Marine Mollusk Aplysia Research Articles

Urotensin II in Invertebrates: From Structure to Function in Aplysia californica

Neuropeptides are ancient signaling molecules that are involved in many aspects of organism homeostasis and function. Urotensin II (UII), a peptide with a range of hormonal functions, previously has been reported exclusively in vertebrates. Here, we provide the first direct evidence that UII-like peptides are also present in an invertebrate, specifically, the marine mollusk Aplysia californica. The presence of UII in the central nervous system (CNS) of Aplysia implies a more ancient gene lineage than vertebrates. Using representational difference analysis, we identified an mRNA of a protein precursor that encodes a predicted neuropeptide, we named Aplysia urotensin II (apUII), with a sequence and structural similarity to vertebrate UII. With in-situ hybridization and immunohistochemistry, we mapped the expression of apUII mRNA and its prohormone in the CNS and localized apUII-like immunoreactivity to buccal sensory neurons and cerebral A-cluster neurons. Mass spectrometry performed on individual isolated neurons, and tandem mass spectrometry on fractionated peptide extracts, allowed us to define the posttranslational processing of the apUII neuropeptide precursor and confirm the highly conserved cyclic nature of the mature neuropeptide apUII. Electrophysiological analysis of the central effects of a synthetic apUII suggests it plays a role in satiety and/or aversive signaling in feeding behaviors. Finding the homologue of vertebrate UII in the numerically small CNS of an invertebrate animal model is important for gaining insights into the molecular mechanisms and pathways mediating the bioactivity of UII in the higher metazoan.

Spatial and temporal variability in response to hybrid electro-optical stimulation

Hybrid electro-optical neural stimulation is a novel paradigm combining the advantages of optical and electrical stimulation techniques while reducing their respective limitations. However, in order to fulfill its promise, this technique requires reduced variability and improved reproducibility. Here we used a comparative physiological approach to aid the further development of this technique by identifying the spatial and temporal factors characteristic of hybrid stimulation that may contribute to experimental variability and/or a lack of reproducibility. Using transient pulses of infrared light delivered simultaneously with a bipolar electrical stimulus in either the marine mollusk Aplysia californica buccal nerve or the rat sciatic nerve, we determined the existence of a finite region of excitability with size altered by the strength of the optical stimulus and recruitment dictated by the polarity of the electrical stimulus. Hybrid stimulation radiant exposures yielding 50% probability of firing (RE50) were shown to be negatively correlated with the underlying changes in electrical stimulation threshold over time. In Aplysia, but not in the rat sciatic nerve, increasing optical radiant exposures (J cm–2) beyond the RE50 ultimately resulted in inhibition of evoked potentials. Accounting for the sources of variability identified in this study increased the reproducibility of stimulation from 35% to 93% in Aplysia and 23% to 76% in the rat with reduced variability.

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.

Sequential recruitment of identified I1/I3 motor neurons enhances muscle contraction through modulatory interactions

Event Abstract Back to Event Sequential recruitment of identified I1/I3 motor neurons enhances muscle contraction through modulatory interactions Hui Lu1*, Jeffrey M. McManus1, Miranda J. Cullins1 and Hillel J. Chiel2 1 Case Western Reserve University, Biology, United States 2 Case Western Reserve University, Biology, Neurosciences, Biomedical Engineering, United States Previous studies have shown that motor neurons for a single muscle are recruited in an orderly fashion based on a ‘size principle’ to produce movements with different force and speed (Henneman, 1957; 1965a; 1965b; Mendell, 2005). Although sequential recruitment of motor neurons has been extensively studied in vertebrates during the past 50 years (Zajac and Faden, 1985; Gabriel et al, 2011; Mantilla and Sieck, 2011), it is difficult to study in vivo at the individual neuron level. To explore the behavioral significance and force outputs of motor neuron recruitment sequences, we studied it in the marine mollusk Aplysia californica, in which individual motor neurons can be identified, monitored and controlled. Aplysia use the same feeding apparatus, the buccal mass, to perform multiple feeding behaviors: biting, swallowing and rejection. All feeding behaviors have two phases, protraction and retraction of the food grasper (radula/odontophore). Previous magnetic resonance imaging evidence (Neustadter et al, 2007) and our current data both suggest that the retraction phase is a major difference between biting and swallowing, and is primarily mediated by the contraction of the I1/I3 muscle. We found that buccal nerve 2 (BN2), which contains the axons of multiple I1/I3 motor neurons (e.g., B3, B6, B9, B10, B38, B39 and B43), is activated more intensely during the retraction phase in swallowing than in biting in vivo. In addition, identified I1/I3 motor neurons B3, B6 and B9 are more active in swallowing than in biting. Thus, we hypothesize that the increased retraction force during swallowing is due to the sequential recruitment of the identified I1/I3 motor neurons B6 and B9, followed by B3. We found that BN2 neural activity of the I1/I3 motor neurons during retraction in vivo and in vitro can be divided into four sub-phases (blocks) based on the BN2 unit sizes, relative timing, bursting features, and muscle innervation. The B3, B6 and B9 neurons are activated during the middle blocks of the retraction phase in all three feeding behaviors. More specifically, B6 and B9 fire in both the second and third blocks of retraction, whereas B3 only fires in the third block. The duration and force of retraction are both primarily determined by the second and third blocks containing B3, B6 and B9. In addition, we found that the frequency range (10-15 Hz) of B3 firing in vivo is not high enough to generate large forces. However, the activation of B6 and B9 prior to B3 can enhance the I1/I3 muscle contraction evoked by B3, consistent with the modulatory effects from B9 to B3 (Keating and Lloyd, 1999), which could be caused by the small cardioactive peptide (SCP) released by B6 and B9 (Church and Lloyd, 1991; Weiss et al, 1992). Moreover, we found that repetitive activation of a single motor neuron, B3, B6, or B9, can also increase its own forces. Therefore, the sequential recruitment of B6 and B9, prior to B3, enhances muscle contraction through modulatory interactions both within single responses and from response to response. Acknowledgements This research was funded by NIH Grant NS047073 and NSF Grant DMS1010434.

Neural control of slow muscle mechanics during feeding in Aplysia californica

Event Abstract Back to Event Neural control of slow muscle mechanics during feeding in Aplysia californica Gregory P. Sutton1, Jeffrey M. McManus2, Miranda J. Cullins3 and Hillel Chiel2, 3, 4 1 University of Bristol, Biology, United Kingdom 2 Case Western Reserve University, Biology, United States 3 Case Western Reserve University, Biomedical Engineering, United States 4 Case Western Reserve University, Neurosciences, United States Variability in the neural firing patterns that generate behaviour pose a difficult question: “How do changes in neural patterns reflect changes in output behaviours?” Often this question is addressed using purely statistical methods. We propose to address this question by applying a biomechanical approach, which can then direct subsequent statistical analyses. We demonstrate this principle using the feeding apparatus of the marine mollusc Aplysia californica. Aplysia’s feeding apparatus, the buccal mass, uses a muscular grasper, the odontophore, to move seaweed past the jaws and into the esophagus. As it does so, the buccal mass moves at a maximum velocity of 1 cm/s – so slowly that the inertial and damping forces are three orders of magnitude smaller than passive elastic and active muscle forces. System analysis can therefore be simplified by ignoring inertial and damping forces. The system is dominated by the position dependent elastic forces and the active forces generated by the muscles. Most importantly, motion of the odontophore is dominated by active forces in two muscles: protractor muscle I2 and jaw muscle I1/I3. Activation of the motor neurons of these two muscles generate forces whose balance moves the odontophore through a series of positions. We designate the predicted outcome of a pattern of motor neuron activity and the mechanical forces a neuromechanical equilibrium point. Analysis of neuromechanical equilibrium points predicts that the amount of motion of the odontophore towards the jaws will be correlated with the duration of I2 activity and with the amount of overlap between the I2 and I1/I3 activity. By measuring the neural activation times and odontophore motion from 37 feeding behaviours in a semi-intact preparation, we found that the correlations predicted by the biomechanical analysis were validated. Analyzing control of the buccal mass in terms of manipulation of neuromechanical equilibrium points provides concrete and testable predictions that, in this case, are verified by analysis of experimental data. Control by manipulation of equilibrium points is a direct consequence of any system in which inertial forces and viscous forces are orders of magnitude smaller than the elastic forces, and thus represents a framework for how any slow moving system could be controlled. Keywords: Aplysia, Biomechanics, Neural control Conference: Tenth International Congress of Neuroethology, College Park. Maryland USA, United States, 5 Aug - 10 Aug, 2012. Presentation Type: Poster (but consider for Participant Symposium) Topic: Motor Systems Citation: Sutton GP, McManus JM, Cullins MJ and Chiel H (2012). Neural control of slow muscle mechanics during feeding in Aplysia californica. Conference Abstract: Tenth International Congress of Neuroethology. doi: 10.3389/conf.fnbeh.2012.27.00245 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Apr 2012; Published Online: 07 Jul 2012. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Gregory P Sutton Jeffrey M McManus Miranda J Cullins Hillel Chiel Google Gregory P Sutton Jeffrey M McManus Miranda J Cullins Hillel Chiel Google Scholar Gregory P Sutton Jeffrey M McManus Miranda J Cullins Hillel Chiel PubMed Gregory P Sutton Jeffrey M McManus Miranda J Cullins Hillel Chiel Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Critical role of the circadian clock in memory formation: lessons from Aplysia

Unraveling the complexities of learning and the formation of memory requires identification of the cellular and molecular processes through which neural plasticity arises as well as recognition of the conditions or factors through which those processes are modulated. With its relatively simple nervous system, the marine mollusk Aplysia californica has proven an outstanding model system for studies of memory formation and identification of the molecular mechanisms underlying learned behaviors, including classical and operant associative learning paradigms and non-associative behaviors. In vivo behavioral studies in Aplysia have significantly furthered our understanding of how the endogenous circadian clock modulates memory formation. Sensitization of the tail-siphon withdrawal reflex represents a defensive non-associative learned behavior for which the circadian clock strongly modulates intermediate and long-term memory formation. Likewise, Aplysia exhibit circadian rhythms in long-term memory, but not short-term memory, for an operant associative learning paradigm. This review focuses on circadian modulation of intermediate and long-term memory and the putative mechanisms through which this modulation occurs. Additionally, potential functions and the adaptive advantages of time of day pressure on memory formation are considered. The influence of the circadian clock on learning and memory crosses distant phylogeny highlighting the evolutionary importance of the circadian clock on metabolic, physiological, and behavioral processes. Thus, studies in a simple invertebrate model system have and will continue to provide critical mechanistic insights to complementary processes in higher organisms.

Developmental transcriptome of Aplysia californica.

Genome-wide transcriptional changes in development provide important insight into mechanisms underlying growth, differentiation, and patterning. However, such large-scale developmental studies have been limited to a few representatives of Ecdysozoans and Chordates. Here, we characterize transcriptomes of embryonic, larval, and metamorphic development in the marine mollusc Aplysia californica and reveal novel molecular components associated with life history transitions. Specifically, we identify more than 20 signal peptides, putative hormones, and transcription factors in association with early development and metamorphic stages-many of which seem to be evolutionarily conserved elements of signal transduction pathways. We also characterize genes related to biomineralization-a critical process of molluscan development. In summary, our experiment provides the first large-scale survey of gene expression in mollusc development, and complements previous studies on the regulatory mechanisms underlying body plan patterning and the formation of larval and juvenile structures. This study serves as a resource for further functional annotation of transcripts and genes in Aplysia, specifically and molluscs in general. A comparison of the Aplysia developmental transcriptome with similar studies in the zebra fish Danio rerio, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, and other studies on molluscs suggests an overall highly divergent pattern of gene regulatory mechanisms that are likely a consequence of the different developmental modes of these organisms.

Synthesis, accumulation, and release of d-aspartate in the Aplysia californica CNS.

d-Aspartate (d-Asp) is an endogenous molecule that is often detected in CNS and endocrine tissues. Using capillary electrophoresis and a variety of radionuclide detection techniques, we examine the synthesis, release, and uptake/accumulation of d-Asp in the CNS of the marine mollusk Aplysia californica. We observe the preferential synthesis and accumulation of d-Asp over l-aspartate (l-Asp) in neuron-containing ganglia compared to surrounding sheath tissues. Little conversion of d-Asp to l-Asp is detected. The Ca(2+) ionophore ionomycin and elevated extracellular potassium stimulates release of d-Asp from the cerebral ganglia. Lastly, radioactive d-Asp in the extracellular media is efficiently taken up and accumulated by individual F-cluster neurons. These observations point to a role for d-Asp in cell-to-cell signaling with many characteristics similar to classical transmitters.

Differential Evolutionary Rates of Neuronal Transcriptome in Aplysia kurodai and Aplysia californica as a Tool for Gene Mining

:The marine mollusk Aplysia is a fascinating model organism for studying molecular mechanisms underlying learning and memory. However, evolutionary studies about Aplysia have been limited by the lack of its genomic information. Recently, large-scale expressed sequence tag (EST) databases have been acquired by sequencing cDNA libraries from A. californica and A. kurodai. The closeness between the two species allowed us to investigate rapidly evolving genes by comparing their orthologs. Using this method, we found that a subset of signal transduction genes in neurons showed rates of protein evolution higher than those of housekeeping genes. Moreover, we were also able to find several candidate genes that may be involved in learning and memory or synaptic plasticity among genes showing relatively higher Ka/Ks ratios. We also investigated the relationship between evolutionary rates and tissue distribution of Aplysia genes. They propose that the estimation of evolutionary rates cannot be a good marker to assess neuronal expression; however, it still can be an efficient way to narrow down the pool of candidate genes involved in neuronal functions for the further studies.

PolyADP‐ribosylation is required for long‐term memory formation in mammals

PolyADP-ribosylation is a post-translational modification of nuclear proteins, catalyzed by polyADP-ribose polymerases (PARPs). In the nucleus, polyADP-ribosylation catalyzed by PARP-1 alters protein-protein and protein-DNA interactions, and is implicated in chromatin remodeling, DNA transcription, and repair. Previous results linked the activation of PARP-1 with long-term memory formation during learning in the marine mollusk Aplysia ( Science 2004, 304:1820-1822). Furthermore, PARP-1 was highly activated in mammalian cerebral neurons treated with neurotrophins and neurotrophic peptides promoting neurite outgrowth and synaptic plasticity. Here, we examine the possibility that PARP-1 activation is required for memory formation during learning in mammals. Mice were tested in two learning paradigms, object recognition and fear conditioning. PolyADP-ribosylation of PARP-1 and histone H1 were detected in their cerebral cortex and hippocampus immediately after their training session. Moreover, in both behavioral paradigms, suppression of PARP activity in the CNS during learning impaired their long-term memory formation, without damaging their short-term memory. These findings implicate PARP-1 activation in molecular processes underlying long-term memory formation during learning.

Molecular identification of candidate chemoreceptor genes and signal transduction components in the sensory epithelium ofAplysia

An ability to sense and respond to environmental cues is essential to the survival of most marine animals. How water-borne chemical cues are detected at the molecular level and processed by molluscs is currently unknown. In this study, we cloned two genes from the marine mollusk Aplysia dactylomela which encode multi-transmembrane proteins. We have performed in situ hybridization that reveals expression and spatial distribution within the long-distance chemosensory organs, the rhinophores. This finding suggests that they could be receptors involved in binding water-borne chemicals and coupling to an intracellular signal pathway. In support of this, we found expression of a phospholipase C and an inositol trisphosphate receptor in the rhinophore sensory epithelia and possibly distributed within outer dendrites of olfactory sensory neurons. In Aplysia, mate attraction and subsequent reproduction is initiated by responding to a cocktail of water-borne protein pheromones released by animal conspecifics. We show that the rhinophore contraction in response to pheromone stimulants is significantly altered following phospholipase C inhibition. Overall, these data provide insight into the molecular components of chemosensory detection in a mollusk. An important next step will be the elucidation of how these coordinate the detection of chemical cues present in the marine environment and activation of sensory neurons.

Preparation of <em>Aplysia</em> Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of <em>Aplysia</em> Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy. An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level. In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Transcriptome analysis and identification of regulators for long-term plasticity inAplysia kurodai

The marine mollusk Aplysia is a useful model organism for studying the cellular bases of behavior and plasticity. However, molecular studies of Aplysia have been limited by the lack of genomic information. Recently, a large scale characterization of neuronal transcripts was performed in A. californica. Here, we report the analysis of a parallel set of neuronal transcripts from a closely related species A. kurodai found in the northwestern Pacific. We collected 4,859 nonredundant sequences from the nervous system tissue of A. kurodai. By performing microarray and real-time PCR analyses, we found that ApC/EBP, matrilin, antistasin, and eIF3e clones were significantly up-regulated and a BAT1 homologous clone was significantly down-regulated by 5-HT treatment. Among these, we further demonstrated that the Ap-eIF3e plays a key role in 5-HT-induced long-term facilitation (LTF) as a positive regulator.

Selective extracellular stimulation of individual neurons in ganglia

Selective control of individual neurons could clarify neural functions and aid disease treatments. To target specific neurons, it may be useful to focus on ganglionic neuron clusters, which are found in the peripheral nervous system in vertebrates. Because neuron cell bodies are found primarily near the surface of invertebrate ganglia, and often found near the surface of vertebrate ganglia, we developed a technique for controlling individual neurons extracellularly using the buccal ganglia of the marine mollusc Aplysia californica as a model system. We experimentally demonstrated that anodic currents can selectively activate an individual neuron and cathodic currents can selectively inhibit an individual neuron using this technique. To define spatial specificity, we studied the minimum currents required for stimulation, and to define temporal specificity, we controlled firing frequencies up to 45 Hz. To understand the mechanisms of spatial and temporal specificity, we created models using the NEURON software package. To broadly predict the spatial specificity of arbitrary neurons in any ganglion sharing similar geometry, we created a steady-state analytical model. A NEURON model based on cat spinal motor neurons showed responses to extracellular stimulation qualitatively similar to those of the Aplysia NEURON model, suggesting that this technique could be widely applicable to vertebrate and human peripheral ganglia having similar geometry.

Repetitive Firing Triggers Clustering of Kv2.1 Potassium Channels in Aplysia Neurons

The Kv2.1 gene encodes a highly conserved delayed rectifier potassium channel that is widely expressed in neurons of the central nervous system. In the bag cell neurons of Aplysia, Kv2.1 channels contribute to the repolarization of action potentials during a prolonged afterdischarge that triggers a series of reproductive behaviors. Partial inactivation of Aplysia Kv2.1 during repetitive firing produces frequency-dependent broadening of action potentials during the afterdischarge. We have now found that, as in mammalian neurons, Kv2.1 channels in bag cell neurons are localized to ring-like clusters in the plasma membrane of the soma and proximal dendrites. Either elevation of cyclic AMP levels or direct electrical stimulation of afterdischarge rapidly enhanced formation of these clusters on the somata of these neurons. In contrast, injection of a 13-amino acid peptide corresponding to a region in the C terminus that is required for clustering of Kv2.1 channels produced disassociation of the clusters, resulting in a more uniform distribution over the somata. Voltage clamp recordings demonstrated that peptide-induced dissociation of the Kv2.1 clusters is associated with an increase in the amplitude of delayed rectifier current and a shift of activation toward more negative potentials. In current clamp recording, injection of the unclustering peptide reduced the width of action potentials and reduced frequency-dependent broadening of action potentials. Our results suggest that rapid redistribution of Kv2.1 channels occurs during physiological changes in neuronal excitability.

2/4-Substituted-9-fluorenones and their O-glucosides as potential immunomodulators and anti-herpes simplex virus-2 agents. Part 5

In pursuing a research on the antiviral and immunomodulatory activity of tilorone congeners, two new series of compounds were prepared and pharmacologically explored: 9-fluorenone carboxyhydroxyesters, indicated as AG, and 9-fluorenone carboxyhydroxamides, indicated as MG. Two of them, AG17 and MG3, were used as sugar acceptors in the transglycosylation reactions performed by α- and β-glucosidases extracted from the marine mollusc Aplysia fasciata providing different α- and β-, mono- and oligosaccharides. Then aglycons and saccharides were assayed for cytotoxicity, for anti-herpes virus-2 properties on peripheral blood mononuclear cells (PBMC) and for their capability to trigger human cells to produce antiviral cytokines such as IFNα and TNFα. Some promising compounds were individuated whereas the utility of the biocatalytic procedures in the preparation of pure anomeric material was further focused.

Aplysia temptin − the ‘glue’ in the water-borne attractin pheromone complex

Temptin, a component of the complex of water-borne protein pheromones that stimulate attraction and mating behavior in the marine mollusk Aplysia, has sequence homology to the epidermal growth factor (EGF)-like domains of higher organisms that mediate protein-cell surface contact during fertilization and blood coagulation. In this work, recombinant temptin for structural and functional studies was produced in Escherichia coli using a cold shock promoter and purified by RP-HPLC. CD spectra confirmed a predominantly beta-sheet structure. Two disulfide bonds were determined via limited proteolysis and MS. One internal disulfide (Cys57-Cys77) was predicted from initial alignments with class I EGF-like domains; the second, between Cys18 and Cys103, could protect temptin against proteolysis in seawater and stabilize its interacting surface. A three-dimensional model of temptin was prepared with our MPACK suite, based on the Ca(2+)-binding, EGF-like domain of the extracellular matrix protein fibrillin. Two temptin residues, Trp52 and Trp79, which align with cysteine residues conserved in fibrillins, lie adjacent to and could stabilize the disulfide bonds and a proposed metal-binding loop. The water-borne pheromone attractin in egg cordon eluates is complexed with other proteins. Docking results with our model and the NMR structure of attractin suggest that one face of temptin interacts with the pheromone, perhaps controlling its access to the cellular receptors. Gel shifts confirmed that temptin complexes with wild-type attractin. These results indicate that temptin, analogous to the role of fibrillin in controlling transforming growth factor-beta concentration, modulates pheromone signaling by direct binding to attractin.

Gene identification and evidence for expression of G protein α subunits, phospholipase C, and an inositol 1,4,5-trisphosphate receptor in Aplysia californica rhinophore

In the marine mollusk Aplysia californica, waterborne protein pheromones that are released during egg laying act in concert to stimulate mate attraction. However, molecular information concerning the cellular receptors and signaling mechanisms that may be involved in waterborne peptide and protein pheromonal communication is lacking. As a first step toward examining whether members of the G protein family and phosphoinositide signaling pathway are present in the primary peripheral chemosensory organs (i.e., rhinophores), we isolated five full-length cDNA clones from an A. californica central nervous system cDNA library. These clones encoded (1) the G protein α subunits of the G q, G i, and G o families, (2) a protein with homology to phospholipase C (PLC) isoforms, and (3) an inositol 1,4,5-trisphosphate receptor (IP 3R). The expression of these genes was examined using laser capture microdissection/reverse transcription-polymerase chain reaction and in situ hybridization. All of them are expressed in the rhinophore sensory epithelium, suggesting that Gα q, Gα i, Gα o, PLC-like protein, and IP 3R may be involved in waterborne protein pheromone detection in Aplysia—possibly via a phosphoinositide signaling mechanism.

The kinematics of multifunctionality: comparisons of biting and swallowing inAplysia californica

What are the mechanisms of multifunctionality, i.e. the use of the same peripheral structures for multiple behaviors? We studied this question using the multifunctional feeding apparatus of the marine mollusk Aplysia californica, in which the same muscles mediate biting (an attempt to grasp food) and swallowing (ingestion of food). Biting and swallowing responses were compared using magnetic resonance imaging of intact, behaving animals and a three-dimensional kinematic model. Biting is associated with larger amplitude protractions of the grasper (radula/odontophore) than swallowing, and smaller retractions. Larger biting protractions than in swallowing appear to be due to a more anterior position of the grasper as the behavior begins, a larger amplitude contraction of protractor muscle I2, and contraction of the posterior portion of the I1/I3/jaw complex. The posterior I1/I3/jaw complex may be context-dependent, i.e. its mechanical context changes the direction of the force it exerts. Thus, the posterior of I1/I3 may aid protraction near the peak of biting, whereas the entire I1/I3/jaw complex acts as a retractor during swallowing. In addition, larger amplitude closure of the grasper during swallowing allows an animal to exert more force as it ingests food. These results demonstrate that differential deployment of the periphery can mediate multifunctionality.