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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. [ABSTRACT FROM AUTHOR]

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.

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.

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.

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.

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.

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.

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.

One hundred five patients were treated for slipped capital femoral epiphyses during the period from 1964 to 1976. Attempts were made to evaluate the differences in results of multiple pinning and open epiphysiodesis performed to treat this problem. Pinning in situ was performed in 61 hips, and open epiphysiodesis was performed in 33 hips. The average follow-up period was seven years four months for pinning in situ and six years seven months for open epiphysiodesis. The average slippage was 22 degrees for patients treated by pinning in situ and 30 degrees for patients treated by open epiphysiodesis. At follow-up evaluation 91.7% of the patients treated by pinning in situ had good or excellent results, as compared with 71.6% of the patients treated by epiphysiodesis. For the patients treated by pinning in situ, 5% had poor results, and 3.3% were considered failures. For the patients treated by epiphysiodesis, 3.4% had poor results, while 25% were considered failures. Pinning in situ is the treatment of choice. It is more predictable, has less complications, and provides better long-term results.

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.

Periarticular fibrous muscle contractures in adults from repeated injections in the same site is predictable. The causes of joint contracture in children are many and complex, but in adults it seems certain that this phenomenon is the result of repeated injections of analgesics or other agents into 1 muscle area. Any drug if repeatedly injected locally may cause fibrosis of the muscle and subsequent joint contracture. Five cases of bilateral abduction contracture of the shoulder in adults including the first case of bilateral abduction contractures of shoulder and hip plus bilateral flexion contracture of elbow and extension contracture of a knee are reported. No underlying disease which might predispose to this fibrosis of muscles was noted. The frequency and period of injections were variable over several years. In all patients the interference in activtities of daily living were serious, but the deformities were corrected by release of the fibrous band with relief of discomfort and restoration of joint motion without recurrence. Noting the potential complication of repeated intramuscular injections in one area, this practice should be avoided whenever possible in adults, as well as in children.