Willem C. Wildering

Rapid Communication: Neuropeptide Expression and Processing as Revealed by Direct Matrix-Assisted Laser Desorption Ionization Mass Spectrometry of Single Neurons

Abstract: 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 experients 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.

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.

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

BackgroundThe 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.ResultsIn 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.ConclusionThis 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.

Evidence for age-dependent mating strategies in the simultaneous hermaphrodite snail, Lymnaea stagnalis (L.).

SUMMARY In many mating systems female reproductive capacity is a limiting resource over which males will compete. As a consequence, males and females have usually different fitness optimization strategies which may give rise to sexual conflict. Since simultaneous hermaphrodites have, in theory, the option to mate as male or as female at any time, conflict will occur if partners insist in taking the same role. Several lines of evidence exists that body size influences gender choice. However, growth in many invertebrates is indeterminate and therefore age is generally a covariant of size. We therefore investigated the effect of age on mating choices in the simultaneous hermaphrodite Lymnaea stagnalis. Using fully sexually mature animals sampled from three different age groups we show that copulation frequency declines with age. Specifically, in age-matched couples the frequency of primary and reciprocal copulations declines with age. Furthermore, the younger partner tends to mate as male with greater probability in couples of unequal age. Size was never a factor in the sex role preference of Lymnaea. Thus, young Lymnaea always attempt to copulate as male independent of the age of their partner, whereas senior snails act primarily as female. The sex role choices of middle-aged snails appear to depend on their partners age. In addition, we demonstrate that the likelihood that an animal will copulate as male is not correlated with prostate gland size but correlates with the level of afferent electrical activity recorded in the nerve originating in the prostate gland. Together, our results indicate the existence of an age- and not size-dependent mating system in Lymnaea.

Phospholipase A2 – nexus of aging, oxidative stress, neuronal excitability, and functional decline of the aging nervous system? Insights from a snail model system of neuronal aging and age-associated memory impairment

The aging brain undergoes a range of changes varying from subtle structural and physiological changes causing only minor functional decline under healthy normal aging conditions, to severe cognitive or neurological impairment associated with extensive loss of neurons and circuits due to age-associated neurodegenerative disease conditions. Understanding how biological aging processes affect the brain and how they contribute to the onset and progress of age-associated neurodegenerative diseases is a core research goal in contemporary neuroscience. This review focuses on the idea that changes in intrinsic neuronal electrical excitability associated with (per)oxidation of membrane lipids and activation of phospholipase A2 (PLA2) enzymes are an important mechanism of learning and memory failure under normal aging conditions. Specifically, in the context of this special issue on the biology of cognitive aging we portray the opportunities offered by the identifiable neurons and behaviorally characterized neural circuits of the freshwater snail Lymnaea stagnalis in neuronal aging research and recapitulate recent insights indicating a key role of lipid peroxidation-induced PLA2 as instruments of aging, oxidative stress and inflammation in age-associated neuronal and memory impairment in this model system. The findings are discussed in view of accumulating evidence suggesting involvement of analogous mechanisms in the etiology of age-associated dysfunction and disease of the human and mammalian brain.

Redox agents modulate neuronal activity and reproduce physiological aspects of neuronal aging

The high oxygen consumption and post-mitotic nature of the central nervous system (CNS) makes it particularly susceptible to oxidative stress, the impact of which is widely regarded as a root cause of functional impairment of the aging brain in vertebrates and invertebrates alike. Using an invertebrate model system we demonstrate that the lipid soluble antioxidant α-tocopherol can both reverse 2,2-azobis(2-methylpropion-amidine) dihydrochloride (AAPH) induced decline in excitability in young neurons as well as restore the electrical activity and excitability of aged neurons not unlike the level of their younger equivalents. Furthermore, using two analogs of α-tocopherol where either the acyl chain has been removed (Trolox) or the hydroxyl group of the chromanol ring has been methylated we were able to assert that the restorative effect of α-tocopherol requires both insertion into the plasma membrane as well as an active OH group. Thus, our results indicate peroxidation is an important modulator of neuronal excitability as well as support the growing body of evidence suggesting α-tocopherols actions may extend well beyond its established role as a lipid domain preventative antioxidant.

Impairment of long-term associative memory in aging snails (Lymnaea stagnalis).

Age-dependent impairment in learning and memory functions occurs in many animal species, including humans. Although cell death contributes to age-related cognitive impairment in pathological forms of aging, learning and memory deficiencies develop with age even without substantial cell death. The molecular and cellular basis of this biological aging process is not well understood but seems to involve a decline in the aging brains capacity for experience-dependent plasticity. To aid in resolving this issue, we used a simple snail appetitive classical conditioning paradigm in which the underlying molecular, cellular, and neural network functions can be directly linked to age-associated learning and memory performance (i.e., the Lymnaea stagnalis feeding system). Our results indicate that age does not affect the acquisition of appetitive memory but that retention and/or consolidation of long-term memory become progressively impaired with advancing age. The latter phenomenon correlates with declining electrophysiological excitability in key neurons controlling the feeding behavior. Together, these results present the Lymnaea feeding system as a powerful paradigm for investigations of cellular and molecular foundations of biological aging in the brain.

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.

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.