Untangling Peripheral Sympathetic Neurocircuits

Abstract

The sympathetic nervous system plays a critical role in regulating many autonomic functions, including cardiac rhythm. The postganglionic neurons in the sympathetic chain ganglia are essential components that relay sympathetic signals to target tissues and disruption of their activity leads to poor health outcomes. Despite this importance, the neurocircuitry within sympathetic ganglia is poorly understood. Canonically, postganglionic sympathetic neurons are thought to simply be activated by monosynaptic inputs from preganglionic cholinergic neurons of the intermediolateral cell columns of the spinal cord. Early electrophysiological studies of sympathetic ganglia where the peripheral nerve trunks were electrically stimulated identified excitatory cholinergic synaptic events in addition to retrograde action potentials, leading some to speculate that excitatory collateral projections are present. However, this seemed unlikely since sympathetic postganglionic neurons were known to synthesize and release norepinephrine and expression of dual neurochemical phenotypes had not been well recognized. In vitro studies clearly established the capacity of cultured sympathetic neurons to express and release acetylcholine and norepinephrine throughout development and even in pathophysiological conditions. Given this insight, we believe that the canonical view of ganglionic transmission needs to be reevaluated and may provide a mechanistic understanding of autonomic imbalance in disease. Further studies likely will require genetic models manipulating neurochemical phenotypes within sympathetic ganglia to resolve the function of cholinergic collateral projections between postganglionic neurons. In this perspective article, we will discuss the evidence for collateral projections in sympathetic ganglia, determine if current laboratory techniques could address these questions, and discuss potential obstacles and caveats.

Full text

PERSPECTIVE
published: 10 February 2022
doi: 10.3389/fcvm.2022.842656
Frontiers in Cardiovascular Medicine | www.frontiersin.org 1February 2022 | Volume 9 | Article 842656
Edited by:
Tania Zaglia,
University of Padova, Italy
Reviewed by:
Nazareno Paolocci,
Johns Hopkins University,
United States
Matthew W. Kay,
George Washington University,
United States
Michael Rubart,
Indiana University Bloomington,
United States
*Correspondence:
Beth A. Habecker
habecker@ohsu.edu
Specialty section:
This article was submitted to
Hypertension,
a section of the journal
Frontiers in Cardiovascular Medicine
Received: 24 December 2021
Accepted: 19 January 2022
Published: 10 February 2022
Citation:
Clyburn C, Andresen MC, Ingram SL
and Habecker BA (2022) Untangling
Peripheral Sympathetic Neurocircuits.
Front. Cardiovasc. Med. 9:842656.
doi: 10.3389/fcvm.2022.842656
Untangling Peripheral Sympathetic
Neurocircuits
Courtney Clyburn 1, Michael C. Andresen 1, Susan L. Ingram 2and Beth A. Habecker 1
*
1Department of Chemical Physiology and Biochemistry, Oregon Health and Science University, Portland, OR, United States,
2Department of Neurological Surgery, Oregon Health and Science University, Portland, OR, United States
The sympathetic nervous system plays a critical role in regulating many autonomic
functions, including cardiac rhythm. The postganglionic neurons in the sympathetic
chain ganglia are essential components that relay sympathetic signals to target tissues
and disruption of their activity leads to poor health outcomes. Despite this importance,
the neurocircuitry within sympathetic ganglia is poorly understood. Canonically,
postganglionic sympathetic neurons are thought to simply be activated by monosynaptic
inputs from preganglionic cholinergic neurons of the intermediolateral cell columns of the
spinal cord. Early electrophysiological studies of sympathetic ganglia where the peripheral
nerve trunks were electrically stimulated identified excitatory cholinergic synaptic events
in addition to retrograde action potentials, leading some to speculate that excitatory
collateral projections are present. However, this seemed unlikely since sympathetic
postganglionic neurons were known to synthesize and release norepinephrine and
expression of dual neurochemical phenotypes had not been well recognized. In vitro
studies clearly established the capacity of cultured sympathetic neurons to express
and release acetylcholine and norepinephrine throughout development and even in
pathophysiological conditions. Given this insight, we believe that the canonical view
of ganglionic transmission needs to be reevaluated and may provide a mechanistic
understanding of autonomic imbalance in disease. Further studies likely will require
genetic models manipulating neurochemical phenotypes within sympathetic ganglia to
resolve the function of cholinergic collateral projections between postganglionic neurons.
In this perspective article, we will discuss the evidence for collateral projections in
sympathetic ganglia, determine if current laboratory techniques could address these
questions, and discuss potential obstacles and caveats.
Keywords: sympathetic ganglia, neurocircuits, synaptic inputs, co-transmission, collaterals
INTRODUCTION
Technical advances have long driven new insights into the mechanistic basis of neurophysiology.
From the identification of the action potential in the nineteenth century (1) to the work of
Joseph Erlanger and Herbert Gasser who revolutionized neurophysiological research by developing
sensitive oscilloscopes that allowed for the visualization and analysis of nerve impulses previously
below the threshold of detection (2,3). With these technological advancements, researchers set
out to decode the complex autonomic signals that regulate visceral functions, including cardiac

Clyburn et al. Neurocircuitry of the Sympathetic Ganglia
activity (4–6). Several important hypotheses arose from this
work, but were untested due to experimental limitations that
existed at the time. These questions include whether or
not collateral projections are present between postganglionic
neurons in sympathetic ganglia and what neurotransmitters
may be involved in sympathetic signaling pathways. Since these
first studies, significant advancements in neurophysiological
equipment and scientific methods have allowed for substantial
progress in understanding the neurophysiology of peripheral
sympathetic activity (7–11), but the neurocircuitry within
sympathetic ganglia remains poorly understood. The purpose
of this article is to review the primary data that support
these untested hypotheses, determine if current technology and
experimental methods can be used to answer these important
questions, and discuss any potential obstacles that may arise.
Canonical Neurocircuitry of Cervical
Sympathetic Ganglia
Several well-written review articles are available that thoroughly
summarize our current understanding of the sympathetic
neurocircuitry that regulates visceral functions and cardiac
activity (12–16). To briefly summarize, sympathetic signals
originate in the hypothalamus and brainstem and travel to the
preganglionic neurons in the intermediolateral cell column of
the spinal cord. These preganglionic neurons send cholinergic
projections to postganglionic neurons in the cervical sympathetic
chain ganglia. These neurons in the sympathetic ganglia then
send noradrenergic projections through the postganglionic nerve
trunk to innervate the target tissue. This general organization
is conserved across species. However, there is significant
biological variability in the anatomy and physiology of the
cervical sympathetic ganglia which clouds our understanding of
peripheral sympathetic neurocircuits. The cervical sympathetic
chain includes the superior, middle, and inferior cervical
ganglia and exhibits significant anatomical variability between
species, between individuals, and even between the left and
right ganglia. In approximately 80% of humans, for example,
the inferior cervical ganglion is fused with the first thoracic
ganglion to form the cervicothoracic (commonly known as
stellate) ganglion. Additionally, the middle cervical ganglion
is completely absent in ∼20% of the population (17–20). In
considering the variability in the sympathetic nervous system
in humans and animal models as well as the murkiness
that surrounds the functional organization of the cervical
sympathetic ganglia, we will consider data from all cervical
sympathetic ganglia interchangeably as we discuss peripheral
sympathetic neurocircuitry.
Evidence for Collateral Projections in
Sympathetic Ganglia
In addition to the ambiguity surrounding the anatomy of
the sympathetic ganglia, the functional organization of the
sympathetic neurocircuitry within these ganglia remains poorly
defined. Current literature usually describes and illustrates the
neurocircuitry within cervical sympathetic ganglia as simple
monosynaptic connections from cholinergic preganglionic
neurons to the noradrenergic postganglionic neurons that
innervate visceral targets and the heart (21,22). However,
Erulkar and Woodward (23) made intracellular recordings
of postganglionic sympathetic neurons from rabbit superior
cervical ganglia (SCG) in situ which suggest this neurocircuitry
may be more complicated. In these early experiments, they found
that stimulation of the external carotid nerve produced single,
short latency spikes followed by long-lasting hyperpolarizations
(presumably action potentials) in some neurons. In these
experiments, electrical stimulation of the postganglionic nerve
trunk evoked antidromic, or retrograde, action potentials that
traveled up the axon to the cell body in the SCG. Surprisingly,
the majority of neurons exhibited an early spike followed by a
long-lasting depolarization. This depolarization suggested that
stimulation of the postganglionic nerve trunk elicited excitatory
synaptic activity within the SCG. Erulkar and Woodward
proposed several hypotheses that would account for this
phenomenon, but state that the simplest explanation would be
that recurrent excitatory collateral fibers from postganglionic
axons project to neighboring cells within the SCG (Figure 1).
Upon stimulation of the preganglionic nerve trunk, Erulkar and
Woodward also observed that a significant proportion of SCG
neurons exhibited multi-spike responses which were uncovered
at increasing stimulation intensities. While they acknowledge
these results may be caused by different populations of
preganglionic fibers with different thresholds and conduction
velocities that converge onto the same cell, this explanation
seems highly unlikely. Instead, they note that the multi-spike
responses are consistent with the collateral hypothesis and that
the late-arriving impulses may have traveled through another
indirect pathway.
Excitatory synaptic events in postganglionic sympathetic
neurons following electrical stimulation of the postganglionic
nerve trunk have also been observed in the cat, rat and guinea
pig (24–26). In 1970, Perri et al. extended the observations by
Erulkar and Woodward by investigating the neurotransmitter
that may be involved in these synaptic events. Curare, a
nicotinic acetylcholine (ACh) receptor antagonist, applied to
the ganglionic preparation abolished these synaptic events,
indicating they were likely cholinergic in nature (25). If
these excitatory events originated from postganglionic collateral
projections, this would mean that these sympathetic neurons
express ACh and norepinephrine (NE). Although we now know
that postganglionic sympathetic neurons and other neurons
can express a dual neurochemical phenotype (27–31), at the
time this was thought to be unlikely. Thus, researchers sought
alternative explanations. These included the possibility that
errant preganglionic fibers may be running up the postganglionic
nerve trunk and forming excitatory synaptic connections with
postganglionic neurons (Figure 1) (25). A second possibility
raised was that sensory afferent neurons were the source of
synaptic excitation. Sympathetic ganglia have long been linked
to sensory afferents of the cardiac region of the thoracic viscera
(6) but on the bases of such anatomical findings broadly
dismissed as only passing through the ganglia on their way
to the spinal cord. Thus, sketches commonly depict that a
portion of these afferent paths include sympathetic ganglia
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Clyburn et al. Neurocircuitry of the Sympathetic Ganglia
FIGURE 1 | Schematic diagram illustrating the errant preganglionic fiber and collateral hypotheses. In the preganglionic hypothesis (left), mis-routed cholinergic (black)
preganglionic fibers travel back up the efferent nerves through which sympathetic axons travel to the target tissue. In the collateral hypothesis (right) postganglionic
sympathetic neurons send noradrenergic projections (red) to the target tissue and cholinergic projections (black) to other sympathetic neurons in the sympathetic
chain ganglia.
(e.g., cervical and stellate) (32). However, functional studies
related to cardiovascular regulation, for example, indicate that
activation of cardiac sensory afferents traveling via the stellate
ganglia act to increase blood pressure by augmenting sympathetic
motor neuron activity directed to the systemic vasculature and
such responses are abolished by severing ganglion connections
to the spinal cord (33). Dye tracing and electrical activity
recordings indicate that such afferent neurons arise from the
dorsal root ganglia and traverse peripheral sympathetic ganglia
to their visceral targets (34). Although it seemed unlikely at
the time, our current understanding of dual neurochemical
phenotypes in sympathetic neurons has persuaded us that
further studies are needed to investigate the potential presence
of cholinergic collateral projections between postganglionic
neurons in sympathetic ganglia.
Evidence for Dual Neurochemical
Phenotypes in Sympathetic Neurons
In 1935, Henry Dale put forth the “one neuron, one
neurotransmitter” hypothesis which was later termed as “Dale’s
Principle” (35,36) and stated that one neuron was only
capable of releasing one neurotransmitter at any one time
[reviewed by: (37)]. By the mid-1970s, evidence disputing
Dale’s Principle began to emerge when Jan and colleagues
made intracellular recordings of sympathetic frog neurons and
observed slow synaptic potentials which were mediated by the
peptide, luteinizing hormone-releasing hormone, in addition to
the canonical cholinergic transmission (38,39). Since these initial
studies, co-transmission of several neuromodulators, including
ATP and neuropeptide Y (NPY) (40–44), as well as the co-
transmission of fast primary neurotransmitters have been well
established throughout the central and peripheral nervous
system (45–47).
Some of the earliest evidence supporting co-expression
of primary neurotransmitters comes from cardiac myocyte—
sympathetic neuron co-cultures (48). In 1976, Furshpan
et al. used electrophysiological recordings of sympathetic SCG
principal neurons cultured with cardiac myocytes from newborn
rats and described a population of neurons that secreted
ACh and NE (48–51). This work sparked a series of studies
to determine if sympathetic neurons produced ACh in vivo
and to identify the differentiation factors that determine
the fates of sympathetic neurons (52). Several “cholinergic
differentiation factors” are now known to induce ACh expression
in noradrenergic sympathetic neurons including leukemia
inhibitor factor (LIF), ciliary neurotrophic factor (CNTF),
cardiotrophin-1 (CT-1), neurotrophin-3 (NT-3) and glial cell
line-derived neurotrophic factor (GDNF) (53–61). While the
cholinergic transdifferentiation of postganglionic sympathetic
neurons in vivo was first thought to be confined to developmental
periods, more recent studies have shown that inflammatory
cytokines can induce cholinergic transdifferentiation in cardiac
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Clyburn et al. Neurocircuitry of the Sympathetic Ganglia
disease (30,62). Early studies have revealed key aspects
of the neurochemical phenotype of sympathetic neurons:
dual and malleable neurochemical capacity. These studies of
cultured neurons indicated that these postganglionic neurons
could form synapses between neurons and could display
adrenergic/cholinergic dual function (50). In culture, the
neurotrophin brain-derived neurotrophic factor (BDNF) rapidly
shifted sympathetic neuron release of norepinephrine to
ACh indicating the dual neurochemical phenotype of these
postganglionic neurons (31). Subsequent work also in cultured
neurons supported both segregation as well as plasticity of
functional release sites of NE and ACh (63). In light of this
evidence, it appears that the cholinergic hypothesis originally
proposed by Erulkar and Woodward in 1968 was dismissed
prematurely before fully testing the idea. Gaining a clear insight
into the neurocircuitry within sympathetic ganglia can provide
better understanding of the mechanisms underlying autonomic
imbalance in disease and may highlight novel therapeutic targets
for cardiac patients.
Challenges and Potential Caveats
The dual expression of ACh and NE in sympathetic neurons
provides support for the collateral hypothesis, but there are
still many questions that remain. When dual neurochemical
phenotypes were first described, these neurons were generally
classified into two mechanistic subtypes, co-release and co-
transmission (37,64). Co-release describes the packaging of
multiple neurotransmitters into the same synaptic vesicle
so they there are released together and co-transmission
originally described different neurotransmitters packaged into
different vesicles within the same presynaptic terminal (37,64).
Dale’s Principle was updated in 1986 to state that neurons
release the same group of neurotransmitters/peptides from
all presynaptic terminals (64,65). However, the collateral
hypothesis challenges this view because the primary data
indicates that ACh is released from the collateral terminals.
The excitatory synaptic events that are observed following
stimulation of the postganglionic nerve trunk are completely
blocked by the application of nicotinic acetylcholine antagonists
(24,25). In the collateral model, this could suggest the
biased expression of ACh and NE at different synapses within
the same neuron. Based on the updated version of Dale’s
hypothesis, we would expect that both ACh and NE would
be released from collateral synapses. As adrenergic receptors
are G-protein coupled receptors, synaptic release of NE cannot
be detected with the electrophysiological techniques used in
these previous studies (25). Future experiments could utilize
new biosensor technology to determine if NE is also released
from collateral projections (66–68). If NE is not released
from collateral projections, there are several mechanisms
that have been described in the CNS that could explain
biased release of different vesicular pools. These include
differing release probabilities and altered coupling to presynaptic
Ca2+channels (69,70), “kiss-and-run” release mechanisms,
and even neurotransmitter segregation to separate axons
(64,71–74). Studies in cultured sympathetic neurons have
observed segregation of the vesicular NE and ACh transporters
(VMAT2 and VAChT, respectively) in distinct varicosities,
suggesting release of each transmitter is independent and
spatially segregated (63). Considering how target-derived factors
and cytokines can induce cholinergic transdifferentiation in
postganglionic sympathetic neurons, it is conceivable that factors
in the microenvironment are involved in determining different
neurochemical identities of synaptic terminals in the ganglia and
target tissues.
Putative afferent fibers could have an important physiological
role in allowing sensory feedback to regulate the activity of
postganglionic sympathetic neurons in the event that inputs
from the central nervous system are disrupted. This sensory
feedback loop would also allow for the fast adaptation of
sympathetic neuron activity to rapidly changing stimuli (24).
However, the synaptic events that are observed following
stimulation of the postganglionic nerve trunk are mediated
by acetylcholine and, therefore, unlikely to be mediated by
sensory fibers, which utilize glutamatergic signaling. The
physiological consequence of putative cholinergic collateral
projections is less obvious. Collateral projections between
postganglionic sympathetic neurons may also have a significant
physiological role in maintaining autonomic control of the
heart and other tissues. Excitatory collateral projections
between postganglionic sympathetic neurons would increase
the probability that these neurons fire synchronously and may
amplify preganglionic signals from the spinal cord. Given the
importance of neural timing for cardiopulmonary integration,
the synchronization of sympathetic inputs could potentially
play a critical role in maintaining appropriate cardiac activity.
Furthermore, disruption of this synchronization following
injury or disease could exacerbate autonomic imbalance.
Early studies hypothesized that single spikes in recordings
of cardiac sympathetic nerve activity represent synchronized
activity of multiple postganglionic neurons (75), but the
potential role of collateral synapses in this synchronization is
purely speculative.
It was not possible in the past to distinguish preganglionic
cholinergic transmission from putative post-ganglionic collateral
projections, but transgenic mouse models and genetic tools
provide the means to address this issue now. Targeted deletion
of ACh production (chat gene) or release (slc18A3 gene)
from neurons expressing tyrosine hydroxylase (th gene),
dopamine beta hydroxylase (dbh gene), or norepinephrine
transporter (slc6a2 gene) would remove cholinergic transmission
selectively from post-ganglionic neurons within the ganglion.
Likewise, targeting expression of channelrhodopsin-2 or
other stimulatory optogenetic tools to noradrenergic neurons
would allow selective stimulation of those cells, coupled
with recordings of resulting synaptic activity. Combining
these approaches with the use of genetically encoded calcium
sensors would allow testing the functional impact of putative
cholinergic collateral transmission in situ. Mouse lines to
facilitate these studies, which directly address the issue of
potential cholinergic collaterals, are available from public
repositories. If cholinergic collaterals are an important aspect
of transmission by post-ganglionic sympathetic neurons then
we would expect them to be present and detectable throughout
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Clyburn et al. Neurocircuitry of the Sympathetic Ganglia
the sympathetic chain, and lead to changes in transmission to
target tissues.
DISCUSSION
While some of the first studies investigating sympathetic
nerve impulses proposed that collateral projections may be
present in the sympathetic ganglia, this model was dismissed
because of our incomplete understanding of dual neurochemical
phenotypes. In hindsight, given our understanding of co-
transmission of ACh and NE in postganglionic sympathetic
neurons, it is clear that studies should be carried out to
clarify the neurocircuitry within the sympathetic ganglia.
The postganglionic neurons within sympathetic ganglia are
critical components of the neurocircuitry responsible for
relaying autonomic signals from the central nervous system
to several visceral targets, including the heart. The activity
of postganglionic sympathetic neurons is disrupted in many
pathologies and sympathetic hyperactivity elevates risk for
cardiac arrhythmias and sudden cardiac death and contributes
to the development of heart failure. A detailed understanding
sympathetic neurocircuitry is needed to understand how
peripheral sympathetic activity is disrupted in pathophysiological
conditions and to develop novel therapeutic strategies.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included
in the article, further inquiries can be directed to the
corresponding author.
AUTHOR CONTRIBUTIONS
CC, MA, SI, and BH contributed to drafting the work and
revising it critically for important intellectual content. All authors
approved the final version of the manuscript and agree to be
accountable for all aspects of the work. All persons designated
as authors qualify for authorship, and all those who qualify for
authorship are listed.
FUNDING
This work was supported by the National Institutes of Health
Grant HL146833.
ACKNOWLEDGMENTS
The figure in this article was created with BioRender.com.
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