Crustacean cardiac ganglion model reveals constraints on morphology and conductances

The crustacean cardiac ganglion (CG) network coordinates the rhythmic contractions of the heart muscle to control the circulation of blood. The network consists of 9 cells, 5 large motor cells (LC1-5) and 4 small endogenous pacemaker cells (SCs). We report a new three-compartmental biophysical model of an LC that is morphologically realistic and includes provision for inputs from the SCs via a gap-junction coupled spike-initiation-zone (SIZ) compartments. To determine physiologically viable LC models in this realistic configuration, maximal conductances in three compartments of an LC are determined by random sampling from a biologically-characterized 9D-parameter space, followed by a three stage rejection protocol that checks for conformity with electrophysiological features from single cell traces. LC models that pass the single cell rejection protocol are then incorporated into a network model which is then used in a final rejection protocol stage. Using disparate experimental data, the study provides hitherto unknown structure-function insights related to the crustacean cardiac ganglion large cell, including predictions about morphology including the role of its SIZ, and the differential roles of active conductances in the three compartments. Further, we extend analyses of emergent conductance relationships and correlations in model neurons relative to their biological counterparts, allowing us to make inferences both with respect to the biological system as well as the implications of the ability to detect such relationships in populations of model neurons going forward.

Coupling, membrane conductance, and ion channel mRNA profiles in the establishment and maintenance of network activity in the crustacean cardiac ganglion

Neural networks produce critical rhythmic behaviors throughout an animal's lifespan, despite growth, differing environments, and changes in physiological state. This requires networks which balance stability in their properties with the plasticity necessary to respond to altered demands or perturbations. Studying the mechanisms which confer these properties requires a well characterized system with a known network topology and identifiable neurons that are amenable to both electrophysiological and molecular characterization and manipulation. Here, we use two networks from Cancer borealis to explore activity dependent regulation of cell connectivity, changes in cell properties with prolonged perturbation, and reliability of gene expression as a means for cell identification. For the first two topics we use the cardiac ganglion alone. The cardiac ganglion consists of a kernel of four interneurons that drive five motor neurons (termed large cells, LCs) which innervate the heart musculature. LCs burst synchronously due to simultaneous stimulation and electrical coupling through gap junctions. Depolarizing pharmacological perturbations have been shown to result in hyperexcitability (Ransdell et al., 2012a) and disrupt synchrony between LCs (Lane et al., 2016) eliciting rapid plasticity in ionic currents and electrical coupling which restores synchrony and excitability (Ransdell et al., 2012a; Lane et al., 2016). The salient electrophysiological signal which elicits coupling plasticity has not been identified. Using voltage clamp we directly control LC depolarizations to vary amplitude and timing of activity between LCs. We find that timing between cells, rather than depolarization elicits plasticity with the direction, i.e., potentiation or depression, being determined by the degree of desynchronization. With dynamic clamp we artificially couple networks from two animals and show that strong coupling with sufficient desynchronization can compromise a cell's output. These results suggest that coupling strength is tuned promoting synchrony or baseline cellular activity in a degree dependent manner. While rapid compensatory plasticity to hyperexcitability has been shown, it is unknown whether the changes are solely post-transcriptional and whether the short-term changes persist over longer time scales. We perturb networks for one or twenty-four hours and compare LCs' excitability, membrane properties, and abundances of ion channel and gap junction transcripts. We find evidence of rapid transcriptional changes at one hour, which may be maintained or regress at twenty-four hours. Additionally, we find that membrane properties and excitability are not maintained from one to twenty-four hours, suggesting a failure to maintain homeostasis or that additional compensatory changes are occurring at the network level. To address our third topic, we use LCs in addition to neurons collected form the stomatogastric ganglion which coordinates mastication and filtering in the digestive track. Both systems allow for unambiguous identification of cells based on anatomy or neuronal projections. We use this to evaluate the efficacy of cluster estimation procedures, clustering methods, and classification algorithms to determine the number of cell types present, group like cells together, and identify cells based on gene expression alone. We use single cell RNA-seq and single cell qRT-PCR to measure all contigs or a select set of ion channel, receptor, and gap junction mRNAs. We find these methods do not reproduce the known number of cell types present. Furthermore, although clustering and classification both outperform chance, we are unable to recapitulate cell type with complete accuracy from these data. These results indicate that, while promising, determining cell type by molecular profiling should not be relied on as the sole metric of cell type determination.

Differential neuropeptide modulation of premotor and motor neurons in the lobster cardiac ganglion

Premotor and motor neurons of the Homarus americanus cardiac ganglion (CG) are normally electrically and chemically coupled, and generate rhythmic bursting that drives cardiac contractions; we show that they can establish independent bursting patterns when physically decoupled by a ligature. The neuropeptide myosuppressin modulates different aspects of the bursting pattern in these neuron types to determine the overall modulation of the intact CG. Differential distribution of myosuppressin receptors may underlie the observed responses to myosuppressin.

Cardiac neural crest contributes to cardiomyocytes in amniotes and heart regeneration in zebrafish

Cardiac neural crest cells contribute to important portions of the cardiovascular system including the aorticopulmonary septum and cardiac ganglion. Using replication incompetent avian retroviruses for precise high-resolution lineage analysis, we uncover a previously undescribed neural crest contribution to cardiomyocytes of the ventricles in Gallus gallus, supported by Wnt1-Cre lineage analysis in Mus musculus. To test the intriguing possibility that neural crest cells contribute to heart repair, we examined Danio rerio adult heart regeneration in the neural crest transgenic line, Tg(−4.9sox10:eGFP). Whereas the adult heart has few sox10+ cells in the apex, sox10 and other neural crest regulatory network genes are upregulated in the regenerating myocardium after resection. The results suggest that neural crest cells contribute to many cardiovascular structures including cardiomyocytes across vertebrates and to the regenerating heart of teleost fish. Thus, understanding molecular mechanisms that control the normal development of the neural crest into cardiomyocytes and reactivation of the neural crest program upon regeneration may open potential therapeutic approaches to repair heart damage in amniotes.

Dopamine maintains network synchrony via direct modulation of gap junctions in the crustacean cardiac ganglion

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.