Molecular and functional characterization of the mouse intracardiac nervous system

Background: The intracardiac nervous system (ICNS) refers to clusters of neurons, located within the heart, that participate to the neuronal regulation of cardiac functions and are involved in the initiation of cardiac arrhythmias. Therefore, deciphering the role of the ICNS in cardiac physiology and physiopathology is mandatory. Whereas transgenic mouse models represent powerful tools to reach this goal, the mouse ICNS is still poorly characterized. Objective: The objective of the present study was to provide a phenotypic, electrophysiological and pharmacological characterization of the mouse ICNS. Methods: Global cardiac innervation and phenotypic diversity was investigated by performing immunohistochemistry on cleared murine heart and on tissue sections. Patch clamp technique was used for electrophysiological and pharmacological characterization of isolated mouse intracardiac neurons. Results: We identified the expression of 7 distinct neuronal markers within mouse intracardiac neurons demonstrating the neurochemical diversity of this network. Of note, we described for the first time in mouse, the existence of neuron expressing the calcium binding protein calbindin, the neuropeptide Y (NPY) and the cocain and amphetamine regulated transcript (CART) peptide. Electrophysiological studies also revealed the existence of two different neuronal population based on their electrical behavior. Finally, we demonstrated that these neurons can be modulated by several neuromodulators. Conclusion: This study demonstrated that mouse ICNS shares similar molecular and functional complexity to that of other species and therefore is a suitable model to decipher the role of individual neuronal subtypes in the modulation of cardiac function and in the initiation of cardiac arrhythmias.

Structural Identification of the Pacemaker Cells and Expression of Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channels in the Heart of the Wild Atlantic Cod, Gadus morhua (Linnaeus, 1758)

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are proteins that contain highly conserved functional domains and sequence motifs that are correlated with their unique biophysical activities, to regulate cardiac pacemaker activity and synaptic transmission. These pacemaker proteins have been studied in mammalian species, but little is known now about their heart distribution in lower vertebrates and c-AMP modulation. Here, we characterized the pacemaker system in the heart of the wild Atlantic cod (Gadus morhua), with respect to primary pacemaker molecular markers. Special focus is given to the structural, ultrastructural and molecular characterization of the pacemaker domain, through the expression of HCN channel genes and the immunohistochemistry of HCN isoforms, including the location of intracardiac neurons that are adjacent to the sinoatrial region of the heart. Similarly to zebrafish and mammals, these neurons are immunoreactive to ChAT, VAChT and nNOS. It has been shown that cardiac pacemaking can be modulated by sympathetic and parasympathetic pathways, and the existence of intracardiac neurons projecting back to the central nervous system provide a plausible link between them.

Ganglionic and non-ganglionic neurons in frog heart: do they differ?

This study was designed to compare the morphology of neurons in relation to their distance from the major nerve trunks in the heart of the frog Rana temporaria. Seventy-nine intracardiac neurons were labelled intracellularly with fluorescent markers Lucifer Yellow CH and Alexa Fluor 568. The neurons located on the extensions of the vagus nerve were considered as ganglionic, while neurons spread loosely at further distance from these extensions were considered as non-ganglionic. The mean area of the soma in ganglionic neurons was about 25% larger than in non-ganglionic neurons. Ganglionic neurons had a higher soma area/nucleus area ratio than non-ganglionic neurons. Although both the total number and the total length of dendrite-like processes was similar between the two groups, ganglionic neurons had significantly fewer dendrite-like processes from the soma (1.5±0.3 vs. 3.9±1.0; P<0.05) and shorter total length of these processes from the soma (63±18 μm vs. 178±51 μm; P<0.05). In conclusion, ganglionic and non-ganglionic frog intracardiac neurons exhibit substantial morphological differences. We hypothesize that these differences may indicate different projections or variations in the number of their preganglionic inputs.

Quantitative study of synaptic boutons on frog intracardiac neurons

The frog is a useful model to study the structure and function of intracardiac neurons. The goal of this study was to evaluate the size and distribution of synaptic boutons on the intracardiac neurons in the frog Rana temporaria. Interatrial septa from four animals were double-labelled immunohistochemically for the cholinergic marker choline acetyltransferase (ChAT) and the marker of synaptic vesicles synaptophysin (SYP). One hundred intracardiac neurons were analysed by confocal microscopy. Terminals of preganglionic axons were strongly positive for ChAT, while synaptic boutons were strongly positive for both ChAT and SYP. The number of synaptic boutons per neuron ranged from 2 to 121 and was 10±2 (mean±SE). The total area of synaptic boutons ranged from 6 μm2 to 270 μm2 and was 98±6 μm2. The largest total area of synaptic boutons was found on the axonal half of neuronal soma (59±4 μm2). The total areas of synaptic boutons on both the non-axonal half of soma and the proximal axon were smaller (36±3 μm2; 6±2 μm2; P<0.001). Synaptic boutons occupied 13±1% of the area of the neuronal soma profile. Conclusions: 1) The axonal half of the soma of the frog intracardiac neuron is more densely innervated than the non-axonal half of the soma. 2) The axosomatic and axoaxonic synapses are present on frog intracardiac neurons. The study provides a framework for further experimental studies on the formation and rearrangement of synapses on frog intracardiac neurons.

Large-conductance calcium-activated potassium current modulates excitability in isolated canine intracardiac neurons

We studied principal neurons from canine intracardiac (IC) ganglia to determine whether large-conductance calcium-activated potassium (BK) channels play a role in their excitability. We performed whole cell recordings in voltage- and current-clamp modes to measure ion currents and changes in membrane potential from isolated canine IC neurons. Whole cell currents from these neurons showed fast- and slow-activated outward components. Both current components decreased in the absence of calcium and following 1–2 mM tetraethylammonium (TEA) or paxilline. These results suggest that BK channels underlie these current components. Single-channel analysis showed that BK channels from IC neurons do not inactivate in a time-dependent manner, suggesting that the dynamic of the decay of the fast current component is akin to that of intracellular calcium. Immunohistochemical studies showed that BK channels and type 2 ryanodine receptors are coexpressed in IC principal neurons. We tested whether BK current activation in these neurons occurred via a calcium-induced calcium release mechanism. We found that the outward currents of these neurons were not affected by the calcium depletion of intracellular stores with 10 mM caffeine and 10 μM cyclopiazonic acid. Thus, in canine intracardiac neurons, BK currents are directly activated by calcium influx. Membrane potential changes elicited by long (400 ms) current injections showed a tonic firing response that was decreased by TEA or paxilline. These data strongly suggest that the BK current present in canine intracardiac neurons regulates action potential activity and could increase these neurons excitability.