The objective of the study was to determine if chronic interruption of all extrinsic nerve inputs to the heart alters cholinergic-mediated responses within the intrinsic cardiac nervous system (ICN). Extracardiac nerve inputs to the ICN were surgically interrupted (ICN decentralized). Three weeks later, the intrinsic cardiac right atrial ganglionated plexus (RAGP) was removed and intrinsic cardiac neuronal responses were evaluated electrophysiologically. Cholinergic receptor abundance was evaluated using autoradiography. In sham controls and chronic decentralized ICN ganglia, neuronal postsynaptic responses were mediated by acetylcholine, acting at nicotinic and muscarinic receptors. Muscarine- but not nicotine-mediated synaptic responses that were enhanced after chronic ICN decentralization. After chronic decentralization, muscarine facilitation of orthodromic neuronal activation increased. Receptor autoradiography demonstrated that nicotinic and muscarinic receptor density associated with the RAGP was unaffected by decentralization and that muscarinic receptors were tenfold more abundant than nicotinic receptors in the right atrial ganglia in each group. After chronic decentralization of the ICN, intrinsic cardiac neurons remain viable and responsive to cholinergic synaptic inputs. Enhanced muscarinic responsiveness of intrinsic cardiac neurons occurs without changes in receptor abundance.

abstract: Intracellular voltage recordings from single neurons in vitro and in vivo have been fundamental to our understanding of neuronal function. Conventional electrodes and associated positioning systems for intracellular recording in vivo are large and bulky, which has largely restricted their use to single-channel recording from anesthetized animals. Further, intracellular recordings are very cumbersome, requiring a high degree of skill not readily achieved in a typical laboratory. This dissertation presents a robotic, head-mountable, MEMS (Micro-Electro-Mechanical Systems) based intracellular recording system to overcome the above limitations associated with form-factor, scalability and highly skilled and tedious manual operations required for intracellular recordings. This system combines three distinct technologies: 1) novel microscale, polycrystalline silicon-based electrode for intracellular recording, 2) electrothermal microactuators for precise microscale navigation of the electrode and 3) closed-loop control algorithm for autonomous movement and positioning of electrode inside single neurons. First, two distinct designs of polysilicon-based microscale electrodes were fabricated and tested for intracellular recordings. In the first approach, tips of polysilicon microelectrodes were milled to nanoscale dimensions (<300 nm) using focused ion beam (FIB) to develop polysilicon nanoelectrodes. Polysilicon nanoelectrodes recorded >1.5 mV amplitude, positive-going action potentials and synaptic potentials from neurons in the abdominal ganglion of Aplysia Californica. In the second approach, polysilicon microelectrodes were integrated with miniaturized glass micropipettes filled with electrolyte to fabricate glass-polysilicon microelectrodes. These electrodes consistently recorded high fidelity intracellular potentials from neurons in the abdominal ganglion of Aplysia Californica (Resting Potentials < -35 mV, Action Potentials > 60 mV) as well as the rat motor cortex (Resting Potentials < -50 mV). Next, glass-polysilicon microelectrodes were coupled with microscale electrothermal actuators and controller for autonomous intracellular recordings from single neurons in the abdominal ganglion. Consistent resting potentials (< -35 mV) and action potentials (> 60 mV) were recorded after each successful penetration attempt with the controller and microactuated glass-polysilicon microelectrodes. The success rate of penetration and quality of recordings achieved using electrothermal microactuators were comparable to that of conventional positioning systems. Finally, the feasibility of this miniaturized system to obtain intracellular recordings from single neurons in the motor cortex of rats in vivo is also demonstrated. The MEMS-based system offers significant advantages: 1) reduction in overall size for potential use in behaving animals, 2) scalable approach to potentially realize multi-channel recordings and 3) a viable method to fully automate measurement of intracellular recordings. / Dissertation/Thesis / Doctoral Dissertation Biomedical Engineering 2018

Chronic myocardial infarction (CMI) is associated with remodeling of the ventricle and evokes adaption in the cardiac neurohumoral control systems. To evaluate the remodeling of the intrinsic cardiac nervous system following myocardial infarction, the dorsal descending coronary artery was ligated in the guinea pig heart and the animals were allowed to recover for 7-9 wk. Thereafter, atrial neurons of the intrinsic cardiac plexus were isolated for electrophysiological and immunohistochemical analyses. Intracellular voltage recordings from intrinsic cardiac neurons demonstrated no significant changes in passive membrane properties or action potential configuration compared with age-matched controls and sham-operated animals. The intrinsic cardiac neurons from chronic infarcted hearts did demonstrate an increase in evoked action potential (AP) frequency (as determined by the number of APs produced with depolarizing stimuli) and an increase in responses to exogenously applied histamine compared with sham and age-matched controls. Conversely, pituitary adenylate cyclase-activating polypeptide (PACAP)-induced increases in intrinsic cardiac neuron-evoked AP frequency were similar between control and CMI animals. Immunohistochemical analysis demonstrated a threefold increase in percentage of neurons immunoreactive for neuronal nitric oxide synthase (NOS) in CMI animals compared with control and the additional expression of inducible NOS by some neurons, which was not evident in control animals. Finally, the density of mast cells within the intrinsic cardiac plexus was increased threefold in preparations from CMI animals. These results indicate that CMI induces a differential remodeling of intrinsic cardiac neurons and functional upregulation of neuronal responsiveness to specific neuromodulators.

Chronic pressure overload (PO) is associated with cardiac hypertrophy and altered autonomic control of cardiac function, in which the latter may involve adaptations in central and/or peripheral cardiac neural control mechanisms. To evaluate the specific remodeling of the intrinsic cardiac nervous system following pressure overload, the descending thoracic aorta artery of the guinea pig was constricted ∼20%, and the animals recovered for 9 wk. Thereafter, atrial neurons of the intrinsic cardiac plexus were isolated for electrophysiological and immunohistochemical analyses. Intracellular voltage recordings from intrinsic cardiac neurons demonstrated no significant changes in passive membrane properties or action potential depolarization compared with age-matched controls and sham-operated animals, but afterhyperpolarization duration was increased in PO animals. Neuronal excitability, as determined by the number of action potentials produced with depolarizing stimuli, was differentially increased in phasic neurons derived from PO animals in response to exogenously applied histamine compared with sham and age-matched controls. Conversely, pituitary adenylate cyclase-activating polypeptide-induced increases in intrinsic cardiac neuron evoked AP frequency were similar between control and PO animals. Immunohistochemical analysis demonstrated a two-fold increase in the percentage of neurons immunoreactive for neuronal nitric oxide synthase in PO animals compared with control. The density of mast cells within the intrinsic cardiac plexus from PO animals was also increased twofold compared with preparations from control animals. These results indicate that congestive heart failure associated with chronic pressure overload induces a differential remodeling of intrinsic cardiac neurons and upregulation of neuronal responsiveness to specific neuromodulators.

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