Aplysia Nervous System Research Articles

Host response to Aplysia Abyssovirus 1 in nervous system and gill

The California sea hare (Aplysia californica) is a model for age associated cognitive decline. Recent researched identified a novel nidovirus, Aplysia Abyssovirus 1, with broad tropism enriched in the Aplysia nervous system. This virus is ubiquitous in wild and maricultured, young and old animals without obvious pathology. Here we re-evaluated gene expression data from several previous studies to investigate differential expression in the nervous system and gill in response to virus and aging as well as the mutational spectrum observed in the viral sequences obtained from these datasets. Viral load and age were highly correlated, indicating persistent infection. Upregulated genes in response to virus were enriched for immune genes and signatures of ER and proteostatic stress, while downregulated genes were enriched for mitochondrial metabolism. Differential expression with respect to age suggested increased iron accumulation and decreased glycolysis, fatty acid metabolism, and proteasome function. Interaction of gene expression trends associated with viral infection and aging suggest that viral infection likely plays a role in aging in the Aplysia nervous system. Mutation analysis of viral RNA identified signatures suggesting ADAR and AID/APOBEC like deaminase act as part of Aplysia anti-viral defense.

Distribution, cellular localization, and colocalization of several peptide neurotransmitters in the central nervous system of Aplysia.

Neuropeptides are widely used as neurotransmitters in vertebrates and invertebrates. In vertebrates, a detailed understanding of their functions as transmitters has been hampered by the complexity of the nervous system. The marine mollusk Aplysia, with a simpler nervous system and many large, identified neurons, presents several advantages for addressing this question and has been used to examine the roles of tens of peptides in behavior. To screen for other peptides that might also play roles in behavior, we observed immunoreactivity in individual neurons in the central nervous system of adult Aplysia with antisera raised against the Aplysia peptide FMRFamide and two mammalian peptides that are also found in Aplysia, cholecystokinin (CCK) and neuropeptide Y (NPY), as well as serotonin (5HT). In addition, we observed staining of individual neurons with antisera raised against mammalian somatostatin (SOM) and peptide histidine isoleucine (PHI). However, genomic analysis has shown that these two peptides are not expressed in the Aplysia nervous system, and we have therefore labeled the unknown peptides stained by these two antibodies as XSOM and XPHI There was an area at the anterior end of the cerebral ganglion that had staining by antisera raised against many different transmitters, suggesting that this may be a modulatory region of the nervous system. There was also staining for XSOM and, in some cases, FMRFamide in the bag cell cluster of the abdominal ganglion. In addition, these and other studies have revealed a fairly high degree of colocalization of different neuropeptides in individual neurons, suggesting that the peptides do not just act independently but can also interact in different combinations to produce complex functions. The simple nervous system of Aplysia is advantageous for further testing these ideas.

Phylogenetic analysis of ionotropic L-glutamate receptor genes in the Bilateria, with special notes on Aplysia californica

BackgroundThe neurotransmitter L-Glutamate (L-Glu) acting at ionotropic L-Glu receptors (iGluR) conveys fast excitatory signal transmission in the nervous systems of all animals. iGluR-dependent neurotransmission is a key component of the synaptic plasticity that underlies learning and memory. During learning, two subtypes of iGluR, α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) and N-methyl-D-aspartate receptors (NMDAR), are dynamically regulated postsynaptically in vertebrates. Invertebrate organisms such as Aplysia californica (Aplysia) are well-studied models for iGluR-mediated function, yet no studies to date have analyzed the evolutionary relationships between iGluR genes in these species and those in vertebrates, to identify genes that may mediate plasticity. We conducted a thorough phylogenetic analysis spanning Bilateria to elucidate these relationships. The expression status of iGluR genes in the Aplysia nervous system was also examined.ResultsOur analysis shows that ancestral genes for both NMDAR and AMPAR subtypes were present in the common bilaterian ancestor. NMDAR genes show very high conservation in motifs responsible for forming the conductance pore of the ion channel. The number of NMDAR subunits is greater in vertebrates due to an increased number of splice variants and an increased number of genes, likely due to gene duplication events. AMPAR subunits form an orthologous group, and there is high variability in the number of AMPAR genes in each species due to extensive taxon specific gene gain and loss. qPCR results show that all 12 Aplysia iGluR subunits are expressed in all nervous system ganglia.ConclusionsOrthologous NMDAR subunits in all species studied suggests conserved function across Bilateria, and potentially a conserved mechanism of neuroplasticity and learning. Vertebrates display an increased number of NMDAR genes and splice variants, which may play a role in their greater diversity of physiological responses. Extensive gene gain and loss of AMPAR genes may result in different physiological properties that are taxon specific. Our results suggest a significant role for L-Glu mediated responses throughout the Aplysia nervous system, consistent with L-Glu’s role as the primary excitatory neurotransmitter.

<em>Aplysia</em> Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Development of an in vitro analog concomitantly expressing the cellular correlates of sensitization and suppression of feeding in Aplysia californica

When presented with aversive stimuli, the marine mollusk Aplysia californica shows increased defensive responses (sensitization) and suppression of feeding. The cellular correlates of sensitization and suppression of feeding include increased excitability of the tail sensory neurons (TSNs, which mediate defensive responses) and decreased excitability in neuron B51 (a cell critical to the Aplysia feeding circuit), respectively. The goal of this project was to develop a reduced preparation of the isolated Aplysia nervous system (buccal, pleural‐pedal, and cerebral ganglia) in which the effects of aversive stimuli within the defense and feeding neural circuits could be mimicked in vitro by electrical stimulation of afferent nerves. We successfully observed significant concomitant increase in excitability of the TSNs (measured by spikes evoked) and decrease in excitability of B51 (increase in the burst threshold) 15 min post stimulation. Currently, we are examining whether this in vitro analog expresses long term (24 h) correlates. This in vitro analog will facilitate further analyses of cellular and molecular modifications resulting from aversive stimulation, as it would allow manipulations not feasible in vivo. Supported by NSF Grant IOS 1120304 to RM.

Physiological evidence that d-aspartate activates a current distinct from ionotropic glutamate receptor currents in Aplysia californica neurons

D-Aspartate (D-Asp) activates an excitatory current in neurons of Aplysia californica. Although D-Asp is presumed to activate a subset of L-glutamate (L-Glu) channels, the identities of putative d-Asp receptors and channels are unclear. Whole cell voltage- and current-clamp studies using primary cultures of Aplysia buccal S cluster (BSC) neurons were executed to characterize D-Asp-activated ion channels. Both D-Asp and L-Glu evoked currents with similar current-voltage relationships, amplitudes, and relatively slow time courses of activation and inactivation when agonists were pressure applied. D-Asp-induced currents, however, were faster and desensitized longer, requiring 40 s to return to full amplitude. Of cells exposed to both agonists, 25% had D-Asp- but not L-Glu-induced currents, suggesting a receptor for D-Asp that was independent of l-Glu receptors. D-Asp channels were permeable to Na(+) and K(+), but not Ca²⁺, and were vulnerable to voltage-dependent Mg²⁺ block similarly to vertebrate NMDA receptor (NMDAR) channels. d-Asp may activate both NMDARs and non-l-Glu receptors in the nervous system of Aplysia.

Protein Kinase M Maintains Long-Term Sensitization and Long-Term Facilitation inAplysia

How the brain maintains long-term memories is one of the major outstanding questions in modern neuroscience. Evidence from mammalian studies indicates that activity of a protein kinase C (PKC) isoform, protein kinase Mζ (PKMζ), plays a critical role in the maintenance of long-term memory. But the range of memories whose persistence depends on PKMζ, and the mechanisms that underlie the effect of PKMζ on long-term memory, remain obscure. Recently, a PKM isoform, known as PKM Apl III, was cloned from the nervous system of Aplysia. Here, we tested whether PKM Apl III plays a critical role in long-term memory maintenance in Aplysia. Intrahemocoel injections of the pseudosubstrate inhibitory peptide ZIP (ζ inhibitory peptide) or the PKC inhibitor chelerythrine erased the memory for long-term sensitization (LTS) of the siphon-withdrawal reflex (SWR) as late as 7 d after training. In addition, both PKM inhibitors disrupted the maintenance of long-term (≥ 24 h) facilitation (LTF) of the sensorimotor synapse, a form of synaptic plasticity previously shown to mediate LTS of the SWR. Together with previous results (Bougie et al., 2009), our results support the idea that long-term memory in Aplysia is maintained via a positive-feedback loop involving PKM Apl III-dependent protein phosphorylation. The present data extend the known role of PKM in memory maintenance to a simple and well studied type of long-term learning. Furthermore, the demonstration that PKM activity underlies the persistence of LTF of the Aplysia sensorimotor synapse, a form of synaptic plasticity amenable to rigorous cellular and molecular analyses, should facilitate efforts to understand how PKM activity maintains memory.

Unique ionotropic receptors for D-aspartate are a target for serotonin-induced synaptic plasticity in Aplysia californica

The non- l-glutamate ( l-Glu) receptor component of d-aspartate ( d-Asp) currents in Aplysia californica buccal S cluster (BSC) neurons was studied with whole cell voltage clamp to differentiate it from receptors activated by other well-known agonists of the Aplysia nervous system and investigate modulatory mechanisms of d-Asp currents associated with synaptic plasticity. Acetylcholine (ACh) and serotonin (5-HT) activated whole cell excitatory currents with similar current voltage relationships to d-Asp. These currents, however, were pharmacologically distinct from d-Asp. ACh currents were blocked by hexamethonium (C6) and tubocurarine (d-TC), while d-Asp currents were unaffected. 5-HT currents were blocked by granisetron and methysergide (MES), while d-Asp currents were unaffected. Conversely, while (2S,3R)-1-(Phenanthren-2-carbonyl)piperazine-2,3-dicarboxylic acid(PPDA) blocked d-Asp currents, it had no effect on ACh or 5-HT currents. Comparison of the charge area described by currents induced by ACh or 5-HT separately from, or with, d-Asp suggests activation of distinct receptors by all 3 agonists. Charge area comparisons with l-Glu, however, suggested some overlap between l-Glu and d-Asp receptors. Ten minute exposure to 5-HT induced facilitation of d-Asp-evoked responses in BSC neurons. This effect was mimicked by phorbol ester, suggesting that protein kinase C (PKC) was involved.

Changes in d-aspartate ion currents in the Aplysia nervous system with aging

d-Aspartate (D-Asp) can substitute for l-glutamate (L-Glu) at excitatory Glu receptors, and occurs as free D-Asp in the mammalian brain. D-Asp electrophysiological responses were studied as a potential correlate of aging in the California sea hare, Aplysia californica. Whole cell voltage- and current clamp measurements were made from primary neuron cultures of the pleural ganglion (PVC) and buccal ganglion S cluster (BSC) in 3 egg cohorts at sexual maturity and senescence. D-Asp activated an inward current at the hyperpolarized voltage of − 70 mV, where molluscan NMDA receptors open free of constitutive block by Mg 2+. Half of the cells responded to both D-Asp and L-Glu while the remainder responded only to D-Asp or L-Glu, suggesting that D-Asp activated non-Glu channels in a subpopulation of these cells. The frequency of D-Asp-induced currents and their density were significantly decreased in senescent PVC cells but not in senescent BSC cells. These changes in sensory neurons of the tail predict functional deficits that may contribute to an overall decline in reflexive movement in aged Aplysia.

Transcriptome and protein domain analyses in Aplysia nervous system with evolutionary implications

The sea hare Aplysia is a powerful model organism for studying the structure and function of the nervous system. Recently, the genomic characterization of Aplysia has been facilitated: A large scale EST sequences was acquired by sequencing cDNA libraries from A. californica and a parallel EST database of the closely related species A. kurodai was reported. These EST databases provide useful tools for both molecular biology and bioinformatics. In our previous report, we demonstrated the utility of the database by screening the candidate genes for the synaptic plasticity and the behavioral sensitization using the microarray containing A. kurodai ESTs. In this Addendum, we have expanded our study to show that the protein domain repertoire and the abundance of regulatory genes displayed a linear relationship with the evolution of the complex brains in different lineages. This distinct set of protein domains may play critical roles in evolution of the nervous systems.

Habituation in Aplysia: The Cheshire Cat of neurobiology

The marine snail, Aplysia californica, is a valuable model system for cell biological studies of learning and memory. Aplysia exhibits a reflexive withdrawal of its gill and siphon in response to weak or moderate tactile stimulation of its skin. Repeated tactile stimulation causes this defensive withdrawal reflex to habituate. Both short-term habituation, lasting <30 min, and long-term habituation, which can last >24 h, have been reported in Aplysia. Habituation of the withdrawal reflex correlates with, and is in part due to, depression of transmission at the monosynaptic connection between mechanoreceptive sensory neurons and motor neurons within the abdominal ganglion. Habituation-related short-term depression of the sensorimotor synapse appears to be due exclusively to presynaptic changes. However, changes within the sensory neuron, by themselves, do not account for more persistent depression of the sensorimotor synapse. Recent behavioral work suggests that long-term habituation in Aplysia critically involves postsynaptic processes, specifically, activation of AMPA- and NMDA-type receptors. In addition, long-term habituation requires activity of protein phosphatases, including protein phosphatases 1, 2A, and 2B, as well as activity of voltage-dependent Ca 2+ channels. Cellular work has succeeded in demonstrating long-term, homosynaptic depression (LTD) of the sensorimotor synapse in dissociated cell culture and, more recently, LTD of the glutamate response of isolated motor neurons in culture (“hemisynaptic” LTD). These in vitro forms of LTD have mechanistic parallels to long-term habituation. In particular, homosynaptic LTD of the sensorimotor synapse requires elevated intracellular Ca 2+ within the motor neuron, and hemisynaptic LTD requires activity of AMPA- and NMDA-type receptors. In addition, activation of group I and II metabotropic glutamate receptors (mGluRs) can induce hemisynaptic LTD. The demonstration of LTD in vitro opens up a promising new avenue for attempts to relate long-term habituation to cellular changes within the nervous system of Aplysia.

Ancient rules of memory

The first kiss, the smell of mother's cooking or the vision of a beautiful landscape; memories such as these are etched firmly into our brains for recall in vivid detail, even decades later. Memories and experience influence our decisions throughout life, both consciously and subconsciously—in a sense, our memories make us what we are. Yet, understanding how information is encoded in our brains and how memories are maintained, both at the conceptual and molecular levels, remains one of the greatest challenges in the life sciences. Through decades of research, biologists have answered many fundamental questions about the brain, and more recent work has revealed the identity of several crucial molecules involved in the storage and retrieval of information. This has at last begun to shed light on the major scientific puzzle: how do we form and maintain long‐term memories? ![][1] The field of neurobiology had made relatively little progress toward understanding memory since the Canadian psychologist Donald Hebb (1904–1985) introduced the idea of synaptic plasticity nearly 60 years ago (Hebb, 1949). Hebb explained the establishment of memory by changes in the number and strength of synaptic connections between neurons. According to his theory, synapses respond to stimulation by neurotransmitters in a cumulative manner, and repeated stimulation—for example, by learning—leads to ever‐stronger connections between specific neurons until they become permanent. Over time, neuroscientists accepted Hebb's idea of synaptic plasticity, despite a lack of direct molecular evidence. In fact, it was not until recently that scientists—in large part Eric Kandel at Columbia University in New York, NY, USA—discovered several biochemical mechanisms that now support Hebb's idea. Kandel himself won the Nobel Prize for medicine in 2000 for his discovery that the syntheses of different proteins relate to short‐term and long‐term memory in the sea slug Aplysia . The foundations for this work and … [1]: /embed/graphic-1.gif

Aplysia Bag Cells Function as a Distributed Neurosecretory Network

The anatomical organization of many neuroendocrine systems implies multiple sites of hormone release in areas mediating multiple aspects of physiology and behavior, yet this neurosecretory complexity has not often been verified. Here we probe the well-characterized hormonal model, the reproductive bag cell neuroendocrine system of the sea slug Aplysia californica. The bag cell neurons of Aplysia mediate egg-laying behavior through the coordinated secretion of a suite of peptides derived from a single gene product, the egg-laying prohormone (proELH). Although the majority of bag cell neurons are located within two major abdominal bag cell clusters, smaller groups of egg-laying hormone-expressing cells have been observed in specific pleural and cerebral ganglia regions, some of which have been reported to be electrically connected to the abdominal bag cell clusters. Releasates are sampled from discrete locations within the Aplysia CNS before and during stimulation of afterdischarge activity and subsequently analyzed with matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Site-specific release profiles are observed at bag cell cluster, pleural, and genital ganglion sites after site-specific electrophysiological activation of bag cell afterdischarges. These data demonstrate that the bag cell network has multiple neurohemal release sites, exhibits descending activation that travels from the cerebral and pleural ganglia down to the abdominal bag cell clusters, and releases spatially distinct profiles of proELH-derived peptides within the Aplysia nervous system. Such distributed neurosecretory organization may be a common feature of neuroendocrine systems across higher order organisms linking multiple behavioral aspects to a single neuronal network.

Localization of putative nitrergic neurons in peripheral chemosensory areas and the central nervous system ofAplysia californica

The distribution of putative nitric oxide synthase (NOS)-containing cells in the opisthobranch mollusc Aplysia californica was studied by using NADPH-diaphorase (NADPH-d) histochemistry in the CNS and peripheral organs. Chemosensory areas (the mouth area, rhinophores, and tentacles) express the most intense staining, primarily in the form of peripheral highly packed neuropil regions with a glomerular appearance as well as in epithelial sensory-like cells. These epithelial NADPH-d-reactive cells were small and had multiple apical ciliated processes exposed to the environment. NADPH-d processes were also found in the salivary glands, but there was no or very little staining in the buccal mass and foot musculature. In the CNS, most NADPH-d reactivity was associated with the neuropil of the cerebral ganglia, with the highest density of glomeruli-like NADPH-d-reactive neurites in the areas of the termini and around F and C clusters. A few NADPH-d-reactive neurons were also found in other central ganglia, including paired neurons in the buccal, pedal, and pleural ganglia and a few asymmetrical neurons in the abdominal ganglion. The distribution patterns of NADPH-d-reactive neurons did not overlap with other known neurotransmitter systems. The highly selective NADPH-d labeling revealed here suggests the presence of NOS in sensory areas both in the CNS and the peripheral organs of Aplysia and implies a role for NO as a modulator of chemosensory processing.

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.

Differential regulation of proteasome activity in the nucleus and the synaptic terminals

Proteasome is a multi-subunit proteolytic complex that degrades proteins covalently linked to multiple molecules of ubiquitin. Earlier studies showed a role for the ubiquitin-proteasome pathway in several models of long-term memory and other forms of synaptic plasticity. In Aplysia, the ubiquitin-proteasome pathway has been shown to contribute to the induction of long-term facilitation. In other model systems, ubiquitin-proteasome-mediated proteolysis has also been shown to play a role in synapse development. Previous studies of synaptic plasticity focused on changes in components or the substrates of the ubiquitin-proteasome pathway in whole neurons. Modification of specific synapses would require precise spatial and temporal regulation of the components of the ubiquitin-proteasome pathway within the subcellular compartments of neurons during learning. As a first step towards testing the idea of local regulation of the ubiquitin-proteasome pathway in neurons, we investigated proteasome activity in nuclear and synaptosomal fractions. Here we show that proteasome activity in the synaptic terminals is higher compared to the activity in the nucleus in the Aplysia nervous system as well as in the mouse brain. Furthermore, the proteasome activity in the two neuronal compartments is differentially modulated by protein kinases. Differential regulation of proteasome activity in neuronal compartments such as the synaptic terminals is likely to be a key mechanism underlying synapse-specific plasticity.

Comparative localization of two serotonin receptors and sensorin in the central nervous system ofAplysia californica

Aplysia californica is a powerful model for understanding the cellular and molecular mechanisms underlying modulation of neuronal plasticity and learning. In the central nervous system of Aplysia, serotonin is associated with various behaviors. For example, it induces short-, intermediate-, and long-term synaptic changes in sensory neurons during learning and inhibits the afterdischarge of the bag cells that initiate egg-laying behavior. Little is known about the nature and contribution of serotonin receptors involved in the numerous serotonin-mediated physiological responses in Aplysia. Recently, two G(i)-coupled serotonin receptors (5-HT(ap1) and 5-HT(ap2)) were cloned. We now report that, by using in situ hybridization to express the profile of these receptors, we are able to gain critical insight into their roles in the behavior of Aplysia. We compared their distribution to that of sensorin-A, a peptide specifically found in sensory neurons. We wished to determine their involvement in some simple forms of behavioral modifications. 5-HT(ap1) and 5-HT(ap2) mRNAs are expressed in all ganglia of the Aplysia central nervous system. Stronger signal was observed with the 5-HT(ap2) antisense probe than with the 5-HT(ap1) antisense probe. Notably, mRNA coding for the receptors was found in several identified neurons, in the bag cells, in characterized serotonergic neurons, and in neurons of the mechanosensory clusters that expressed sensorin. We also observed heterogeneity of receptor expression between R2 and LPl1 and among neurons of a single cluster of sensory neurons. These results suggest that 5-HT(ap1) and 5-HT(ap2) receptors may regulate the response to serotonin and/or its release in several neurons.

PolyADP‐ribose polymerase‐1 (PARP‐1) and the evolution of learning and memory

PARP-1 is a multifunctional enzyme that can modulate gene expression. Cohen-Armon et al.(1) found that a homologue of PARP-1 is activated in the Aplysia nervous system as the animal responds to an aversive stimulus, which leads to sensitization, and during a more complex form of learning that involves feeding behavior. Significantly, inhibiting PARP-1 activation blocked the learning. Several key pathways in Aplysia neurons are activated both during learning and after injury, suggesting that mechanisms of learning evolved from primitive responses to injury. Since PARP-1 is evolutionarily conserved as a responder to various forms of stress, the finding that PARP-1 is activated during learning supports this idea.

PRQFVamide, a novel pentapeptide identified from the CNS and gut of Aplysia.

We have purified a novel pentapeptide from the Aplysia nervous system using bioassay on gut contractions. The structure of the peptide is Pro-Arg-Gln-Phe-Val-amide (PRQFVa). The precursor for PRQFVa was found to code for 33 copies of PRQFVamide and four related pentapeptides. Peaks corresponding to the predicted masses of all five pentapeptides were detected in Aplysia neurons by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Northern analysis revealed that expression of the precursor is abundant in the abdominal ganglion, much less in the pedal and cerebral ganglia, and rarely seen in the buccal and pleural ganglia. PRQFVa-positive neurons, mapped by immunohistochemistry and in situ hybridization, were present in all the central ganglia. PRQFVa immunopositive processes were observed in the gut, particularly in association with the vasculature. Some arteries and other highly vascularized tissues, such as the gill and the kidney, also contain numerous PRQFVa immunopositive processes. Application of synthetic PRQFVa suppresses not only contractions of the gut but also contractions of vasculature. PRQFVa is expressed in some of the neurons within the feeding circuitry and application of synthetic PRQFVa was found to decrease the excitability of some (B4/5 and B31/32) but not all (B8) neurons of the buccal feeding circuit. Our findings suggest that PRQFVa may act as a modulator within the feeding system as well as in other systems of Aplysia.

Serotonin persistently activates the extracellular signal-related kinase in sensory neurons of Aplysia independently of cAMP or protein kinase C

Activation of the extracellular signal-related kinase is important for long-term increases in synaptic strength in the Aplysia nervous system. However, there is little known about the mechanism for the activation of the kinase in this system. We examined the activation of Aplysia extracellular signal-related kinase using a phosphopeptide antibody specific to the sites required for activation of the kinase. We found that phorbol esters led to a prolonged activation of extracellular signal-related kinase in sensory cells of the Aplysia nervous system. Surprisingly, inhibitors of protein kinase C did not block this activation. Serotonin, the physiological transmitter involved in long-term synaptic facilitation, also led to prolonged activation of extracellular signal-related kinase, but inhibitors of protein kinase A or protein kinase C did not block this activation. We examined whether the protein synthesis-dependent increase in excitability stimulated by phorbol esters was dependent on phorbol ester activation of extracellular signal-related kinase, but increases in excitability were still seen in the presence of inhibitors of extracellular signal-related kinase activation. Our results suggest that prolonged phosphorylation of extracellular signal-related kinase in the Aplysia system is not mediated by either of the classic second messenger activated kinases in this system, protein kinase A or protein kinase C and that extracellular signal-related kinase is not important for phorbol ester induced long-term effects on excitability.