We recently developed novel AAV capsids for efficient and noninvasive gene transfer across the central and peripheral nervous systems. In this protocol, we describe how to produce and systemically administer AAV-PHP viruses to label and/or genetically manipulate cells in the mouse nervous system and organs including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans eight days, excluding the time required to assess gene expression, and can be readily adopted by laboratories with standard molecular and cell culture capabilities. We provide guidelines for experimental design and choosing the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing. 1). The recombinant AAV (rAAV) genome contains the components required for gene expression including promoters, transgenes, protein trafficking signals, and recombinasedependent expression schemes. Hence, different capsid-cargo combinations create a versatile AAV toolbox for genetic manipulation of diverse cell populations in wild-type and transgenic animals. Here, we provide researchers, especially those new to working with AAVs or systemic delivery, with resources to utilize AAV-PHP viruses in their own research. Overview of the protocol We provide an instruction manual for users of AAV-PHP variants. The procedure includes three main stages (Fig. 1): AAV production (Steps 1-42), intravenous delivery (Steps 43-49), and evaluation of transgene expression (Step 50). The AAV production protocol is adapted from established methods. First, HEK293T cells are transfected with three plasmids 4-6 (Steps 1-3) (Figs. 1 and 6): (1) pAAV, which contains the rAAV genome of interest (Fig. 5 and Table 1); (2) AAV-PHP Rep-Cap, which encodes the viral replication and capsid proteins; and (3) pHelper, which encodes adenoviral proteins necessary for replication. Using this triple transfection approach, the rAAV genome is packaged into an AAV-PHP capsid in HEK293T cells. AAV-PHP viruses are then harvested 7 (Steps 4-14), purified 8,9 (Steps 15-31), and titered 10 (Steps 32-42) (Fig. 6). Purified viruses are intravenously delivered to mice via retro-orbital injection 11 (Steps 43-49) and gene expression is later assessed using molecular, histological, or functional methods relevant to the experimental aims (Step 50). This protocol is optimized to produce AAVs at high titer (over 10 13 vector genomes/ml) and with high transduction efficiency in vivo 2,3. Experimental design Before proceeding with the protocol, a number of factors should be considered, namely the expertise and resources available in the lab; the capsid and rAAV genome to be used; the dose for intravenous administration; and the method(s) available for assessing transgene expression. Each of these topics is discussed below and intended to guide users in de...

Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we develop a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs.

Intracellular recordings were made in vitro from guinea-pig cardiac ganglia to determine whether endogenous neuropeptides such as pituitary adenylate cyclase-activating polypeptide (PACAP) or substance P released during tetanic neural stimulation modulate cardiac neurone excitability and/or contribute to slow excitatory postsynaptic potentials (sEPSPs). When nicotinic and muscarinic receptors were blocked by hexamethonium and atropine, 20 Hz stimulation for 10 s initiated a sEPSP in all innervated neurones. In 40% of the cells, excitability was enhanced after termination of the sEPSP. This suggested that non-cholinergic receptor-mediated mechanisms contributed to the sEPSP and modulated neuronal excitability. Exogenous PACAP and substance P initiated a slow depolarization in the neurones whereas neuronal excitability was only increased by PACAP. When ganglia were treated with the PAC 1 antagonist PACAP6-38 (500 nM), the sEPSP evoked by 20 Hz stimulation was reduced by ∼50% and an enhanced excitability occurred in only 10% of the cells. These observations suggested that PACAP released from preganglionic nerve terminals during tetanic stimulation enhanced neuronal excitability and evoked sEPSPs. After addition of 1 nM PACAP to the bath, 7 of 9 neurones exhibited a tonic firing pattern whereas in untreated preparations, the neurons had a phasic firing pattern. PACAP6-38 (500 nM) diminished the increase in excitability caused by 1 nM PACAP so that only 4 of 13 neurones exhibited a tonic firing pattern and the other 9 cells retained a phasic firing pattern. These findings indicate that PACAP can be released by tetanic neural stimulation in vitro and increase the excitability of intrinsic cardiac neurones. We hypothesize that in vivo PACAP released during preganglionic firing may modulate neurotransmission within the intrinsic cardiac ganglia.

Rationale: Cardiac function is under exquisite intrinsic cardiac neural control. Neuroablative techniques to modulate control of cardiac function are currently being studied in patients, albeit with variable and sometimes deleterious results. Objective: Recognizing the major gaps in our understanding of cardiac neural control, we sought to evaluate neural regulation of impulse initiation in the sinoatrial node as an initial discovery step. Methods and Results: We report an in-depth, multi-scale structural and functional characterization of the innervation of the sinoatrial node (SAN) by the right atrial ganglionated plexus (RAGP) in porcine and human hearts. Combining intersectional strategies including tissue clearing, immunohistochemical and ultrastructural techniques, we have delineated a comprehensive neuroanatomic atlas of the RAGP-SAN complex. The RAGP shows significant phenotypic diversity of neurons while maintaining predominant cholinergic innervation. Cellular and tissue-level electrophysiologic mapping and ablation studies demonstrate interconnected ganglia with synaptic convergence within the RAGP to modulate SAN automaticity, atrioventricular (AV) conduction and left ventricular (LV) contractility. Using this approach, we comprehensively demonstrate that intrinsic cardiac neurons influence the pacemaking site in the heart. Conclusions: This report provides an experimental demonstration of a discrete neuronal population controlling a specific geographic region of the heart (SAN) that can serve as a framework for further exploration of other parts of the intrinsic cardiac nervous system (ICNS) in mammalian hearts and for developing targeted therapies.