The organization of the sympathetic nervous system: shining new light on historic views

The organization and spatial distribution of neurons within the sympathetic nervous system (SNS) have been long-standing questions in autonomic neuroscience, particularly concerning the functional innervation of target organs. First understood as an ‘alarm reaction’ system turned on en masse, advancements in nerve recording techniques have revealed sympathetic outflow to be differentially controlled to distinct functional subunits within (DiBona 2000; Incognito et al. 2019) and between (Rundqvist et al. 1997) organ systems. Our modern understanding of sympathetic control is now region- and function-specific, governed by an intricate topography within central autonomic nuclei (e.g. rostral ventrolateral medulla; RVLM). Despite intensive efforts to elucidate the regulation of sympathetic outflow, a consensus amongst investigators regarding how topographic arrangements mediate unique activation patterns remains to be reached. Expanding the organizational framework of the SNS is crucial for understanding pathophysiological manifestations. Specifically, cardiovascular disease states present with hallmark sympathetic overactivation, predictive of disease severity (Rundqvist et al. 1997), and with characteristic region-specific alterations in sympathetic outflow to morphologically distinct target organs (e.g. heart, kidney, muscle; Rundqvist et al. 1997). Thus, investigating how the central autonomic circuitry mediates sympathoexcitatory patterns may elucidate cardiovascular (or other) disease mechanisms of neurogenic origin. In a recent article in The Journal of Physiology, Farmer & colleagues (2019) used viral vector tracing and optogenetic manipulation, in vivo, of spinally projecting RVLM neurons to test if these neurons possess the anatomical chassis and functional capacity to elicit sympathoexcitation to regionally and morphologically distinct target organs. The primary objectives were to (1) identify RVLM neurons synapsing at both rostral and caudal thoracic spine regions (i.e. axon collaterals at T2 and T10 spinal segments) and (2) identify if selective stimulation of RVLM neurons with known projections to the caudal thoracic spine (i.e. T12) elicits parallel activation of post-ganglionic nerves in regionally (i.e. rostral thoracic spine) and/or morphologically (i.e. non-vascular) distinct target organs. The authors suggest that evidence of RVLM neurons with functioning axon collaterals would revive older views of generalized/global SNS activation properties, adding an intriguing supplement to the well-established modern view of differential control. Using healthy Sprague–Dawley rats, the first objective was to define the anatomical organization of RVLM projections using retrograde viral tracing. Investigators performed bilateral pressure injections of recombinant herpes simplex retrograde viral vectors into the interomediolateral column (IML) at T2 and T10. Differences in fluorescent tags enabled neurons projecting from the two sites of injection (green fluorescent protein (GFP) at T2 and mCherry at T10) to be distinguished. Of the labelled RVLM neurons, 53% expressed GFP (projecting to T2), 26% expressed mCherry (projecting to T10), and 21% co-expressed GFP and mCherry (projecting to T2 and T10) – the latter being evidence for axon collateralization, and therefore the required substrate for generalized sympathetic activation, in a subgroup of spinally projecting sympathetic RVLM neurons. The second objective of Farmer and colleagues was to investigate if post-ganglionic nerves arising from the upper thoracic spine are activated in response to optogenetic stimulation of select RVLM neurons with known projections to the lower thoracic spine. The optogenetic methods involved an intersectional approach whereby the AAV-ChAR2 vector was injected into the RVLM, and the CAV2-CMV-Cre promoter was injected into the lower thoracic spine to ensure Channelrhodopsin expression in the RVLM would be only in neurons projecting to the IML at the level of T12. After 11–50 days post-injection, simultaneous recordings from post-ganglionic nerve pairings were obtained from (1) forelimb muscle sympathetic nerve activity (SNA) and hindlimb muscle SNA, and (2) left cardiac SNA and hindlimb muscle SNA. Upon optogenetic stimulation of these select RVLM neurons, there was an increase in hindlimb muscle SNA, as expected, but also in forelimb muscle SNA and cardiac SNA. These results demonstrate the functional capacity of RVLM neurons to evoke generalized sympathoexcitation to distant, yet morphologically similar, target organs (i.e. forelimb and hindlimb muscle vasculature), as well as to morphologically distinct, yet potentially functionally similar, target organs (i.e. muscle vasculature and heart). These findings demonstrate the existence of axon collateralization in a subpopulation of spinally projecting RVLM neurons and their capacity to mediate generalized sympathoexcitation. The results of the current study well support the primary conclusions and have sparked important physiological and pathophysiological considerations. A critical point raised by the authors is whether collateralized RVLM neurons regulate functionally similar subsets of pre-ganglionic neurons. From post-ganglionic recordings, there exist single-unit subgroups that are differentially controlled in response to stress (DiBona 2000; Incognito et al. 2019). Differential control within post-ganglionic nerves (likely driven by pre-ganglionic innervation) is required for selective control of functionally distinct target-organ subgroups. This is particularly apparent in the kidney, where there is sympathetic innervation to the renal tubules, vasculature and juxtaglomerular granular cells (DiBona 2000). Similarly, in humans differential stress responses of post-ganglionic muscle sympathetic single units can be observed (Incognito et al. 2019), hypothesized to govern the release of different neurotransmitters (i.e. noradrenaline, ATP, neuropeptide Y) and/or differentially control skeletal muscle arterioles and venules. The functional subunits within the heart are also plentiful, and include the sinoatrial node, myocardium and coronary vasculature, all known to have innervation from RVLM neurons (Goodson et al. 1993). Thus, one worthwhile consideration is whether RVLM neurons with axon collaterals controlling sympathetic outflow to the skeletal muscle and heart predominantly influence vascular constriction (i.e. muscle and coronary vasculature). In another light, defining function from a systemic physiological standpoint, sympathetic innervation to the sinoatrial node and myocardium of the heart and of the peripheral vasculature are all under strong influence of arterial baroreceptors. It can be hypothesized, then, that single RVLM neurons may influence chronotropy and/or inotropy of the heart in synergy with peripheral vasoconstriction to permit coordinated control of a single physiological consequence – increasing blood pressure. Therefore, the notion that sympathetic neurons can elicit generalized activation to the target organs examined in the present study should not be outstretched to generalized functional capacities, as cautioned by the authors. Investigations into the existence of axon collaterals to post-ganglionic nerves innervating target organs with distinct functional subunits and systemic physiological functions (i.e. cardiac and non-vasomotor splanchnic nerves) are warranted. Along this line of inquiry, it is of crucial importance for future investigations to examine the magnitude of influence of RVLM neuron subgroups on target-organ function, not measured in the present study. When performing these investigations, however, the influence of anaesthesia is a concern which cannot be overlooked. In this preparation, anaesthesia may dampen the excitation threshold of the entire sympathetic network and neural activation not representative of the conscious state may occur. Specifically, anaesthesia could attenuate pre-ganglionic recruitment and thus target-organ excitation, which would underestimate the level of influence of RVLM neuron subgroups over target-organ function. Additionally, the level of post-ganglionic nerve excitation can alter vesicular release of ATP, noradrenaline and/or neuropeptide Y, which could also change target-organ excitation, as well as functional responses (Svennson et al. 2018). Until multiple sympathetic recordings become feasible in conscious animals, anaesthetized preparations are the best possible method to assess functional sympathoexcitation. Better understanding the regulation of this RVLM neuron subgroup is also of clinical relevance. In human heart failure, there are alterations in regional sympathetic outflow compared to the healthy state, such that cardiac SNA has been observed to be selectively elevated in mild to moderate disease stages before observable increases in renal SNA and skeletal muscle SNA in severe stages (Rundqvist et al. 1997). In relation to the present study, given the selective excitation of cardiac SNA and unchanged skeletal muscle SNA in early stage heart failure, it is unlikely that RVLM neurons with axon collaterals to these distinct spinal segments are primarily responsible for this sympathetic outflow pattern. In end-stage heart failure, however, there appears to be more systemic activation. It can be speculated that RVLM neurons with axon collaterals to pre-ganglionic neurons controlling sympathetic outflow to the heart and skeletal muscle may be recruited with worsening disease, further representing the downward spiral of activation – weakened heart requiring augmented sympathoexcitation to maintain driving pressure. Extending this notion, one acknowledged question is whether populations of such neurons contribute to resting levels of vasomotor tone or if they are recruited in emergency conditions (e.g. end-stage disease). The increases in signal traffic following optogenetic stimulation within forelimb muscle and cardiac sympathetic nerves suggests that these RVLM neurons are inactive at rest (or under anaesthesia). This showcases a sympathetic nerve recruitment reserve for skeletal muscle and cardiac SNA in the healthy state, a reserve known to be vastly diminished in heart failure conditions. The regulation of this RVLM neuron subgroup and their relation to disease-specific sympathetic outflow patterns warrant further investigation. The present investigation has advanced the organizational framework of the SNS, and the execution of the technical methodology is highly commendable. The results have expanded the current knowledge of sympathetic control in physiological states and has raised new ideas of how sympathetic arrangements may be morphologically distinct yet functionally similar. This study highlights the need for further investigation into the autonomic circuitry underlying autonomic control of target-organ function to inform the development of neurally focused strategies to prevent and/or treat diseases involving autonomic dysregulation. None. Both authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. A.V.I. is supported by a Natural Science and Engineering Research Council of Canada (NSERC) Canada Graduate Scholarship. N.G.J. is a Parker B. Francis Fellow and also supported by an Alberta Innovates Postdoctoral Fellowship. The authors recognize that not all relevant articles were cited due to reference limitations. We would like to thank Drs Philip J. Millar (University of Guelph) and Richard J. A. Wilson (University of Calgary) for their guidance and critical insights on the presented viewpoints.

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