Molluscan Neurons Research Articles

The distribution and evolutionary dynamics of dopaminergic neurons in molluscs.

Dopamine is one of the most versatile neurotransmitters in invertebrates. It's distribution and plethora of functions is likely coupled to feeding ecology, especially in Euthyneura (the largest clade of molluscs), which presents the broadest spectrum of environmental adaptations. Still, the analyses of dopamine-mediated signaling were dominated by studies of grazers. Here, we characterize the distribution of dopaminergic neurons in representatives of two distinct ecological groups: the sea angel - obligate predatory pelagic mollusc Clione limacina (Pteropoda, Gymnosomata) and its prey - the sea devil Limacina helicina (Pteropoda, Thecosomata) as well as the plankton eater Melibe leonina (Nudipleura, Nudibranchia). By using tyrosine hydroxylase-immunoreactivity (TH-ir) as a reporter, we showed that the dopaminergic system is moderately conservative among euthyneurans. Across all studied species, small numbers of dopaminergic neurons in the central ganglia contrast to significant diversification of TH-ir neurons in the peripheral nervous system, primarily representing sensory-like cells, which predominantly concentrated in the chemotactic areas and projecting afferent axons to the central nervous system. Combined with α-tubulin immunoreactivity, this study illuminates the unprecedented complexity of peripheral neural systems in gastropod molluscs, with lineage-specific diversification of sensory and modulatory functions.

Tyrosine hydroxylase messenger RNA corroborates protein localization in the nervous system of the pond snail, Lymnaea stagnalis

AbstractCatecholaminergic neurons are abundant in molluscs and are involved in a variety of behaviors such as feeding, respiration, learning, and locomotion. However, previous identification of these neurons has relied almost exclusively on immunohistochemistry using antibodies, which have not been fully validated for use in molluscs. We employed tissue‐specific quantitative PCR in adults of Lymnaea stagnalis (a pulmonate gastropod) and whole‐mount in situ hybridization in larvae to both quantify and visualize messenger RNA of the catecholamine synthesis enzyme, tyrosine hydroxylase (TH). TH messenger RNA was found to localize primarily in the foot and the central nervous system, with smaller quantities present in the cephalic sensory organs. Additionally, we performed western blots that validated a popular antibody used as a marker for catecholaminergic neurons in molluscs. Taken together, these data indicate that TH messenger RNA is present in the central and peripheral nervous system of L. stagnalis and support the specificity of past immunohistochemical labeling of the TH protein. These findings have potentially broad implications, given the wide range of biological processes that have been studied in L. stagnalis.

Protonophoric action of BAM15 on planar bilayers, liposomes, mitochondria, bacteria and neurons

Small molecules capable of uncoupling respiration and ATP synthesis in mitochondria are protective towards various cell malfunctions. Recently (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine (BAM15), a new compound of this type, has become popular as a potent mitochondria-selective depolarizing agent producing minimal adverse effects. To validate protonophoric mechanism of BAM15 action, we examined its behavior in bilayer lipid membranes (BLM). BAM15 proved to be a typical anionic protonophore with the activity on planar membranes being suppressed upon decreasing membrane dipole potential. In both planar BLM and liposomes, BAM15 induced proton conductance with the potency close to that of the classical protonophoric uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP). In isolated rat liver mitochondria (RLM), BAM15 caused membrane potential collapse, increased respiration rate and induced Ca2+ efflux at concentrations slightly higher than those for CCCP. Surprisingly, the uncoupling action of BAM15 on isolated RLM, in contrast to that of CCCP, was partially reversed by carboxyatractyloside (CATR), an inhibitor of adenine nucleotide translocase, thereby indicating involvement of this protein in the BAM15-induced uncoupling. BAM15 inhibited growth of Bacillus subtilis at micromolar concentrations. In electrophysiological experiments on molluscan neurons, BAM15 caused plasma membrane depolarization and suppression of electrical activity, but the effect developed more slowly than that of CCCP.

Characterisation of d-Conotoxin TxVIA as a Mammalian T-Type Calcium Channel Modulator.

The 27-amino acid (aa)-long δ-conotoxin TxVIA, originally isolated from the mollusc-hunting cone snail Conus textile, slows voltage-gated sodium (NaV) channel inactivation in molluscan neurons, but its mammalian ion channel targets remain undetermined. In this study, we confirmed that TxVIA was inactive on mammalian NaV1.2 and NaV1.7 even at high concentrations (10 µM). Given the fact that invertebrate NaV channel and T-type calcium channels (CaV3.x) are evolutionarily related, we examined the possibility that TxVIA may act on CaV3.x. Electrophysiological characterisation of the native TxVIA on CaV3.1, 3.2 and 3.3 revealed that TxVIA preferentially inhibits CaV3.2 current (IC50 = 0.24 μM) and enhances CaV3.1 current at higher concentrations. In fish bioassays TxVIA showed little effect on zebrafish behaviours when injected intramuscular at 250 ng/100 mg fish. The binding sites for TxVIA at NaV1.7 and CaV3.1 revealed that their channel binding sites contained a common epitope.

The Fusion of Nerve Processes and the Formation of Syncytia in Living Mollusk Neurons in Tissue Culture

The Fusion of Nerve Processes and the Formation of Syncytia in Living Mollusk Neurons in Tissue Culture

Mechanisms of Anesthetic Action and Neurotoxicity: Lessons from Molluscs.

Anesthesia is a prerequisite for most surgical procedures in both animals and humans. Significant strides have been made in search of effective and safer compounds that elicit rapid induction and recovery from anesthesia. However, recent studies have highlighted possible negative effects of several anesthetic agents on the developing brain. The precise nature of this cytotoxicity remains to be determined mainly due to the complexity and the intricacies of the mammalian brain. Various invertebrates have contributed significantly toward our understanding of how both local and general anesthetics affect intrinsic membrane and synaptic properties. Moreover, the ability to reconstruct in vitro synapses between individually identifiable pre- and postsynaptic neurons is a unique characteristic of molluscan neurons allowing us to ask fundamental questions vis-à-vis the long-term effects of anesthetics on neuronal viability and synaptic connectivity. Here, we highlight some of the salient aspects of various molluscan organisms and their contributions toward our understanding of the fundamental mechanisms underlying the actions of anesthetic agents as well as their potential detrimental effects on neuronal growth and synaptic connectivity. We also present some novel preliminary data regarding a newer anesthetic agent, dexmedetomidine, and its effects on synaptic transmission between Lymnaea neurons. The findings presented here underscore the importance of invertebrates for research in the field of anesthesiology while highlighting their relevance to both vertebrates and humans.

Protonophoric action of triclosan causes calcium efflux from mitochondria, plasma membrane depolarization and bursts of miniature end-plate potentials

The formerly widely used broad-spectrum biocide triclosan (TCS) has now become a subject of special concern due to its accumulation in the environment and emerging diverse toxicity. Despite the common opinion that TCS is an uncoupler of oxidative phosphorylation in mitochondria, there have been so far no studies of protonophoric activity of this biocide on artificial bilayer lipid membranes (BLM). Yet only few works have indicated the relationship between TCS impacts on mitochondria and nerve cell functioning. Here, we for the first time report data on a high protonophoric activity of TCS on planar BLM. TCS proved to be a more effective protonophore on planar BLM, than classical uncouplers. Correlation between a strong depolarizing effect of TCS on bacterial membranes and its bactericidal action on Bacillus subtilis might imply substantial contribution of TCS protonophoric activity to its antimicrobial efficacy. Protonophoric activity of TCS, monitored by proton-dependent mitochondrial swelling, resulted in Ca2+ efflux from mitochondria. A comparison of TCS effects on molluscan neurons with those of conventional mitochondrial uncouplers allowed us to ascribe the TCS-induced neuronal depolarization and suppression of excitability to the consequences of mitochondrial deenergization. Also similar to the action of common uncouplers, TCS caused a pronounced increase in frequency of miniature end-plate potentials at neuromuscular junctions. Thus, the TCS-induced mitochondrial uncoupling could alter neuronal function through distortion of Ca2+ homeostasis.

Signatures of chaos in animal search patterns.

One key objective of the emerging discipline of movement ecology is to link animal movement patterns to underlying biological processes, including those operating at the neurobiological level. Nonetheless, little is known about the physiological basis of animal movement patterns, and the underlying search behaviour. Here we demonstrate the hallmarks of chaotic dynamics in the movement patterns of mud snails (Hydrobia ulvae) moving in controlled experimental conditions, observed in the temporal dynamics of turning behaviour. Chaotic temporal dynamics are known to occur in pacemaker neurons in molluscs, but there have been no studies reporting on whether chaotic properties are manifest in the movement patterns of molluscs. Our results suggest that complex search patterns, like the Lévy walks made by mud snails, can have their mechanistic origins in chaotic neuronal processes. This possibility calls for new research on the coupling between neurobiology and motor properties.

Lubinska Phenomenon: Simultaneous Bidirectional Axoplasmic Flow in Nerve Fibers.

Experiments on live mollusk neurons isolated with a neurite fragmentsat its various levels demonstrated that axoplasm is characterized by mechanical strain realized in the form of retraction up to complete invagination of the axoplasm into the soma. Changes in axon geometry were attributed to neuroplasm movement. It was found that the direction of axoplasm movement depends on the location of adhesion points. It was always simultaneous and oppositely directed, as is the case with contractile myofibrils. The formation of distant paired adhesion sites can promote moving away of the axoplasm mass and organelles carried by it. The velocity and activity of axoplasm movement depend on the quantity and intensity of adhesion points along the axon.

Neural Evolution: Marginal Gains through Soma Location

Unlike in most vertebrate neurons, the soma of many arthropod and mollusc neurons is placed at the end of a thin neurite. Multi-compartment computational modelling suggests this strategy may reduce the attenuation of signals from the dendrites, reducing the energy costs of signalling.

Retinoic acid affects calcium signaling in adult molluscan neurons.

Retinoic acid, the active metabolite of vitamin A, is important for nervous system development, regeneration, as well as cognitive functions of the adult central nervous system. These central nervous system functions are all highly dependent on neuronal activity. Retinoic acid has previously been shown to induce changes in the firing properties and action potential waveforms of adult molluscan neurons in a dose- and isomer-dependent manner. In this study, we aimed to determine the cellular pathways by which retinoic acid might exert such effects, by testing the involvement of pathways previously shown to be affected by retinoic acid. We demonstrated that the ability of all-trans retinoic acid (atRA) to induce electrophysiological changes in cultured molluscan neurons was not prevented by inhibitors of protein synthesis, protein kinase A or phospholipase C. However, we showed that atRA was capable of rapidly reducing intracellular calcium levels in the same dose- and isomer-dependent manner as shown previously for changes in neuronal firing. Moreover, we also demonstrated that the transmembrane ion flux through voltage-gated calcium channels was rapidly modulated by retinoic acid. In particular, the peak current density was reduced and the inactivation rate was increased in the presence of atRA, over a similar time course as the changes in cell firing and reductions in intracellular calcium. These studies provide further evidence for the ability of atRA to induce rapid effects in mature neurons.

Simultaneous opposite axonal currents in neural process. Retraction hypothesis

Bidirectional axonal current of organelles and molecules in the nerve fibers was demonstrated using radioautography, the horseradish peroxidase and in virology. However, the mechanism of this phenomenon and regulation of the currents direction in axoplasm still remain not entirely understood. In this article we used the model of living single neurons of mollusk isolated with fragment of neural process at its different levels. It was proved that the axoplasm has a mechanical tone, which is realized in the form of retraction up to complete axoplasm invagination in the neuron soma. The geometry changing of the living axon was treated as its transport neuroplasm mass. It turned out that the direction of axoplasm mass depends on the location of its adhesion sites. It is always simultaneous and bidirectional opposite, as it is the case with contractile muscle fibers.

Changes of intracellular potentials and ionic currents of the mollusk neurons under action of solvents dimethylsulfoxide and acetone

Dimethylsulfoxide (DMSO), and dimethyl ketone (acetone) in concentrations up to 100 mM after extracellular application did not change the electrical activity of the identified pedal ganglia neurons of clam Planorbarius corneus in microelectrode studies and voltage clamp technique. DMSO in concentrations of 500 and 1000 mM (4 and 8 % solution), and acetone in smaller concentrations (from 200 mM-1.2% or higher) was shown to depolarize neurons and to increase the frequency and duration of action potentials, their amplitude and the total ionic currents (dV/dt) being decreased. The changes were fully reversible, hyperpolarization of cells was registered after washing, which was caused probably by activation of electrogenic transport of sodium ions. DMSO and acetone were suggested to change the neuron state not only by changes in resting potential but also by direct effects on ion channels. Acetone reduced amplitude of action potentials in neurons because of depolarization and in less degree because of its effects on ion channels. DMSO and acetone in concentrations of 100-200 mM are concluded to be safe for biological objects when they are used for dissolution of pharmacological agents.

Epigenetic modification in neurons of the mollusc Pomacea canaliculata after immune challenge

In human and rodents, the transcriptional response of neurons to stress is related to epigenetic modifications of both DNA and histone proteins. To assess the suitability of simple invertebrate models in studying the basic mechanisms of stress-related epigenetic modifications, we analyzed epigenetic modifications in neurons of the freshwater snail Pomacea canaliculata after the injection of Escherichia coli-derived lipopolysaccharide (LPS). The phospho-acetylation of histone H3, together with the induction of stress-related factors, c-Fos and HSP70, were evaluated in large and small neurons of the pedal ganglia of sham- and LPS-injected snails. Immunocytochemical investigations showed that after LPS injection, the immunopositivity towards phospho (Ser10)-acetyl (Lys14)-histone H3 and c-Fos increases in the nuclei of small gangliar neurons. Western blot analysis confirmed a significant increase of phospho (Ser10)-acetyl (Lys14)-histone H3 in nuclear extracts from 2h LPS-injected animals. c-Fos protein levels were significantly augmented 6h after LPS injection. Immunocytochemistry and western blot indicated that no changes occurred in HSP70 distribution and protein levels. To our knowledge this is the first demonstration of epigenetic changes in molluscan neurons after an immune challenge and indicate the gastropod P. canaliculata as a suitable model for evolutionary and translational studies on stress-related epigenetic modifications.

Effect of high SARs produced by cell phone like radiofrequency fields on mollusk single neuron

During exposure to the cell phone electromagnetic field (EMF), some neurons in the brain at areas of peak specific absorption rate (SAR) absorb more electromagnetic energy than is permitted by existing guidelines. The goal of the present work was to investigate the influence of cell phone-like EMF signal on excitability and memory processes in single neurons. A Transverse Electromagnetic Cell (TEM Cell) was used to expose single neurons of mollusk to the EMF.Finite-Difference Time-Domain (FDTD) method was used for modeling the TEM Cell and the EMF interactions with living nerve ganglion and neurons. Neuron electrophysiology was investigated using standard microelectrode technique. SAR deposited into the single neuron was calculated to be 8.2 W/kg with a temperature increment of 1.21°C. After acute exposure, the threshold of firing of action potentials (AP) was significantly decreased (p ≈ 0.001). Time of habituation to stimulation with the intracellular current injection was increased (p ≈ 0.003). These results indicate that acute exposure to EMF at high SARs impairs the ability of neurons to store information.

A contribution to the history of the proton channel.

The low numbers of hydrogen ions in physiological solutions encouraged the assumption that H+ currents flowing through conductive pathways would be so small as to be unmeasurable even if theoretically possible. Evidence for an H+-based action potential in the luminescent dinoflagellate Noctiluca and for an H+-conducting channel created by the secretions of the bacterium Bacillus brevis, did little to alter this perception. The clear demonstration of H+ conduction in molluscan neurons might have provided the breakthrough but the new pathway was without an easily demonstrable function, and escaped general attention. Indeed the extreme measures that must be taken to successfully isolate H+ currents meant that it was some years before proton channels were identified in mammalian cells. However, with the general availability of patch-clamp techniques and evidence for an important role in mammalian neutrophils, the stage was set for a series of structure/function studies with the potential to make the proton channel the best understood channel of all. In addition, widespread genomic searches have established that proton channels play important roles in processes ranging from fertilization of the human ovum to the progression of breast cancer. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Contraction of Traumatized Nerve Cell Processes

Studies were performed on living isolated mollusk (Lymnaea stagnalis) neurons. The aim was to investigate the contractile activity of traumatized neuron processes. Retraction of process in Ringer’s solution was seen in 90% of cases. The characteristic club-shaped formation, i.e., the retraction bulb, was a marker of contraction. The rate of process contraction ranged in different neurons from 0.03 to 9 μm/min. Increases in process diameter by an average of 35% occurred during normal linear contraction, while increases in cell body volume were by an average of 30%. Three forms of contractile activity were identified: linear contraction, isometric contraction (with decreases in process width with unaltered length), and mixed contraction. It is suggested that the mechanism of retraction is involved in the formation of diastases on nerve transection and in lesions to the conducting pathways of the brain. Nerve diastases formed not only as a result of the elastic properties of its connective tissue sheaths and glia, but also because of retraction of nerve fibers.

Molluscan neurons in culture: shedding light on synapse formation and plasticity

From genes to behaviour, the simple model system approach has played many pivotal roles in deciphering nervous system function in both invertebrates and vertebrates. However, with the advent of sophisticated imaging and recording techniques enabling the direct investigation of single vertebrate neurons, the utility of simple invertebrate organisms as model systems has been put to question. To address this subject meaningfully and comprehensively, we first review the contributions made by invertebrates in the field of neuroscience over the years, paving the way for similar breakthroughs in higher animals. In particular, we focus on molluscan (Lymnaea, Aplysia, and Helisoma) and leech (Hirudo) models and the pivotal roles they have played in elucidating mechanisms of synapse formation and plasticity. While the ultimate goal in neuroscience is to understand the workings of the human brain in both its normal and diseased states, the sheer complexity of most vertebrate models still makes it difficult to define the underlying principles of nervous system function. Investigators have thus turned to invertebrate models, which are unique with respect to their simple nervous systems that are endowed with a finite number of large, individually identifiable neurons of known function. We start off by discussing in vivo and semi-intact preparations, regarding their amenability to simple circuit analysis. Despite the 'simplicity' of invertebrate nervous systems however, it is still difficult to study individual synaptic connections in detail. We therefore emphasize in the next section, the utility of studying identified invertebrate neurons in vitro, to directly examine the development, specificity, and plasticity of synaptic connections in a well-defined environment, at a resolution that it is still unapproachable in the intact brain. We conclude with a discussion of the future of invertebrates in neuroscience in elucidating mechanisms of neurological disease and developing neuron-silicon interfaces.

Impact of Amyloid-β Peptide (1–42) on Voltage-Gated Ion Currents in Molluscan Neurons

Different types of voltage-gated ion currents were recorded in isolated neurons of snail Helix pomatia using the two-microelectrode voltage-clamp technique. Application of amyloid-β peptide (1-42, 1-10 μM) in the bathing solution did not change delayed rectifier K(+)-current and leakage current, but enhanced inactivation of Ca(2+)-current and blocked Ca(2+)-dependent K(+)-current.

Acute Effect of Exposure of Mollusk Single Neuron to 900-MHz Mobile Phone Radiation

The goal of the present work was to explore the influence of commercially available cell phone irradiation on the single neuron excitability and memory processes. A Transverse Electromagnetic Cell (TEM Cell) was used to expose single neurons of mollusk to the electromagnetic field.Finite-Difference Time-Domain (FDTD) method was used for modeling the TEM Cell and the electromagnetic field interactions with living nerve ganglion and neurons. Neuron electrophysiology was investigated using standard microelectrode technique.The specific absorption rate (SAR) deposited into the single neuron was calculated to be 0.63 W/kg with a temperature increment of 0.1°C. After acute exposure, average firing threshold of the action potentials was not changed. However, the average latent period was significantly decreased. This indicates that together with latent period the threshold and the time of habituation might be altered during exposure. However, these alterations are transient and only latent period remains on the changed level.