Petra M. Hermann

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.

Attenuation of knee joint inflammation by peripherally administered endomorphin-1.

Endomorphin-1 is a selective endogenous ligand for the μ-opioid receptor, and this study investigated the effect of endomorphin-1 on rat knee joint inflammation by examining the ability of the neuropeptide to modulate synovial protein extravasation. Acute joint inflammation was induced by intraarticular injection of 2% kaolin followed by 2% carrageenan and the animals allowed to recover for 3 h. Immunohistochemical examination of these inflamed joints revealed endomorphin-1-like immunoreactive nerves in deep synovium with a proportion of the nerve fibers occurring in close proximity to synovial blood vessels. Perfusion of inflamed knees with exogenous endomorphin-1 across the dose range 10−9−10−6M produced a significant reduction in synovial vascular permeability with the 10−7M dose producing the greatest fall in protein exudation (approx 55%). These effects were blocked by the specific μ-opioid receptor antagonist CTOP. Destruction of knee joint unmyelinated afferent nerve fibers by capsaicin treatment significantly attenuated the anti-inflammatory effects of endomorphin-1, suggesting that the peptide is acting via a neurogenic mechanism. The findings of this study indicate that endomorphin-1 acts peripherally in knee joints to reduce synovial protein extravasation. These anti-inflammatory effects are mediated by μ-opioid receptors located on capsaicin-sensitive afferent nerves.

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.

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.

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.

Developmental plasticity of respiratory behavior in Lymnaea.

The authors investigated the contribution of experience to development and maintenance of pulmonary respiration in Lymnaea stagnalis. Respiration in L. stagnalis is bimodal via both the skin and the lung. Rearing snails from eggs to adulthood while preventing lung respiration (differentially reared snails) showed that L. stagnalis can develop and survive without pulmonary respiration. These snails were able to open and close their pneumostome when given the opportunity as adults. However, quantitative aspects of their respiratory behavior were significantly altered. Prevention of pulmonary respiration in adult, normally reared snails also induced behavioral changes. Comparison of these changes with those in differentially reared snails revealed specific developmental effects, which were reversible. Thus, this is a suitable model system for studying questions related to behavioral plasticity.

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 ∼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.

Epidermal growth factor‐dependent enhancement of axonal regeneration in the pond snail Lymnaea stagnalis: Role of phagocyte survival

Peripheral nerve injury triggers complex responses from neuronal as well as from multiple nonneuronal cell types. These responses are coordinated by a wide spectrum of secreted and nonsecreted factors, including growth factors, cytokines, and cell adhesion molecules. These molecules originate from different sources and act both locally at the site of injury as well as centrally at the location of the neuronal cell bodies. One of the signal systems frequently implicated in this process is the epidermal growth factor (EGF) family and its receptors. Expression of members of this family as well as that of EGF‐receptors is upregulated in different cell types after peripheral nerve injury. However, the functional significance of this response is unclear. Using a simple invertebrate model system (Lymnaea stagnalis), the present study implicates the EGF/EGF‐receptor system in the survival of ionized calcium‐binding adaptor molecule 1 (Iba1)‐positive phagocytes that reside in the nervous system. We show that inhibiting the EGF‐signaling pathway enhances cell death in this type of cell, an effect paralleled by a substantial reduction in axonal regeneration. Therefore, complementing our previous observation that Lymnaea EGF provides trophic support to axotomized neurons, the present results emphasize the significance of nonneuronal actions of EGF receptor ligands in axonal regeneration. Thus, we add a novel perspective to the ongoing discussion on the functional significance of the EGF signaling system in the injury responses of the nervous system. J. Comp. Neurol. 492:383–400, 2005.

Failure of delayed nonsynaptic neuronal plasticity underlies age-associated long-term associative memory impairment

BackgroundCognitive impairment associated with subtle changes in neuron and neuronal network function rather than widespread neuron death is a feature of the normal aging process in humans and animals. Despite its broad evolutionary conservation, the etiology of this aging process is not well understood. However, recent evidence suggests the existence of a link between oxidative stress in the form of progressive membrane lipid peroxidation, declining neuronal electrical excitability and functional decline of the normal aging brain. The current study applies a combination of behavioural and electrophysiological techniques and pharmacological interventions to explore this hypothesis in a gastropod model (Lymnaea stagnalis feeding system) that allows pinpointing the molecular and neurobiological foundations of age-associated long-term memory (LTM) failure at the level of individual identified neurons and synapses.ResultsClassical appetitive reward-conditioning induced robust LTM in mature animals in the first quartile of their lifespan but failed to do so in animals in the last quartile of their lifespan. LTM failure correlated with reduced electrical excitability of two identified serotonergic modulatory interneurons (CGCs) critical in chemosensory integration by the neural network controlling feeding behaviour. Moreover, while behavioural conditioning induced delayed-onset persistent depolarization of the CGCs known to underlie appetitive LTM formation in this model in the younger animals, it failed to do so in LTM-deficient senescent animals. Dietary supplementation of the lipophilic anti-oxidant α-tocopherol reversed the effect of age on CGCs electrophysiological characteristics but failed to restore appetitive LTM function. Treatment with the SSRI fluoxetine reversed both the neurophysiological and behavioural effects of age in senior animals.ConclusionsThe results identify the CGCs as cellular loci of age-associated appetitive learning and memory impairment in Lymnaea and buttress the hypothesis that lipid peroxidation-dependent depression of intrinsic excitability is a hallmark of normal neuronal aging. The data implicate both lipid peroxidation-dependent non-synaptic as well as apparently lipid peroxidation-independent synaptic mechanisms in the age-dependent decline in behavioural plasticity in this model system.