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Peptides are the largest and most diverse class of molecules used for neurochemical communication, playing key roles in the control of essentially all aspects of physiology and behavior. The American lobster, Homarus americanus, is a crustacean of commercial and biomedical importance; lobster growth and reproduction are under neuropeptidergic control, and portions of the lobster nervous system serve as models for understanding the general principles underlying rhythmic motor behavior (including peptidergic neuromodulation). While a number of neuropeptides have been identified from H. americanus, and the effects of some have been investigated at the cellular/systems levels, little is currently known about the molecular components of neuropeptidergic signaling in the lobster. Here, a H. americanus neural transcriptome was generated and mined for sequences encoding putative peptide precursors and receptors; 35 precursor- and 41 receptor-encoding transcripts were identified. We predicted 194 distinct neuropeptides from the deduced precursor proteins, including members of the adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin C, bursicon, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptide, diuretic hormone 31, diuretic hormone 44, eclosion hormone, FLRFamide, GSEFLamide, insulin-like peptide, intocin, leucokinin, myosuppressin, neuroparsin, neuropeptide F, orcokinin, pigment dispersing hormone, proctolin, pyrokinin, SIFamide, sulfakinin and tachykinin-related peptide families. While some of the predicted peptides are known H. americanus isoforms, most are novel identifications, more than doubling the extant lobster neuropeptidome. The deduced receptor proteins are the first descriptions of H. americanus neuropeptide receptors, and include ones for most of the peptide groups mentioned earlier, as well as those for ecdysis-triggering hormone, red pigment concentrating hormone and short neuropeptide F. Multiple receptors were identified for most peptide families. These data represent the most complete description of the molecular underpinnings of peptidergic signaling in H. americanus, and will serve as a foundation for future gene-based studies of neuropeptidergic control in the lobster.

The postdevelopmental basis of cellular identity and the unique cellular output of a particular neuron type are of particular interest in the nervous system because a detailed understanding of circuits responsible for complex processes in the brain is impeded by the often ambiguous classification of neurons in these circuits. Neurons have been classified by morphological, electrophysiological, and neurochemical techniques. More recently, molecular approaches, particularly microarray, have been applied to the question of neuronal identity. With the realization that proteins expressed exclusively in only one type of neuron are rare, expression profiles obtained from neuronal subtypes are analyzed to search for diagnostic patterns of gene expression. However, this expression profiling hinges on one critical and implicit assumption: that neurons of the same type in different animals achieve their conserved functional output via conserved levels and quantitative relationships of gene expression. Here we exploit the unambiguously identifiable neurons in the crab stomatogastric ganglion to investigate the precise quantitative expression profiling of neurons at the level of single-cell ion channel expression. By measuring absolute mRNA levels of six different channels in the same individually identified neurons, we demonstrate that not only do individual cell types possess highly variable levels of channel expression but that this variability is constrained by unique patterns of correlated channel expression.

To what extent do identified neurons from different animals vary in their expression of ion channel genes? In neurons of the same type, is ion channel expression highly variable and/or is there any relationship between ion channel expression that is conserved? To address these questions we measured ion channel mRNA in large cells (LCs) of the crab cardiac ganglion. We cloned a calcium channel, caco, and a potassium channel, shaker. Using single-cell quantitative PCR, we measured levels of mRNA for these and 6 other different ion channels in cardiac ganglion LCs. Across the population of LCs we measured 3-9 fold ranges of mRNA levels, and we found correlations in the expression of many pairs of conductances In previous measurements from the crab stomatogastric ganglion (STG), ion channel expression was variable, but many pairs of channels had correlated expression. However, each STG cell type had a unique combination of ion channel correlations. Our findings from the crab cardiac ganglion are similar, but the correlations in the LCs are different from those in STG neurons, supporting the idea that such correlations could be markers of cell identity or activity.

The medicinal leech is one of the most venerated model systems for the study of fundamental nervous system principles, ranging from single-cell excitability to complex sensorimotor integration. Yet, molecular analyses have yet to be extensively applied to complement the rich history of electrophysiological study that this animal has received. Here, we generated the first de novo transcriptome assembly from the entire central nervous system of Hirudo verbana, with the goal of providing a molecular resource, as well as to lay the foundation for a comprehensive discovery of genes fundamentally important for neural function. Our assembly generated 107,704 contigs from over 900 million raw reads. Of these 107,704 contigs, 39,047 (36%) were annotated using NCBI's validated RefSeq sequence database. From this annotated central nervous system transcriptome, we began the process of curating genes related to nervous system function by identifying and characterizing 126 unique ion channel, receptor, transporter, and enzyme contigs. Additionally, we generated sequence counts to estimate the relative abundance of each identified ion channel and receptor contig in the transcriptome through Kallisto mapping. This transcriptome will serve as a valuable community resource for studies investigating the molecular underpinnings of neural function in leech and provide a reference for comparative analyses.

The Large Cell (LC) motor neurons of the crab cardiac ganglion have variable membrane conductance magnitudes even within the same individual, yet produce identical synchronized activity in the intact network. In a previous study we blocked a subset of K+ conductances across LCs, resulting in loss of synchronous activity (Lane et al., 2016). In this study, we hypothesized that this same variability of conductances makes LCs vulnerable to desynchronization during neuromodulation. We exposed the LCs to serotonin (5HT) and dopamine (DA) while recording simultaneously from multiple LCs. Both amines had distinct excitatory effects on LC output, but only 5HT caused desynchronized output. We further determined that DA rapidly increased gap junctional conductance. Co-application of both amines induced 5HT-like output, but waveforms remained synchronized. Furthermore, DA prevented desynchronization induced by the K+ channel blocker tetraethylammonium (TEA), suggesting that dopaminergic modulation of electrical coupling plays a protective role in maintaining network synchrony.

Experiments on neurons in the heart system of the leech reveal why rhythmic behaviors differ between individuals.Experiments on neurons in the heart system of the leech reveal why rhythmic behaviors differ between individuals.

Motor neurons of the crustacean cardiac ganglion generate virtually identical, synchronized output despite the fact that each neuron uses distinct conductance magnitudes. As a result of this variability, manipulations that target ionic conductances have distinct effects on neurons within the same ganglion, disrupting synchronized motor neuron output that is necessary for proper cardiac function. We hypothesized that robustness in network output is accomplished via plasticity that counters such destabilizing influences. By blocking high-threshold K+ conductances in motor neurons within the ongoing cardiac network, we discovered that compensation both resynchronized the network and helped restore excitability. Using model findings to guide experimentation, we determined that compensatory increases of both GA and electrical coupling restored function in the network. This is one of the first direct demonstrations of the physiological regulation of coupling conductance in a compensatory context, and of synergistic plasticity across cell- and network-level mechanisms in the restoration of output. Neurons can communicate with each other by releasing chemicals called neurotransmitters, or by forming direct connections with each other known as gap junctions. These direct connections allow electrical impulses to flow from one neuron to another via pores in the membranes between the cells. Unlike communication via neurotransmitters, gap junctions are usually thought to be hard-wired and unchanging over the life of the animal. Lane et al. recorded electrical activity in a network of neurons that generates rhythmic heart contractions in the Jonah crab. Neurons in this network usually all fire an electrical impulse at the same time, which is crucial to make sure that the whole heart contracts at the same time. The experiments show that drugs that block potassium channel pores in the membrane cause the neurons to fire too much and at different times to each other. However, the network of neurons soon adapted to the changes caused by the drugs and returned to working as normal. Mimicking these changes in a computer model of the neuron network, together with experimental data, showed that changes to the gap junctions play a major role in restoring normal activity to the network. The next step following on from this research is to understand how a network of neurons ‘senses’ that it is not working normally and changes its electrical activity.

The authors found considerable preparation-to-preparation variability in the strength of two identified synapses, the amplitude of a modulator-evoked current and the expression of six ion channel genes in the pyloric circuit of the crab stomatogastric ganglion. These parameters correlated with circuit performance. Circuits produced similar outputs because of compensatory and coordinated changes among the parameters. How different are the neuronal circuits for a given behavior across individual animals? To address this question, we measured multiple cellular and synaptic parameters in individual preparations to see how they correlated with circuit function, using neurons and synapses in the pyloric circuit of the stomatogastric ganglion of the crab Cancer borealis . There was considerable preparation-to-preparation variability in the strength of two identified synapses, in the amplitude of a modulator-evoked current and in the expression of six ion channel genes. Nonetheless, we found strong correlations across preparations among these parameters and attributes of circuit performance. These data illustrate the importance of making multidimensional measurements from single preparations for understanding how variability in circuit output is related to the variability of multiple circuit parameters.

It is often assumed that all neurons of the same cell type have identical intrinsic properties, both within an animal and between animals. We exploited the large size and small number of unambiguously identifiable neurons in the crab stomatogastric ganglion to test this assumption at the level of channel mRNA expression and membrane currents (measured in voltage-clamp experiments). In lateral pyloric (LP) neurons, we saw strong correlations between measured current and the abundance of Shal and BK-KCa mRNAs (encoding the Shal-family voltage-gated potassium channel and large-conductance calcium-activated potassium channel, respectively). We also saw two- to fourfold interanimal variability for three potassium currents and their mRNA expression. Measurements of channel expression in the two electrically coupled pyloric dilator (PD) neurons showed significant interanimal variability, but copy numbers for IH (encoding the hyperpolarization-activated, inward-current channel) and Shal mRNA in the two PD neurons from the same crab were similar, suggesting that the regulation of some currents may be shared in electrically coupled neurons. Note: The PDF version of this article was corrected on 05 February 2006. Please see the PDF for details.

Background Crustaceans have been studied extensively as model systems for nervous system function from single neuron properties to behavior. However, lack of molecular sequence information and tools have slowed the adoption of these physiological systems as molecular model systems. In this study, we sequenced and performed de novo assembly for the nervous system transcriptomes of two decapod crustaceans: the Jonah crab ( Cancer borealis ) and the American lobster ( Homarus americanus ). Results Forty-two thousand, seven hundred sixty-six and sixty thousand, two hundred seventy-three contigs were assembled from C. borealis and H. americanus respectively, representing 9,489 and 11,061 unique coding sequences. From these transcripts, genes associated with neural function were identified and manually curated to produce a characterization of multiple gene families important for nervous system function. This included genes for 34 distinct ion channel types, 17 biogenic amine and 5 GABA receptors, 28 major transmitter receptor subtypes including glutamate and acetylcholine receptors, and 6 gap junction proteins – the Innexins. Conclusion With this resource, crustacean model systems are better poised for incorporation of modern genomic and molecular biology technologies to further enhance the interrogation of fundamentals of nervous system function.