Electrophysiological characterization of mouse intracardiac calbindin neurons

Introduction: Neural control of the heart involves central and peripheral neurons that act interdependently to modulate cardiac parameters such as heart rate, conduction velocity or contractility. Within this cardiac neuronal regulation, the intrinsic cardiac nervous system (ICNS), which correspond to clusters of neurons found on the dorsal atrial surface of the heart, is receiving growing attention. Indeed, whereas they were initially considered as simple parasympathetic postganglionic neurons, studies conducted over the past 30 years suggested a more complex organization, involving the existence of sensory, local regulatory and motor neurons within intracardiac ganglia. Moreover, growing evidence suggest the implication of this neural network in the initiation and maintenance of cardiac arrhythmias. However, the functional organization of this intracardiac neural network, as well as its involvement in cardiac diseases have not been fully elucidated.Objective: This study aims to decipher the complexity of this intracardiac nervous system by examining the phenotypic and electrophysiological properties of mouse intracardiac neurons.Method: Global cardiac innervation and phenotypic diversity of mouse intracardiac neurons were investigated by performing immunohistochemistry on cleared heart and on tissue sections. The patch clamp technique (current and voltage clamp) was used for the electrophysiological characterization of these neurons. Calbindin expressing neurons were specifically studied by using cre transgenic mice and targeted viral transduction strategy.Results: We identified 6 distinct neuronal markers within mouse intracardiac neurons revealing the neurochemical diversity of this intracardiac neural network. The characterization of passive and active electrical membrane properties of these neurons gave rise to the identification of two distinct firing profiles within intracardiac neurons. The first group was classified as phasic due to its limited firing activity while the second was defined as adapting. Phasic neurons were also characterized by a higher rheobase compared to the adapting one. In addition, intracardiac neurons could also be distinguished by the presence or absence of an afterhyperpolarization. We also identified calbindin expressing neurons as a population with a distinct electrophysiological signature. This could be explained by the differential expression of several ionic channels including the N-type voltage-gated calcium channel.