ADENYLATE CYCLASE IN HELIX AND APLYSIA GANGLIA: CHARACTERISTICS OF ITS STIMULATION BY A PEPTIDE-CONTAINING NERVOUS SYSTEM EXTRACT

Abstract— A crude particulate fraction, prepared from the central ganglia of Helix or Aplysia , contains levels of adenylate cyclase activity comparable to those in mammalian brain. This activity can be stimulated up to 50-fold by NaF, and 4- to 10-fold by guanyl nucleotides such as GTP and guanylylimidodiphosphate (Gpp(NH)p). A peptide-containing extract from Helix or Aplysia nervous system also stimulates the adenylate cyclase, by 50-400°. In contrast, a number of peptides known to occur in vertebrate and invertebrate nervous system are without effect. The adenylate cylase stimulation by the endogenous molluscan peptide-containing extract may be receptor-mediated, but the effect is not enhanced in the presence of guanyl nucleotides: in this respect it differs from many other hormone-sensitive adenylate cyclases. The endogenous extracts prepared from Helix and Aplysia each stimulate both Helix and Aplysia adenylate cyclases, suggesting that the putative cyclase-linked receptors may be similar in the two species. Furthermore, the active components in the extracts from Helis and Aplysia appear to be similar, since preliminary evidence suggests that they may interact with the same adenylate cyclase-linked receptor in particulate fraction from Helix ganglia.     

Molecular approaches to the study of long-term sensitization in Aplysia californica

In order to gain insight into the molecular mechanisms underlying the establishment and maintenance of long-term sensitization in Aplysia new tools are being used to study the synaptic facilitation in the sensory-motor connection which mediates the gill-and-siphon withdrawal reflex. The supposition that long-term changes in neuronal properties share molecular pathways with other cells during development and differentiation points towards specific candidate genes as well as towards a general experimental strategy designed to find proteins which might mediate these changes. The unique properties of Aplysia cell biology may, in addition, provide a means to examine their specific roles in the triggering of the changes, as well as the nature of the changes themselves.     

Newly identified water-borne protein pheromones interact with attractin to stimulate mate attraction in Aplysia

The water-borne protein attractin is a potent sex pheromone involved in forming and maintaining mating and egg-laying aggregations in the marine mollusk Aplysia. Binary blends of attractin and either enticin, temptin, or seductin, three other Aplysia protein pheromones, stimulate mate attraction. The four pheromones are thought to act in concert during egg-laying. The new data presented here show that: (1) the water-borne odor of non-laying Aplysia brasiliana further increases the attractiveness of attractin and of eggs in T-maze bioassays. This suggests that individual Aplysia release additional factors that enhance the effects of attractin, enticin, temptin, and seductin during egg-laying; (2) the N-terminal region of enticin aligns well with the conserved epidermal growth factor (EGF)-like domain of mammalian reproductive proteins known as fertilins, which may mediate intercellular adhesion interactions between eggs and sperm; (3) temptin, according to fold recognition servers, may also have an EGF-like fold. Enticin and temptin also have conserved metal binding sequences that may play a role in their signaling behavior. These results suggest that aspects of mammalian egg-sperm interactions (fertilins) may have evolved from pheromonal signaling mechanisms. We also review the structure, expression, localization, release, and behavioral actions of attractin, enticin, temptin, and seductin.     

Mnk is a negative regulator of cap-dependent translation in Aplysia neurons

To investigate the mechanisms underlying regulation of eukaryotic initiation factor 4E (eIF4E) phosphorylation in Aplysia neurons, we have cloned the Aplysia homolog of the vertebrate eIF4E kinases, Mnk1 and -2. Aplysia Mnk shares many conserved regions with vertebrate Mnk, including putative eukaryotic initiation factor 4G binding regions, activation loop phosphorylation sites, and a carboxy-terminal anchoring site for MAP kinases. As expected, purified Aplysia Mnk phosphorylated Aplysia eIF4E at a conserved carboxy-terminal serine and over-expression of Aplysia Mnk in sensory neurons led to increased phosphorylation of endogenous eIF4E. Over-expression of Aplysia Mnk led to strong decreases in cap-dependent translation, while generally sparing internal ribosomal entry site (IRES)-dependent translation. However, decreases in cap-dependent translation seen after expression of Aplysia Mnk could only be partly explained by increases in eIF4E phosphorylation. In Aplysia sensory neurons, phosphorylation of eIF4E is reduced during intermediate memory formation. However, we found that this physiological regulation of eIF4E phosphorylation was independent of changes in Aplysia Mnk phosphorylation. We propose that changes in eIF4E phosphorylation in Aplysia neurons are a consequence of changes in cap-dependent translation that are independent of regulation of Aplysia Mnk.     

In search of general mechanisms for long-lasting plasticity: Aplysia and the hippocampus

Long-term synaptic plasticity is thought to underlie many forms of long-lasting memory. Long-lasting plasticity has been most extensively studied in the marine snail Aplysia and in the mammalian hippocampus, where Bliss and Lømo first described long-term potentiation 30 years ago. The molecular mechanisms of plasticity in these two systems have proven to have many similarities. Here, we briefly describe some of these areas of overlap. We then summarize recent advances in our understanding of the mechanisms of long-lasting synaptic facilitation in Aplysia and suggest that these may prove fruitful areas for future investigation in the mammalian hippocampus and at other synapses in the mammalian brain.     

Structural changes and the storage of long-term memory in Aplysia

Long-term memory for sensitization of the gill-withdrawal reflex in Aplysia is associated with the growth of new synaptic connections between sensory and motor neurons. The duration of this structural change parallels the behavioral retention of the memory. Such changes can be reconstituted in dissociated cell culture by repeated presentations of the modulatory neurotransmitter serotonin (5HT) and are associated with an activity-dependent downregulation of NCAM-related cell adhesion molecules thought to contribute to cell recognition and axonal outgrowth during development. Thus, aspects of the mechanisms utilized for learning-related synaptic growth initiated by experience in the adult may eventually be understood in the context of the molecular logic that shapes synaptic circuitry during the later stages of neuronal development.     

An antibody to the Drosophila period protein recognizes circadian pacemaker neurons in Aplysia and Bulla

The molecular mechanisms of the pacemakers underlying circadian rhythms are not well understood. One molecule that presumably functions in the circadian clock of Drosophila is the product of the period (per) gene, which dramatically affects biological rhythms when mutated. An antibody specific for the per protein labels putative circadian pacemaker neurons and fibers in eyes of two marine gastropods, Aplysia and Bulla. As was found for the Drosophila per protein, there is a daily rhythm in the levels of the per-like antigen in Aplysia eyes. Thus, certain molecular features of the per protein, as well as aspects of the temporal regulation of its expression, may be conserved in circadian pacemakers of widely divergent species.     

The cellular mechanisms of learning in Aplysia: of blind men and elephants

Until recently, investigations of the neurobiological substrates of simple forms of learning and memory in the marine snail Aplysia have focused mostly on plastic changes that occur within the presynaptic sensory neurons. Here, I summarize the results of recent studies that indicate that exclusively presynaptic processes cannot account for simple forms of learning in Aplysia. In particular, I present evidence that postsynaptic mechanisms play a far more important role in nonassociative learning in Aplysia than has been appreciated before now. Moreover, I describe recent data that suggests the intriguing hypothesis that the persistent, learning-induced changes in Aplysia sensory neurons might depend critically on postsynaptic signals for their induction. Finally, I discuss the potential applicability of this hypothesis to learning-related synaptic plasticity in the mammalian brain.     

Long-term sensitization training in Aplysia leads to an increase in calreticulin, a major presynaptic calcium-binding protein.

Long-term memory for sensitization in Aplysia requires new protein and RNA synthesis. Here, we identify a late protein as calreticulin, the major Ca(2+)-binding protein of the lumen of the endoplasmic reticulum. An antiserum against Aplysia calreticulin reveals an enrichment of calreticulin immunoreactivity in presynaptic varicosities. Quantitative S1 nuclease analysis indicates that the steady-state level of calreticulin mRNA in Aplysia sensory neurons increases during the maintenance phase of long-term sensitization. The finding that this mRNA increases in expression late, some time after training, is consistent with the idea that long-term neuromodulatory changes underlying sensitization may depend on a cascade of gene expression in which the induction of early regulatory genes leads to the expression of late effector genes.     

A conserved heptapeptide sequence in the waterborne attractin pheromone stimulates mate attraction in Aplysia

Mate attraction in the marine mollusk Aplysia involves long-distance waterborne chemical signaling via the release of the peptide pheromone attractin during egg laying. Aplysia californica attractin attracts conspecifics, reduces the latency to mating, and stimulates hermaphroditic mating. Four additional members of the Aplysia attractin family have recently been characterized from Aplysia brasiliana, Aplysia fasciata, Aplysia depilans, and Aplysia vaccaria. The five sequences differ significantly, but share six cysteine residues and the strictly conserved sequence Ile30-Glu-Glu-Cys-Lys-Thr-Ser36. Attractin is attractive to geographically and evolutionarily distant species, suggesting that the conserved heptapeptide region may be important for mate attraction. Consistent with this prediction, a synthetic constrained cyclic peptide that contains the conserved heptapeptide sequence is significantly attractive in T-maze bioassays. The attractins are the first family of waterborne peptide pheromones characterized in invertebrates and are unique in that family members are not species-specific pheromonal attractants.     

Gene Expression and Regulation of Aplysia californica Prohormone Convertases aPC1B and aPC2

Abstract: Peptide diversity can be regulated in many ways. One of the possible mechanisms of such a regulation may lie in the differential processing of a precursor in a tissue-specific manner or in a modulated processing within a single cell. To address these questions the enzymes responsible for the release of active neuropeptides first need to be well characterized. In this study, we have established the cellular distributions of the recently cloned prohormone convertases aPC2 and aPC1B in Aplysia californica and found the distribution of these enzymes to be mainly neuronal and endocrine. We then investigated in two sets of neurons (bag cell neurons and sensory neurons) the possible regulation of these two prohormone convertases by a neurotransmitter known to modulate behavior in Aplysia . In these neurons, we found that only aPC2 is regulated by serotonin possibly via the cyclic AMP cascade. Finally, a study of the biosynthesis of the bag cell egg-laying hormone precursor after treatment with serotonin suggests that such a treatment may increase its processing.     

METABOLISM OF PUTATIVE TRANSMITTERS IN INDIVIDUAL NEURONS OF APLYSIA CALIFORNICA: AROMATIC AMINO ACID DECARBOXYLASE

Abstract— The activities of aromatic amino acid decarboxylase (EC 4.1.1.26) in various ganglia, nerve trunks, and individual identifiable neurons of Aplysia culifornica were measured. The distribution of the decarboxylase enzyme is ubiquitous throughout the central nervous system of the Aplysia . Every Aplysia neuron tested contained some decarboxylase activity. The presence of this particular synthetic enzyme in an Aplysia neuron, therefore, cannot be used to classify these neurons as 'aminergic'.     

The first gamma-carboxyglutamate-containing neuropeptide

A key factor in the characterization of peptide transmitters used in neuronal signaling is the correct elucidation of post-translational modifications, especially as they are often required to confer biological activity. A rare carboxylation modification is described on the D-peptide from the insulin prohormone in the sea slug, Aplysia californica. Using liquid chromatography purification coupled with electrospray ionization and nanoelectrospray ionization-ion trap-mass spectrometry (ESI- and nanoESI-MS), the presence of this D-peptide within Aplysia insulin (AI)-producing neurons is confirmed. Further detailed mass spectrometric analyses demonstrate that the Aplysia insulin D-peptide is carboxylated on the single glutamate residue within the sequence. This gamma-carboxy D-peptide, along with other identified AI-related peptides, is secreted from the central nervous system in response to ionophore stimulation, thus suggesting a signaling role within the nervous system. Although carboxylated peptides have been described previously, the Aplysia gamma-carboxy D-peptide appears to be the first reported carboxylated neuropeptide.     

Identification and molecular cloning of a neuropeptide Y homolog that produces prolonged inhibition in Aplysia neurons.

The neuroendocrine bag cell neurons of the marine mollusk Aplysia produce prolonged inhibition that lasts for more than 2 hr. We purified a peptide from the abdominal ganglion that mimics this inhibition. Mass spectrometry and microsequence analysis indicate that the peptide is 40 aa long and is amidated at its carboxyl terminus. It is highly homologous to vertebrate neuropeptide Y (NPY) and other members of the pancreatic polypeptide family. As determined from cloned cDNA, the gene coding for the precursor protein shares a common structural organization with genes encoding precursors of the vertebrate family. The peptides may therefore have arisen from a common ancestral gene. Bag cell neurons are immunoreactive for Aplysia NPY, and Northern blot analysis indicates that as with its vertebrate counterparts, the peptide is abundantly expressed in the CNS. This suggests that peptides related to NPY may have important functions in the nervous system of Aplysia as well as in other invertebrates.     

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.     

The development of central nervous system control of the gill withdrawal reflex evoked by siphon stimulation in Aplysia

In older Aplysia, the central nervous system (CNS) (abdominal ganglion) exerts suppressive and facilitatory control over the peripheral nervous system (PNS) which initially mediates the gill withdrawal reflex and its subsequent habituation evoked by tactile stimulation of the siphon. In young animals, both the suppressive and facilitatory CNS control were found to be absent. In older animals, removal of branchial nerve (Br) input to the gill resulted in a significantly reduced reflex latency and, with ctenidial (Ct) and siphon (Sn) nerves intact, a significantly increased reflex amplitude and an inability of the reflex to habituate with repeated siphon stimulation. In young animals, removal of Br had no effect on reflex latency and with Ct and Sn intact, the reflex amplitude latency was not increased and the reflex habituated. Older animals can easily discriminate between different intensity stimuli applied to the siphon as evidenced by differences in reflex amplitude, rates of habituation, and evoked neural activity. On the other hand, young animals cannot discriminate well between different stimulus intensities. The lack of CNS control in young animals was found to be due to incompletely developed neural processes within the abdominal ganglion and not the PNS. The lack of CNS control in young Aplysia results in gill reflex behaviours being less adaptive in light of changing stimulus conditions, but may be of positive survival value in that the young will not habituate as easily. The fact that CNS control is present in older animals strengthens the idea that in any analysis of the underlying neural mechanisms of habituation the entire integrated CNS-PNS must be taken into account.