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/.

Different receptors mediate the electrophysiological and growth cone responses of an identified neuron to applied dopamine.

Neurotransmitters are among the many cues that may guide developing axons toward appropriate targets in the developing nervous system. We have previously shown in the mollusk Lymnaea stagnalis that dopamine, released from an identified pre-synaptic cell, differentially affects growth cone behavior of its target and non-target cells in vitro. Here, we describe a group of non-target cells that also produce an inhibitory electrophysiological response to applied dopamine. We first determined, using pharmacological blockers, which receptors mediate this physiological response. We demonstrated that the dopaminergic electrophysiological responses of non-target cells were sensitive to a D2 receptor antagonist, as are known target cell responses. However, the non-target cell receptors were linked to different G-proteins and intracellular signaling pathways than the target cell receptors. Despite the presence of a D2-like receptor at the soma, the growth cone collapse of these non-target cells was mediated by D1-like receptors. This study shows that different dopamine receptor sub-types mediated the inhibitory physiological and growth cone responses of an identified cell type. We therefore not only provide further evidence that D2- and D1-like receptors can be present on the same neuron in invertebrates, but also show that these receptors are likely involved in very different cellular functions.

Transmitter-receptor interactions between growth cones of identified Lymnaea neurons determine target cell selection in vitro.

In addition to their involvement in transsynaptic communication in the adult nervous system, neurotransmitters also participate in many developmental events, such as neurite initiation and outgrowth. Although growth cones can release transmitters and are themselves sensitive to exogenously applied neurotransmitters, a direct causal relationship between the release of transmitter from one growth cone and its effect on another has not yet been demonstrated. In this study, we provide evidence that dopamine release from the growth cones of an identified Lymnaea neuron, right pedal dorsal 1 (RPeD1), differentially regulates the growth cone behavior of its in vivo target and nontarget neurons in vitro. In coculture, RPeD1 growth cones enhanced the rate of growth cone advance from target cells and synaptic connections developed immediately after contact. In contrast, RPeD1 growth cones not only inhibited the rate of growth cone advance from nontarget cells but they also induced growth cone collapse. Using a "sniffer cell" approach, we demonstrated that both RPeD1 growth cones and somata released dopamine, which can be detected at a distance of several hundred micrometers. RPeD1 somata were used to demonstrate that spontaneous release of dopamine also acted as a chemoattractant for target growth cones but as a chemorepellent for nontarget growth cones. These effects were mimicked by exogenous dopamine application, and both RPeD1 growth cone and soma-induced effects were also blocked in the presence of dopamine receptor antagonists. This study emphasizes the importance of transmitter-receptor interactions between growth cones in target cell selection.

Functional implications of neurotransmitter expression during axonal regeneration: serotonin, but not peptides, auto-regulate axon growth of an identified central neuron.

We studied the regenerative properties of one of two electrically coupled molluscan neurons, the serotonergic cerebral giant cells (CGCs) of Lymnaea stagnalis, after axotomy. The CGCs play a crucial role in feeding behavior, and when both cells are disconnected from their target neurons, animals no longer feed. When one CGC was permanently disconnected from its targets and the other was reversibly damaged by a nerve crush, the latter one regenerated over a period of 2 weeks to reform functional synapses with specific target neurons. At the same time, recovery of the feeding behavior was observed. After the crush, neuropeptide gene expression in the CGC was downregulated to approximately 50%. Serotonin synthesis, on the other hand, remained unaffected, suggesting that serotonin might have an active role in regeneration. In primary neuron culture, CGCs failed to extend neurites in the presence of serotonin; in cells that extended neurites in the absence of serotonin, focally applied serotonin, but not neuropeptides, induced growth cone collapse. Using serotonin-sensitive sniffer cells, we show that CGC neurites and growth cones release serotonin in culture. Finally, both the spontaneous and stimulation-induced release of serotonin from CGCs in culture resulted in growth cone collapse responses that could be blocked by the serotonin receptor antagonist methysergide. Our data suggest that auto-released serotonin is inhibitory to CGC neurite outgrowth in vitro. During regeneration in vivo, serotonin release might fine-tune axon guidance and branching by inducing local collapse responses in extending neurites.

Halothane affects both inhibitory and excitatory synaptic transmission at a single identified molluscan synapse, in vivo and in vitro.

In the isolated CNS of Lymnaea, a peptidergic neuron termed VD4 makes monosynaptic connections with identified pedal A cluster neurons. In this study, the pedal A (PeA) neurons were further divided into two subgroups depending upon whether they received an inhibitory or excitatory input from VD4. PeA cells inhibited by VD4 were designated PeA(I), whereas those excited by VD4 were termed PeA(E). Both inhibitory and excitatory effects of VD4 stimulation on the PeA(I) and PeA(E) cells, respectively, were mimicked by exogenous FMRFamide in culture (in vitro), implicating this or a related peptide as the transmitter utilized at the VD4-to-PeA synapses. We tested the ability of the general anesthetic, halothane, to affect either the inhibitory or the excitatory peptidergic synapses between VD4 and the PeA neurons, both in the isolated CNS (in vivo) and at the in vitro reconstructed synapses. In the presence of 1% halothane, the excitatory synaptic potential between VD4 and the PeA(E) cells was either depressed or completely abolished, whereas the inhibitory synaptic potential between VD4 and the PeA(I) cells was unaffected in the presence of 1% halothane. The inhibitory potential between VD4 and the PeA(I) cells was, however, blocked in 2% halothane. In order to determine halothane' 5 site of action, exogenous FMRFamide was applied to both PeA(E) and PeA(I) cells in the presence of 1 or 2% halothane. In 1% halothane, the excitatory responses produced by FMRFamide were substantially reduced or abolished, whereas the inhibitory responses to FMRFamide were maintained and enhanced in duration in 1% halothane. In 2% halothane, the inhibitory responses to exogenous FMRFamide remained unchanged. It, therefore, appears that halothane exerts effects at both the pre- and postsynaptic level of the synapse, although presynaptic transmitter release is probably not substantially affected until a concentration of 2% halothane is reached. Our data provide the first evidence that clinically relevant concentrations of halothane (1-2%) affect both excitatory and inhibitory peptidergic synaptic transmission between identified neurons in the nervous system. Furthermore, excitatory transmission is abolished at lower anesthetic concentrations than inhibitory transmission.

Predicting the success of reambulation in patients with Duchenne muscular dystrophy.

We used biochemical and clinical variables to develop a method to predict the expected duration of independent walking following surgery and bracing in patients with Duchenne muscular dystrophy. Data from the records of fifty patients were analyzed by linear and multiple regression. The most useful factors, applied in combination, in predicting the duration of walking ability after bracing were: percentage of residual muscle strength, vital capacity, creatinine coefficient, motivation of the patient at the time of bracing, and decrease in creatinine coefficient in the two years prior to bracing. This system uses readily available variables to predict the response to bracing in patients with Duchenne muscular dystrophy. Improvement in the criteria for the selection of patients for surgery and bracing is important in view of the economic cost as well as the demands on the time and energy of these children and their parents.

Modulation of reconstructed peptidergic synapses and electrical synapses by general anaesthetics.

1. The actions of clinically relevant concentrations of general anaesthetics on reconstructed peptidergic synapses and electrical synapses in the intact brain of the mollusc Lymnaea stagnalis (L.) are described. 2. At identified, reconstructed, FMRFamidergic synapses, chemical synaptic transmission is completely blocked in 2% halothane. 3. Inhibitory postsynaptic responses to directly applied FMRFamide are maintained in 2% halothane and are enhanced in 1% halothane, unlike excitatory responses which are abolished at this concentration. 4. Met-enkephalin normally produces inhibitory responses on postsynaptic PeA neurones, but these are non-reversibly abolished by halothane, whose presence induces novel, dose-dependent, enkephalinergic depolarising responses. 5. The biophysical effects of volatile anaesthetics and sodium pentobarbital on neuronal membranes have been described and they are shown to have opposite dose-dependent effects on input resistance, input conductance and time constant of the electrically coupled neurones VD1 and RPD2. 6. Volatile anaesthetics decouple the neurones VD1 and RPD2 in a dose dependent manner, whilst sodium pentobarbital either enhances coupling or has no effect, depending on the concentration used.

Children's competency to consent: an ethical dilemma.

The application of the best interests principle in current legislation creates an ethical dilemma in relation to children's consent to treatment. The guiding principle of the UN Convention on the Rights of the Child (1989) symbolises a formal expression of children's participation rights. Children's rights to consent to treatment are granted on socially determined ideals of competency. Children's participation in health care is increasingly advocated in legislation but many barriers remain. Nurses can facilitate children's participation through communicating information and creating partnerships with children.

Perturbation of the activity of a single identified neuron affects long-term memory formation in a molluscan semi-intact preparation.

The aim of this study was to investigate the neural basis of operant conditioning in a semi-intact preparation of the pond snail, Lymnaea stagnalis. Lymnaea learns, via operant conditioning, to reduce its aerial respiratory behaviour in response to an aversive tactile stimulus to its open pneumostome. Here we report the successful conditioning of naïve semi-intact preparations to show ;learning in the dish' and long-term memory that persists for at least 18 h. The neurons that generate this behaviour are readily identifiable and, for the first time, we have recorded from a neuron during a training paradigm that leads to long-term memory formation in the same preparation. Specifically, we recorded from the respiratory neuron Right Pedal Dorsal 1 (RPeD1), which is part of the respiratory central pattern generator and initiates the aerial respiratory behaviour. Previous studies have shown that long-term memory of this behaviour results in reduced RPeD1 activity. In the present study, we demonstrate that preventing RPeD1 impulse activity between training sessions reduces the number of sessions needed to produce long-term memory in our semi-intact preparation.

The molluscan RING-finger protein L-TRIM is essential for neuronal outgrowth.

The tripartite motif proteins TRIM-2 and TRIM-3 have been put forward as putative organizers of neuronal outgrowth and structural plasticity. Here, we identified a molluscan orthologue of TRIM-2/3, named L-TRIM, which is up-regulated during in vitro neurite outgrowth of central neurons. In adult animals, L-Trim mRNA is ubiquitously expressed at low levels in the central nervous system and in peripheral tissues. Central nervous system expression of L-Trim mRNA is increased during postnatal brain development and during in vitro and in vivo neuronal regeneration. In vitro double-stranded RNA knock-down of L-Trim mRNA resulted in a >70% inhibition of neurite outgrowth. Together, our data establish a crucial role for L-TRIM in developmental neurite outgrowth and functional neuronal regeneration and indicate that TRIM-2/3 family members may have evolutionary conserved functions in neuronal differentiation.

Health needs of an industrial population.

PIP: To assess the health needs of an industrial population, the type of industry, its distribution, its number of employees, special work hazards, environment, and other problems need to be considered. Additionally, attention must be given to the age, sex, ethnic distribution, and rural or urban background of the population groups involved. Each of the groups has its own special needs in the health field. The worker along with the physician and management needs to participate in determining what his/her health needs are. Conclusions and recommendations are offered as follows: 1) known and accepted procedures must be refined, expanded, and critically evaluated so that a worker's health is protected within his/her environment and his/her environment is controlled; 2) industries should pool their health data in order to detect early health effects of industrial exposure and to provide a basis for epidemiological studies; 3) industrial medical departments should become involved in community medicine; 4) emotional health needs will play a large role and will include the provision of supportive short-term therapy, birth control counseling, day care services, and preretirement counseling along with other specific needs of various groups; 5) rehabilitation efforts must be expanded; 6) health education efforts are needed to slow the progress of degenerative diseases; 7) additional research on susceptibility to industrial substances of individuals with genetic defects needs to be conducted, and the means of selecting out such individuals before employment should be developed; and 8) the feasibility of developing a health information center for small industrial medical clinics should be explored.^ieng

Destructive monarticular arthritis secondary to anticoagulant therapy.

Hemarthrosis secondary to anticoagulant therapy is a well-known clinical problem. The pathologic process usually occurs in large joints and is reversible with the discontinuation of the anticoagulant medication. The condition presented for consideration here is unusual for two reasons. First, it is relatively uncommon for the ankle joint to be involved. Second, the destructive arthritis progressed after the medication was discontinued. Resting the involved joint until symptoms subside is often adequate treatment for anticoagulant-induced hemarthrosis. In an 84-year-old man, an arthrodesis was necessary to achieve a symptom-free ankle joint. Hemarthrosis secondary to anticoagulant medication may not be a benign disease process.

Dopamine regulation of neurite outgrowth from identified Lymnaea neurons in culture.

1. An identified dopaminergic interneuron (RPeD1) of the snail Lymnaea stagnalis, makes specific synaptic connections with a number of target (VI and VJ) but not non-target (VF and RPB) neurons in vivo. When cultured in vitro with both target and non-target cells, RPeD1 re-establishes synapses with target cells only. 2. To test whether exogenous dopamine exerts effects on the neurite outgrowth of both target and non-target neurons respectively, these cells were cultured in conditioned media (CM) in the presence of dopamine (10(-5) M). The growth of the non-target cells was severely restricted and retarded in the presence of dopamine. These data suggest that dopamine may regulate neurite outgrowth of non-target cells in culture. 3. The growth regulatory effects of dopamine on the non-target cells were blocked in the presence of a dopamine receptor antagonist (R(+) SCH-23390, 10(-4) M). These results indicate that dopamine-induced growth regulation of the non-target cells is mediated via dopamine receptors on these cells. 4. In the absence of conditioned media, dopamine was not sufficient to exert growth promoting effects on either target or non-target cells. 5. Taken together, our data show that dopamine differentially regulates growth of identified Lymnaea neurons in culture. Dopamine alone, however, is not sufficient to initiate and support neurite outgrowth from these cells. Rather, it functions to suppress the neurite outgrowth of the non-target cells, initiated by the conditioned media.

Neurotransmitters and neurodevelopment. Role of dopamine in neurite outgrowth, target selection and specific synapse formation.

Neurotransmitters and their receptors appear early during nervous system development and are thought to play important roles in neurite outgrowth, growth cone motility, target cell selection and synaptogenesis. In vivo studies in both vertebrates and invertebrates have shown that the perturbations of embryonic transmitter expression result in abnormal morphological and synaptic development. In vitro studies have further revealed that transmitters are capable of affecting neurite outgrowth and growth cone behaviour. The precise cellular mechanisms by which neurotransmitters affect these developmental steps are, however, poorly defined. In vitro, a presynaptic neuron from the mollusc Lymnaea stagnalis releases dopamine, which induces both growth cone attraction and growth cone collapse of target and non-target cell growth cones, respectively. We propose that the ability of dopamine to differentially affect growth cone motility of two cell types results from a divergence of the dopamine receptor-activated second messenger pathways at the G-protein level. Such transmitter-receptor interactions between growth cones of specific neurons may not only induce changes in the growth cone motility, but may subsequently play an important role in target cell selection and specificity of synaptogenesis.

Neural changes after operant conditioning of the aerial respiratory behavior in Lymnaea stagnalis.

In this study, we demonstrate neural changes that occurred during operant conditioning of the aerial respiratory behavior of Lymnaea stagnalis. Aerial respiration in Lymnaea occurs at the water interface and is achieved by opening and closing movements of its respiratory orifice, the pneumostome. This behavior is controlled by a central pattern generator (CPG), the neurons of which, as well as the motoneurons innervating the pneumostome, have previously been identified and their synaptic connections well characterized. The respiratory behavior was operantly conditioned by applying a mechanical stimulus to the open pneumostome whenever the animal attempted to breathe. This negative reinforcement to the open pneumostome resulted in its immediate closure and a significant reduction in the overall respiratory activity. Electrophysiological recordings from the isolated CNSs after operant conditioning showed that the spontaneous patterned respiratory activity of the CPG neurons was significantly reduced. This included reduced spontaneous activity of the CPG interneuron involved in pneumostome opening (input 3 interneuron) and a reduced frequency of spontaneous tonic activity of the CPG interneuron [right pedal dorsal 1 (RPeD1)]. The ability to trigger the patterned respiratory activity by electrical stimulation of RPeD1 was also significantly reduced after operant conditioning. This study therefore demonstrates significant changes within a CPG that are associated with changes in a rhythmic homeostatic behavior after operant conditioning.

Halothane-induced synaptic depression at both in vivo and in vitro reconstructed synapses between identified Lymnaea neurons.

1. In the present study we tested the ability of the general anesthetic, halothane, to affect synaptic transmission at in vivo and in vitro reconstructed peptidergic synapses between identified neurons of Lymnaea stagnalis. 2. An identified respiratory interneuron, visceral dorsal 4 (VD4), innervates a number of postsynaptic cells in the central ring ganglia of Lymnaea. Because VD4 has previously been shown to exhibit immunoreactivity for FMRFamide-related peptides, it was hypothesized that these peptides may be utilized by VD4 during synaptic transmission. In the intact, isolated CNS of Lymnaea, we have identified novel connections between VD4 and the pedal A (PeA) cells. We demonstrate that VD4 makes inhibitory connections with the PeA neurons, in particular PeA4, and that these synaptic responses are mimicked by exogenous application of FMRFamide. 3. The synaptic transmission between VD4 and the PeA cells in an intact, isolated CNS preparation was completely blocked in 2%, but not 1% halothanc. Interestingly, the postsynaptic responses (PeA) to exogenous FMRFamide were maintained in the presence of both 1 and 2% halothane. 4. To determine the specificity of the observed responses and to determine the precise synaptic site of anesthetic action, we reconstructed the VD4/PeA synapses in vitro. After isolation from their respective ganglia, both cell types extended processes and established neuritic contact. We demonstrated that not only did the presynaptic neuron reestablish the appropriate inhibitory synapses with the PeA neurons, but that the PeA cells also maintained their responsiveness to exogenous FMRFamide. 5. Superfusion of the in vitro synaptically connected VD4 and PeA cells with 2% halothane completely abolished the synaptic transmission between these cells. However, even higher concentrations of 4% halothane failed to block the responsiveness of the PeA neurons to exogenous FMRFamide. Moreover, both 1 and 2% halothane enhanced the duration of the postsynaptic response to exogenously applied FMRFamide. These data suggest that the halothane-induced depression of synaptic transmission most likely occurred at the presynaptic level. 6. This study provides the first direct evidence that peptidergic transmission in the nervous system may also be susceptible to the actions of general anesthetics. In addition, we utilized a novel approach of in vitro reconstructed synapses for studying the effects of general anesthetics on monosynaptic transmission in the absence of other synaptic influences.

Transmucosal electrical resistance in rabbit isolated gastric mucosa during exposure to acid.

1. Transmucosal electrical resistance (Rt) and short-circuit current (Isc) were determined in rabbit isolated fundic mucosa. Under basal conditions, with a HCO(3-)-free HEPES-buffered solution (pH 7.4) bathing both sides of the mucosae, Rt was 161.5 + 5.0 omega cm2 and Isc 41.8 +/- 1.8 microA cm-2, and these values were not significantly different to values observed in HCO(3-)-buffered Krebs-Hensleit solution. 2. The basal Isc was inhibited by the Cl- channel blocker diphenylamine-2-carboxylate, and ouabain, but unaffected by the Na+ channel blocker amiloride (10(-5) M), consistent with electrogenic chloride secretion dependent upon a sodium gradient. Prostaglandin E2 (10(-7) M) stimulated an increase in Isc which was susceptible to inhibition by diphenylamine-2-carboxylate, but not amiloride, again consistent with Cl- secretion. 3. Stepwise acidification of the mucosal solution to pH 2.8 resulted in an increase in Rt of 43%, as compared with that measured with mucosal pH 7.4. Isc did not change during the acidification to pH 2.8, indicating retention of tissue viability. Increased Rt while Isc remained constant is consistent with an acid-induced decrease in the shunt (paracellular) conductance in this Cl(-)-secreting tissue. At pH less than 2.8, Rt declined rapidly and Isc declined and reversed, consistent with H+ back-diffusion. Scanning electron microscopic investigation of tissue exposed to mucosal pH 2.8 revealed little difference from control (pH 7.4) tissue, but there was considerable evidence of cellular damage and membrane disruption in tissue exposed to pH 1.8. 4. Acidification of the serosal solution did not increase Rt, which was maintained until pH 3.7, and then rapidly declined at pH less than 3.7. Bilateral acidification produced a mixed response; Rt increased, as for mucosal acidification, down to pH 2.8, after which there was a rapid decline in Rt following the pattern observed for serosal acidification. 5. Compared at a mucosal pH approximately 2.8, DIDS (4 x 10(-4) M) and amiloride (10(-3) M) inhibited the acid-induced increase in Rt, suggesting a role for both Cl(-)-HCO3- and Na(+)-H+ exchange in the response. In contrast, the acid-induced increase in Rt was unaffected by a lower concentration of amiloride (10(-5) M), acetazolamide and ouabain. Therefore, neither Na+ channels nor a Na+ gradient appear to be involved in the acid-induced increase in Rt.(ABSTRACT TRUNCATED AT 400 WORDS)