete amino acid sequence of the myoglobin from Aplysia juliana, a species distributed world-wide, has been determined and compared with the sequence of the myoglobin of Aplysia limacina, a Mediterranean species, and of Aplysia kurodai, a Japanese and Asian species. Unlike mammalian myoglobins, Aplysia myoglobins contain only a single histidine residue, lacking the distal one, the homology being 76% between A. juliana and A. limacina, 74% between A. juliana and A. kurodai, and 83% between A. limacina and A. kurodai. The hydropathy profiles of the Aplysia myoglobins are very similar, but completely different from that of sperm whale myoglobin, taken as the reference.     

Proteomics reveals selective regulation of proteins in response to memory-related serotonin stimulation in Aplysia californica ganglia

The marine mollusk Aplysia californica (Aplysia) is a powerful model for learning and memory due to its minimalistic nervous system. Key proteins, identified to be regulated by the neurotransmitter serotonin in Aplysia, have been successfully translated to mammalian models of learning and memory. Based upon a recently published large‐scale analysis of Aplysia proteomic data, the current study investigated the regulation of protein levels 24 and 48 h after treatment with serotonin in Aplysia ganglia using a 2‐D gel electrophoresis approach. Protein spots were quantified and protein‐level changes of selected proteins were verified by Western blotting. Among those were Rab GDP dissociation inhibitor alpha (RabGDIα), synaptotagmin‐1 and deleted in azoospermia‐associated protein (DAZAP‐1) in cerebral ganglia, calreticulin, RabGDIα, DAZAP‐1, heterogeneous nuclear ribonucleoprotein F (hnRNPF), RACK‐1 and actin‐depolymerizing factor (ADF) in pleural ganglia and DAZAP‐1, hnRNPF and ADF in pedal ganglia. Protein identity of the majority of spots was confirmed by a gel‐based mass spectrometrical method (FT‐MS). Taken together, protein‐level changes induced by the learning‐related neurotransmitter serotonin in Aplysia ganglia are described and a role for the abovementioned proteins in synaptic plasticity is proposed.     

Heavy infestation by endoparasitic copepod crustaceans (Poecilostomatoida: Splanchnotrophidae) in Chilean opisthobranch gastropods, with aspects of splanchnotrophid evolution

Copepods of the family Splanchnotrophidae are very significant parasites of shell-less opisthobranchs, but little information exists on their occurrence, infection frequencies, and local or seasonal abundances. Using a quantitative faunistic approach, 2257 potential hosts belonging to 47 opisthobranch species were collected from 1991 to 1996 off the Chilean and Argentinian coasts, mainly by SCUBA. Endoparasitic splanchnotrophids of the genus Ismaila were found in 303 host specimens, corresponding to a 13% prevalence of infection. The opisthobranch hosts were one sacoglossan, three doridoidean and four aeolidoidean nudibranch species. In total, 12 Chilean opisthobranch species are known to be infected with splanchnotrophids. This amounts to about 20% of all shell-less opisthobranch species from Chile, and a remarkable 26% of all splanchnotrophid hosts worldwide. Infection frequencies are low in most host species, but reached 89–100% in certain populations of Thecacera darwini, Okenia luna, Flabellina sp. 1 and Elysia patagonica, representing the highest rates of infestation by splanchnotrophids ever documented. In Thecacera darwini, the prevalence was very low in northern Chile, consistently high in central Chile, and low in the south. High infestation coupled with a high number of sympatric but host-specific species indicate the coast of central Chile is a centre of Ismaila evolution. The biogeography of splanchnotrophid genera is discussed, and a hypothesis on their distributional history is presented.See also Electronic Supplement (Parts 1 and 2) at http://www.senckenberg.de/odes/02-03.htm     

Dietary selectivity for the toxic cyanobacterium Lyngbya majuscula and resultant growth rates in two species of opisthobranch mollusc

Trophodynamics of blooms of the toxic marine cyanobacterium Lyngbya majuscula were investigated to determine dietary specificity in two putative grazers: the opisthobranch molluscs, Stylocheilus striatus and Bursatella leachii. S. striatus is associated with L. majuscula blooms and is known to sequester L. majuscula metabolites. The dietary specificity and toxicodynamics of B. leachii in relation to L. majuscula is less well documented. In this study we found diet history had no significant effect upon dietary selectivity of S. striatus when offered a range of plant species. However, L. majuscula chemotype may alter S. striatus' selectivity for this cyanobacterium. Daily biomass increases between small and large size groups of both species were recorded in no-choice consumption trials using L. majuscula. Both S. striatus and B. leachii preferentially consumed L. majuscula containing lyngbyatoxin-a. Increase in mass over a 10-day period in B. leachii (915%) was significantly greater than S. striatus (150%), yet S. striatus consumed greater quantities of L. majuscula (g day^− 1) and thus had a lower conversion efficiency (0.038) than B. leachii (0.081) based on sea hare weight per gram of L. majuscula consumed day^− 1. Our findings suggest that growth rates and conversion efficiencies may be influenced by sea hare maximum growth potential, acquisition of secondary metabolites or diet type.     

Calcium-dependent increase in spike duration during repetitive firing of Aplysia axon in the presence of TEA

Repetitive stimulation was studied in the axon of the giant neuron, R2, of Aplysia in the presence of TEA. In 25 or 50 mM extracellular TEA, a plateau develops on the axon spike during repetitive stimulation at frequencies greater than 3/sec. The plateau in extracellular TEA is inhibited by 30 mM CoCl₂ or 1 mM CdCl₂, and is enhanced by raising the Ca concentration. Intracellular TEA induces a plateau on the axon spike at frequencies less than 1/30sec. This plateau increases in duration with repetitive stimulation at higher frequencies and is inhibited by 30 mM CoCl₂ or 1 mM CdCl₂. The increase in spike duration during repetitive firing in the presence of TEA is indicative of an increased entry of Ca during the spike train.