Transcriptome analysis of the central nervous system of the mollusc Lymnaea stagnalis.

BACKGROUND: The freshwater snail Lymnaea stagnalis (L. stagnalis) has served as a successful model for studies in the field of Neuroscience. However, a serious drawback in the molecular analysis of the nervous system of L. stagnalis has been the lack of large-scale genomic or neuronal transcriptome information, thereby limiting the use of this unique model. RESULTS: In this study, we report 7,712 distinct EST sequences (median length: 847 nucleotides) of a normalized L. stagnalis central nervous system (CNS) cDNA library, resulting in the largest collection of L. stagnalis neuronal transcriptome data currently available. Approximately 42% of the cDNAs can be translated into more than 100 consecutive amino acids, indicating the high quality of the library. The annotated sequences contribute 12% of the predicted transcriptome size of 20,000. Surprisingly, approximately 37% of the L. stagnalis sequences only have a tBLASTx hit in the EST library of another snail species Aplysia californica (A. californica) even using a low stringency e-value cutoff at 0.01. Using the same cutoff, approximately 67% of the cDNAs have a BLAST hit in the NCBI non-redundant protein and nucleotide sequence databases (nr and nt), suggesting that one third of the sequences may be unique to L. stagnalis. Finally, using the same cutoff (0.01), more than half of the cDNA sequences (54%) do not have a hit in nematode, fruitfly or human genome data, suggesting that the L. stagnalis transcriptome is significantly different from these species as well. The cDNA sequences are enriched in the following gene ontology functional categories: protein binding, hydrolase, transferase, and catalytic enzymes. CONCLUSION: This study provides novel molecular insights into the transcriptome of an important molluscan model organism. Our findings will contribute to functional analyses in neurobiology, and comparative evolutionary biology. The L. stagnalis CNS EST database is available at http://www.Lymnaea.org/.

RGD-mediated adhesive interactions are important for peripheral axon outgrowth in vivo.

We identify an essential role for the RGD (Arg-Gly-Asp tripeptide) moiety in vivo during adult peripheral neuron regenerative growth. Beyond a peripheral nerve transection there were rises in the fibronectin extracellular matrix, and striking rises in the mRNA and protein expression of integrin subunits sensitive to RGD/fibronectin signalling. Neuron perikarya, axons and Schwann cells all expressed RGD/fibronectin sensitive integrins after injury. To evaluate the significance of RGD/fibronectin-integrin interactions, we infused solutions of a pentapeptide including the RGD motif (sRGD) serially and directly within the milieu of early axon growth across rat sciatic transection injuries. While low dose infusions of sRGD facilitated early axon ingrowth, we encountered inhibition of ingrowth and bridge formation with higher doses of sRGD indicating competitive disruption of RGD/fibronectin-integrin signalling. Fibronectin RGD moieties serve a critical and important role during peripheral axon outgrowth.

CRNF, a molluscan neurotrophic factor that interacts with the p75 neurotrophin receptor.

A 13.1-kilodalton protein, cysteine-rich neurotrophic factor (CRNF), was purified from the mollusk Lymnaea stagnalis by use of a binding assay on the p75 neurotrophin receptor. CRNF bound to p75 with nanomolar affinity but was not similar in sequence to neurotrophins or any other known gene product. CRNF messenger RNA expression was highest in adult foot subepithelial cells; in the central nervous system, expression was regulated by lesion. The factor evoked neurite outgrowth and modulated calcium currents in pedal motor neurons. Thus, CRNF may be involved in target-derived trophic support for motor neurons and could represent the prototype of another family of p75 ligands.

Lymnaea epidermal growth factor promotes axonal regeneration in CNS organ culture.

Members of the epidermal growth factor (EGF) family are frequently implicated in the injury response of the mammalian nervous system. Although this implication is supported by extensive molecular evidence, it is not underpinned by conclusive functional data. Recently, we found that expression of an EGF homolog from the pond snail Lymnaea stagnalis (L-EGF) is upregulated after axotomy in the adult CNS, suggesting a role for this molecule in the injury response of the CNS. In the present study we asked whether L-EGF can promote axonal regeneration of three types of identified neurons in organ-cultured CNS. Treatment with purified L-EGF substantially enhanced axonal regeneration of all three types of neurons, an effect inhibited by submicromolar doses of PD153035, a specific EGF receptor (EGFR) tyrosine kinase inhibitor. In addition, PD153035 and K252a, a nonspecific kinase inhibitor, also reduced the degree of axonal regeneration that occurs without L-EGF supplementation, indicating that L-EGF or other EGFR ligands synthesized in the CNS participate in the regenerative response. An intriguing aspect of these results is that axonal regeneration of different, intrinsically L-EGF responsive and unresponsive neurons occurred in a coordinated manner. This observation suggests that indirect in addition to direct actions contribute to the beneficial effect of L-EGF. In conclusion, we provide functional evidence that an EGF homolog can promote axonal regeneration, substantiating existing molecular evidence implicating the EGF family in peripheral nerve regeneration and emphasizes the therapeutic potential of these molecules.

Lymnaea EGF and gigantoxin I, novel invertebrate members of the epidermal growth factor family.

In this review, we compare the sequence and structural relationships of two epidermal growth factor (EGF) family related proteins that have recently been discovered in invertebrate species. The first is L-EGF, a secreted growth factor from the gastropod mollusk Lymnaea stagnalis. The second is a peptide toxin (Gigantoxin I), isolated from the sea anenome Stichodactyla giganteus, which can paralyze crabs. L-EGF and Gigantoxin I share striking sequence similarity with mammalian erbB1 receptor ligands, including most of the essential receptor binding sites. Intriguingly, L-EGF's tertiary structure resembles more the structure of the EGF-like domain of coagulation factors. That is, the secondary and tertiary structure of L-EGF indicates the presence of a double-stranded beta-sheet but also suggests that this protein, in contrast to all other erbB1 ligands, contains a calcium-binding domain. One of the most remarkable features of L-EGF and Gigantoxin I however, is the indication that these protein are synthesized as non-membrane bound secreted peptides. This feature sets L-EGF and Gigantoxin I apart from all other members of the EGF family or EGF-like proteins identified thus far. We discuss sequence similarities and dissimilarities in the light of indications that, despite the more than 600 million years of phylogenetic distance separating both these invertebrates from mammals, Gigantoxin I and L-EGF retain some affinity for the mammalian erbB-family of receptors. Considering that mammalian EGF and its family members are frequently implicated in neoplastic diseases, the increasing number of identified and characterized invertebrate EGF family members may provide valuable leads in the design of erbB receptor antagonists.

Functional recovery of respiratory behavior during axonal regeneration in snails (Lymnaea stagnalis) is experience dependent.

This study investigated the role of experience in recovery of pulmonary respiration during axonal regeneration in Lymnaea stagnalis. Pulmonary respiration occurs when snails break the water surface and open the lung orifice, the pneumostome. It was shown that axotomy of all the axons innervating the pneumostome and surrounding area prevents the occurrence of lung respiration in 69% of snails. In the remaining 31%, lung respiration persisted, indicating that peripheral components alone are capable of initiating pneumostome openings and closures. Five weeks postsurgery, all snails with previous nerve crushes showed opening of the pneumostome with normal latency after breaking the water surface. However, prevention of pulmonary respiration during the recovery period dramatically changed the recovered behavior. Thus, experience in pulmonary respiration during axonal regeneration plays a role in the recovery of this behavior.

Serotonergic modulation of junctional conductance in an identified pair of neurons in the mollusc Lymnaea stagnalis.

Serotonin (5-HT) is shown to modulate electrotonic coupling between two giant peptidergic neurons in the CNS of Lymnaea stagnalis. The primary effect of 5-HT appears to be a rapid and reversible decrease in gap junctional conductance.

The role of pacemaker properties and synaptic input in generation and modulation of spiking activity in a pair of electrically coupled peptidergic neurons.

The origin of patterned electrical activity in two electronically coupled peptidergic neurons, VD1 and RPD2, in the CNS of Lymnaea stagnalis was investigated. VD1 proved to have intrinsic beating pacemaker properties. Hybrid current/voltage clamp experiments demonstrated that in the intact CNS generation of spike activity in the coupled cell system is dominated by VD1. Modulation of spiking activity of VD1/RPD2 appears to originate mainly from chemical synaptic input. The electrical coupling of VD1 and RPD2 proved essential for spike synchronization between the cells.

Age-related changes in junctional and non-junctional conductances in two electrically coupled peptidergic neurons of the mollusc Lymnaea stagnalis.

Age-related changes in electrotonic coupling ratio of two identified neurons in Lymnaea stagnalis were studied together with the underlying changes in the steady-state conductance properties of the network. Two phases were distinguished in the development of coupling ratio across lifespan. During the first phase (age of 3-13 months), coupling ratio decreased from decreased from 60% to 30%. The second phase (age 13-20 months) was characterized by an increase in coupling ratio. Values of up to 60% were reached again in the oldest animals. Voltage clamp measurements showed that the biphasic trend of the age-related changes in coupling ratio is paralleled by changes in conductance properties of the junction between VD1 and RPD2. During the first phase junctional conductance decreased, whereas during the second phase junctional conductance increased. In addition to the decrease in junctional conductance, a growth-related increase in non-junctional conductance of VD1 and RPD2 contributed to the decrease in coupling ratio observed during the first phase. Thus our results indicate that in Lymnaea junctional connections between neurons may undergo considerable and discontinuous changes after sexual maturation. In addition to these changes in steady-state electrical properties, indications were obtained that age-related changes of kinetically slower conductance(s) may occur in the non-junctional membrane of VD1 and RPD2.

Operant conditioning of aerial respiratory behaviour in Lymnaea stagnalis

In this study, we operantly conditioned the aerial respiratory behaviour of the freshwater snail Lymnaea stagnalis. Aerial respiration in Lymnaea stagnalis is accomplished by the spontaneous opening and closing of its respiratory orifice, the pneumostome, at the water surface. Weak tactile stimulation of the pneumostome area, when the pneumostome is open, evoked only the pneumostome closure response, which is one aspect of the escape-withdrawal reflex. Pneumostome stimulation resulted in its closure and the termination of aerial respiratory activity. A contingent tactile stimulation paradigm was used to operantly condition the animals. Stimulation of the pneumostome whenever the animal attempted to breathe resulted in significantly fewer attempts to open the pneumostome as training progressed. The latency of the first breath (subsequent to stimulation), the number of breaths and the total breathing time were measured before and after each training period. Significant, quantifiable changes in these behavioural parameters were observed only in the operant conditioning group animals. Control animals receiving tactile stimulation to their pneumostome not contingent upon pneumostome opening movements (yoked controls) or those that were physically prevented from surfacing to breathe (hypoxic controls), did not exhibit significant changes in these behavioural parameters. Our data provide the first direct evidence for operant conditioning of respiration in any animal.

Patterns of body temperature during feeding in rats under varying ambient temperatures.

Relationships between feeding and body temperature of rats were investigated at three ambient temperatures during the whole light/dark cycle. Basal liver temperature was negatively correlated with ambient temperature. Only at 29 degrees C liver temperature indicated activation of autonomic and locomotory thermoregulatory responses due to heat stress. At 21 degrees C, liver temperature was always higher than skin temperature. Both showed a clear circadian rhythm with higher values during the dark phase. Meal-associated temperature patterns were superimposed on this circadian rhythm. Liver and skin temperatures showed a preprandial and prandial rise. Liver temperature reached an almost similar peak value just above 39 degrees C at the end of a meal, irrespective of meal size and ambient temperatures of 13 degrees C and 21 degrees C. Liver temperature reached this peak about 2 min earlier and dropped sooner than skin temperature. These results indicate a threshold liver temperature at which feeding activity stops. The present study suggests that temperatures do not exceed this value by adaptive autonomic thermoregulatory responses shifting heat flow from core to skin and by stopping all locomotory activities including feeding, thereby avoiding deterioration of vital organs and physiological processes due to hyperthermia.

The role of membrane properties, synaptic input and electrical junction in determination of spike output of a pair of peptidergic neurons in the mollusc Lymnaea stagnalis.

The two electrotonically coupled peptidergic neurons, VD1 and RPD2 show in the isolated central nervous system (CNS) a patterned activity. The cells fire in almost perfect synchrony in CNS's derived from animals of moderate age, while in old animals disturbances in synchrony are observed. The firing pattern varies from beating to bursting. Isolated VD1's show a beating firing pattern, indicating that the cell possesses pacemaker properties, while the isolated RPD2 is almost always silent. Hybrid current/voltage clamp experiments show that in the intact CNS spike generation in the two cells is due to VD1, indicating that the pacemaker properties of VD1 constitute the main driving force for the firing rhythm of the two cell system. In low calcium/high magnesium solutions the rhythm is a beating one, while in solutions which do not suppress synaptic input, sometimes a bursting pattern is observed. The idea that the bursting pattern is due to synaptic modulation of these cells is corroborated by the observation that application of certain transmitters induces a very prominent burst of activity in these neurons. These bursts are very reminiscent of the bursts, observed in semi-intact preparations, which are accompanied by penumostome movements. Increase in coupling resistance, which occurs at older age, is accompanied by irregularities in firing rhythm. Apparently the electrotonic junction is essential for the patterned output of the two cell system.

Voltage-gated potassium channels regulate the response of retinal growth cones to axon extension and guidance cues.

Xenopus retinal ganglion cell growth cones express various voltage-gated potassium (Kv) channels. We showed previously that 4-aminopyridine and tetraethylammonium have different effects on the outward currents of embryonic Xenopus retinal ganglion cells. Therefore, we asked whether these Kv channel inhibitors differentially regulate the response of retinal ganglion cell growth cones to extrinsic cues. First, we tested the role of Kv channels in axon extension mediated by a substrate bound cue and found that 4-aminopyridine blocked, whereas tetraethylammonium enhanced basal extension on laminin. Yet, when the growth cones were stimulated to extend with application of soluble growth factors, both inhibitors resulted in a return to the basal extension rates observed in the presence of laminin alone. Second, we asked if Kv channels modulate the response of retinal ganglion cell growth cones to a guidance cue, the chemorepellent fibroblast growth factor-2. When presented in a gradient to one side of the growth cone, fibroblast growth factor-2 repulsed retinal ganglion cell growth cones in the presence of 4-aminopyridine but not tetraethylammonium. These data argue that tetraethylammonium- and 4-aminopyridine-sensitive Kv channels differ in the manner by which they regulate the response of retinal ganglion cell axons to extension and guidance cues. Non-ratiometric calcium imaging indicated that differences in the ability of tetraethylammonium- and 4-aminopyridine-sensitive Kv channels to regulate calcium activity within the growth cone may underlie their unique modulation of growth cone behaviour.

Neurite outgrowth, RGD-dependent, and RGD-independent adhesion of identified molluscan motoneurons on selected substrates.

The extracellular matrix (ECM) provides structural support to cells and tissues and is involved in the regulation of various essential physiological processes, including neurite outgrowth. Most of the adhesive interactions between cells and ECM proteins are mediated by integrins. Integrins typically recognize short linear amino acid sequences in ECM proteins, one of the most common being Arginine-Glycine-Aspartate (RGD). The present study investigated neurite outgrowth and adhesion of identified molluscan neurons on a selection of substrates in vitro. Involvement of RGD binding sites in adhesion to the different substrates was investigated using soluble synthetic RGD peptides. The cells adhered to native (i.e., nondenatured) laminin and type IV collagen, but not to native plasma fibronectin. Denaturation of fibronectin dramatically enhanced cell adhesion. Only the adhesion to denatured fibronectin was inhibited by RGD peptides, indicating that denaturation uncovers a RGD binding site in the protein. Laminin as well as denatured fibronectin, but not type IV collagen, induced neurite outgrowth from a percentage of the RPA neurons. These results demonstrate that molluscan neurons can attach to various substrates using both RGD-dependent and RGD-independent adhesion mechanisms. This suggests that at least two different cell adhesion receptors, possibly belonging to the integrin family, are expressed in these neurons. Moreover, the results show that vertebrate ECM proteins can induce outgrowth from these neurons, suggesting that the mechanisms involved in adhesion as well as outgrowth promoting are evolutionarily well conserved.

Age-related changes in female reproductive activity and growth in the mollusc Lymnaea stagnalis.

Age-related changes in reproduction and growth of the pulmonate snail Lymnaea stagnalis were studied in animals under standard culture conditions. Longitudinal studies as well as cross-sectional studies were done. Egg-laying activity starts in Lymnaea at an age of about 2 months. Up to an age of about 250 days it increases, and in older snails it decreases with age. Cross-sectional studies showed that the increase in egg-laying activity is due to an increase in the number of animals that lay eggs and to an increase in the egg-laying frequency of individuals. The decrease in egg-laying activity is due to a decrease in the egg-laying frequency of individual animals and to an increase in the number of animals that stop egg laying. Growth (shell length and body weight) ceases at about the same age at which egg laying begins to decrease. The results are discussed in relation to the organization of the neuroendocrine system in Lymnaea and to results of earlier studies on age-related changes in peptidergic neurons in the CNS of Lymnaea.

Nerve growth factor (NGF) acutely enhances high-voltage-activated calcium currents in molluscan neurons.

1. Nerve growth factor (NGF) is a member of a family of molecules (the neurotrophins) that can regulate the survival and/or outgrowth of many vertebrate cells. NGF also induces outgrowth from Lymnaea neurons under experimental conditions. Recent studies have shown that the neurotrophins can also acutely modulate some physiological properties of adult neurons. Here we examined the actions of NGF on high-voltage-activated (HVA) Ca2+ currents in Lymnaea motoneurons. 2. NGF induced a dose-dependent and reversible increase in HVA Ca2+ currents within 2 min. 3. The threshold dose of the NGF-induced enhancement of HVA Ca2+ currents ranged between 1 and 1,000 pg/ml. In the most sensitive cells, the response saturated at doses higher than 1 ng/ml. 4. The results indicate that neurotrophins acutely modulate voltage-gated Ca2+ currents in molluscan neurons through a high affinity signal transduction pathway. The data support the existence of neurotrophins in invertebrates. Moreover, this property of NGF may explain the neuromodulatory actions of neurotrophins observed in various preparations.

Pronase acutely modifies high voltage-activated calcium currents and cell properties of Lymnaea neurons.

Pronase E ('pronase') is one of the proteolytic enzymes that are used in preparative procedures such as cell isolation and to soften the sheath of invertebrate ganglia. Although several effects of proteolytic enzymes on the physiology of non-neuronal tissues have been described, the effects of these enzymes on central neurons have received little attention. We examined the effects of bath-applied pronase on neurons in the Lymnaea central nervous system and in vitro. Pronase caused action potential broadening in neurons that exhibit a shoulder on the repolarization phase of their action potentials. This effect of pronase was accompanied by, although unrelated to, a depolarization and decrease in action potential interval. Some, but not all, effects of pronase in the central nervous system were reversible. For example, the decreases in membrane potential and action potential interval were both reversed after approximately 1 h of washing with saline. However, the effect of pronase on the action potential duration was not reversed after a period of 90 min. The modulation of action potential width prompted us to examine Ca2+ currents. Exposure to pronase resulted in an increase in both peak and late high voltage-activated Ca2+ currents in isolated neurons. Pronase neither changed the inactivation rate nor caused a shift in the current-voltage relationship of the current. The changes in action potential duration could be prevented by application of 0.1 mM Cd2+, indicating that the action potential broadening caused by pronase depends on Ca2+ influx. This is the first systematic study of the acute and direct actions of pronase on Ca2+ currents and cell properties both in the CNS and in vitro.

Neuropeptide expression and processing as revealed by direct matrix-assisted laser desorption ionization mass spectrometry of single neurons.

Neuropeptides were directly detected in single identified neurons and the neurohemal area of peptidergic (neuroendocrine) systems in the Lymnaea brain by using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The samples were placed in matrix solution and ruptured to allow mixing of cell contents with the matrix solution. After formation of matrix crystals, the analytes were analyzed by MALDI-MS. It was surprising that clean mass spectra were produced, displaying extreme sensitivity of detection. In one of the neuroendocrine systems studied, we could demonstrate for the first time, by comparing the peptide patterns of soma and of neurohemal axon terminals, that processing of the complex prohormone expressed in this system occurs entirely in the soma. In the other system studied, novel peptides could be detected in addition to peptides previously identified by conventional molecular biological and peptide chemical methods. Thus, complex peptide processing and expression patterns could be predicted that were not detected in earlier studies using conventional methods. As the first MALDI-MS study of direct peptide fingerprinting in the single neuron, these experiments demonstrate that MALDI-MS forms a new and valuable approach to the study of the synthesis and expression of bioactive peptides, with potential application to single-cell studies in vertebrates, including humans.

Neurotrophic actions of a novel molluscan epidermal growth factor.

The mammalian epidermal growth factor (EGF) is expressed in the developing and adult CNS, and it has been implicated in the control of cell proliferation, differentiation, and neurotrophic events. Despite extensive evolutionary conservation of the EGF motif in a range of different types of proteins, secreted EGF homologs with neurotrophic actions have not been reported in invertebrates. In this study, we present a novel member of the family of EGF-like growth factors, an EGF homolog from the mollusc Lymnaea stagnalis (L-EGF), and we demonstrate that this protein has neurotrophic activity. Purified L-EGF is a 43-residue peptide and retains the typical structural characteristics of the EGF motif. The L-EGF cDNA reveals a unique precursor organization. In contrast to the multidomain mammalian EGFs, it consists of only two domains, a signal peptide and a single EGF motif. Conspicuously, the L-EGF precursor lacks a transmembrane domain, setting it apart from all other members of the EGF-family. L-EGF mRNA is expressed throughout embryonic development, in the juvenile CNS, but not in the normal adult CNS. However, expression in the adult CNS is upregulated after injury, suggesting a role of L-EGF in repair functions. This notion is supported by the observation that L-EGF evokes neurite outgrowth in specific adult Lymnaea neurons in vitro, which could be inhibited by an EGF receptor tyrosine kinase inhibitor. In conclusion, our findings further substantiate the notion that the EGF family has an early phylogenetic origin, and our data support a neurotrophic role for L-EGF during development and injury repair.