Neural crest origin of sympathetic neurons at the dawn of vertebrates.

The neural crest is an embryonic stem cell population unique to vertebrates1 whose expansion and diversification are thought to have promoted vertebrate evolution by enabling emergence of new cell types and structures such as jaws and peripheral ganglia2. Although jawless vertebrates have sensory ganglia, convention has it that trunk sympathetic chain ganglia arose only in jawed vertebrates3-8. Here, by contrast, we report the presence of trunk sympathetic neurons in the sea lamprey, Petromyzon marinus, an extant jawless vertebrate. These neurons arise from sympathoblasts near the dorsal aorta that undergo noradrenergic specification through a transcriptional program homologous to that described in gnathostomes. Lamprey sympathoblasts populate the extracardiac space and extend along the length of the trunk in bilateral streams, expressing the catecholamine biosynthetic pathway enzymes tyrosine hydroxylase and dopamine ß-hydroxylase. CM-DiI lineage tracing analysis further confirmed that these cells derive from the trunk neural crest. RNA sequencing of isolated ammocoete trunk sympathoblasts revealed gene profiles characteristic of sympathetic neuron function. Our findings challenge the prevailing dogma that posits that sympathetic ganglia are a gnathostome innovation, instead suggesting that a late-developing rudimentary sympathetic nervous system may have been characteristic of the earliest vertebrates.

The role of somatosensory innervation of adipose tissues.

Adipose tissues communicate with the central nervous system to maintain whole-body energy homeostasis. The mainstream view is that circulating hormones secreted by the fat convey the metabolic state to the brain, which integrates peripheral information and regulates adipocyte function through noradrenergic sympathetic output1. Moreover, somatosensory neurons of the dorsal root ganglia innervate adipose tissue2. However, the lack of genetic tools to selectively target these neurons has limited understanding of their physiological importance. Here we developed viral, genetic and imaging strategies to manipulate sensory nerves in an organ-specific manner in mice. This enabled us to visualize the entire axonal projection of dorsal root ganglia from the soma to subcutaneous adipocytes, establishing the anatomical underpinnings of adipose sensory innervation. Functionally, selective sensory ablation in adipose tissue enhanced the lipogenic and thermogenetic transcriptional programs, resulting in an enlarged fat pad, enrichment of beige adipocytes and elevated body temperature under thermoneutral conditions. The sensory-ablation-induced phenotypes required intact sympathetic function. We postulate that beige-fat-innervating sensory neurons modulate adipocyte function by acting as a brake on the sympathetic system. These results reveal an important role of the innervation by dorsal root ganglia of adipose tissues, and could enable future studies to examine the role of sensory innervation of disparate interoceptive systems.

A detailed protocol for RNA cleavage assay in sympathetic neurons.

This protocol illustrates the use of an in vitro assay to study the cleavage of the IMPA1 3'UTR by the endonuclease Ago2 in sympathetic neurons. The procedure includes the preparation of cytoplasmic protein extracts and also describes the synthesis and labeling of the RNA probe. The protocol can be applied to other cell systems, RNA transcripts, and endonucleases to confirm the role of known cleavage site(s) and cleavage proteins, or to investigate new ones. For complete details on the use and execution of this protocol, please refer to Andreassi et al. (2021).

The sympathetic nervous system in development and disease.

The sympathetic nervous system prepares the body for 'fight or flight' responses and maintains homeostasis during daily activities such as exercise, eating a meal or regulation of body temperature. Sympathetic regulation of bodily functions requires the establishment and refinement of anatomically and functionally precise connections between postganglionic sympathetic neurons and peripheral organs distributed widely throughout the body. Mechanistic studies of key events in the formation of postganglionic sympathetic neurons during embryonic and early postnatal life, including axon growth, target innervation, neuron survival, and dendrite growth and synapse formation, have advanced the understanding of how neuronal development is shaped by interactions with peripheral tissues and organs. Recent progress has also been made in identifying how the cellular and molecular diversity of sympathetic neurons is established to meet the functional demands of peripheral organs. In this Review, we summarize current knowledge of signalling pathways underlying the development of the sympathetic nervous system. These findings have implications for unravelling the contribution of sympathetic dysfunction stemming, in part, from developmental perturbations to the pathophysiology of peripheral neuropathies and cardiovascular and metabolic disorders.

Neuro-mesenchymal units control ILC2 and obesity via a brain-adipose circuit.

Signals from sympathetic neurons and immune cells regulate adipocytes and thereby contribute to fat tissue biology. Interactions between the nervous and immune systems have recently emerged as important regulators of host defence and inflammation1-4. Nevertheless, it is unclear whether neuronal and immune cells co-operate in brain-body axes to orchestrate metabolism and obesity. Here we describe a neuro-mesenchymal unit that controls group 2 innate lymphoid cells (ILC2s), adipose tissue physiology, metabolism and obesity via a brain-adipose circuit. We found that sympathetic nerve terminals act on neighbouring adipose mesenchymal cells via the ß2-adrenergic receptor to control the expression of glial-derived neurotrophic factor (GDNF) and the activity of ILC2s in gonadal fat. Accordingly, ILC2-autonomous manipulation of the GDNF receptor machinery led to alterations in ILC2 function, energy expenditure, insulin resistance and propensity to obesity. Retrograde tracing and chemical, surgical and chemogenetic manipulations identified a sympathetic aorticorenal circuit that modulates ILC2s in gonadal fat and connects to higher-order brain areas, including the paraventricular nucleus of the hypothalamus. Our results identify a neuro-mesenchymal unit that translates cues from long-range neuronal circuitry into adipose-resident ILC2 function, thereby shaping host metabolism and obesity.

Neuroprosthetic baroreflex controls haemodynamics after spinal cord injury.

Spinal cord injury (SCI) induces haemodynamic instability that threatens survival1-3, impairs neurological recovery4,5, increases the risk of cardiovascular disease6,7, and reduces quality of life8,9. Haemodynamic instability in this context is due to the interruption of supraspinal efferent commands to sympathetic circuits located in the spinal cord10, which prevents the natural baroreflex from controlling these circuits to adjust peripheral vascular resistance. Epidural electrical stimulation (EES) of the spinal cord has been shown to compensate for interrupted supraspinal commands to motor circuits below the injury11, and restored walking after paralysis12. Here, we leveraged these concepts to develop EES protocols that restored haemodynamic stability after SCI. We established a preclinical model that enabled us to dissect the topology and dynamics of the sympathetic circuits, and to understand how EES can engage these circuits. We incorporated these spatial and temporal features into stimulation protocols to conceive a clinical-grade biomimetic haemodynamic regulator that operates in a closed loop. This 'neuroprosthetic baroreflex' controlled haemodynamics for extended periods of time in rodents, non-human primates and humans, after both acute and chronic SCI. We will now conduct clinical trials to turn the neuroprosthetic baroreflex into a commonly available therapy for people with SCI.

Sympathetic innervation of inguinal white adipose tissue in the mouse.

Adipose tissue plays an important role in metabolic homeostasis and its prominent role as endocrine organ is now well recognized. Adipose tissue is controlled via the sympathetic nervous system (SNS). New viral, molecular-genetic tools will soon allow a more detailed study of adipose tissue innervation in metabolic function, yet, the precise anatomical extent of preganglionic and postganglionic inputs to the inguinal white adipose tissue (iWAT) is limited. Furthermore, several viral, molecular-genetic tools will require the use of cre/loxP mouse models, while the available studies on sympathetic iWAT innervation were established in larger species. In this study, we generated a detailed map for the sympathetic innervation of iWAT in male and female mice. We adapted iDISCO tissue clearing to process large, whole-body specimens for an unprecedented view of the natural abdominal SNS. Combined with pseudorabies virus retrograde tracing from the iWAT, we defined the preganglionic and postganglionic sympathetic input to iWAT. We used fluorescence-guided anatomical dissections of sympathetic nerves in reporter mice to further clarify that postganglionic axons connect to iWAT via lateral cutaneous rami (dorsolumbar iWAT portion) and the lumbar plexus (inguinal iWAT portion). Importantly, these rami carry axons that branch to iWAT, as well as axons that travel further to innervate the skin and vasculature, and their functional impact will require consideration in denervation studies. Our study may serve as a comprehensive map for future experiments that employ virally driven neuromodulation techniques to predict anatomy-based viral labeling.

Nociceptive nerves regulate haematopoietic stem cell mobilization.

Haematopoietic stem cells (HSCs) reside in specialized microenvironments in the bone marrow-often referred to as 'niches'-that represent complex regulatory milieux influenced by multiple cellular constituents, including nerves1,2. Although sympathetic nerves are known to regulate the HSC niche3-6, the contribution of nociceptive neurons in the bone marrow remains unclear. Here we show that nociceptive nerves are required for enforced HSC mobilization and that they collaborate with sympathetic nerves to maintain HSCs in the bone marrow. Nociceptor neurons drive granulocyte colony-stimulating factor (G-CSF)-induced HSC mobilization via the secretion of calcitonin gene-related peptide (CGRP). Unlike sympathetic nerves, which regulate HSCs indirectly via the niche3,4,6, CGRP acts directly on HSCs via receptor activity modifying protein 1 (RAMP1) and the calcitonin receptor-like receptor (CALCRL) to promote egress by activating the Gαs/adenylyl cyclase/cAMP pathway. The ingestion of food containing capsaicin-a natural component of chili peppers that can trigger the activation of nociceptive neurons-significantly enhanced HSC mobilization in mice. Targeting the nociceptive nervous system could therefore represent a strategy to improve the yield of HSCs for stem cell-based therapeutic agents.

Cell Types Promoting Goosebumps Form a Niche to Regulate Hair Follicle Stem Cells.

Piloerection (goosebumps) requires concerted actions of the hair follicle, the arrector pili muscle (APM), and the sympathetic nerve, providing a model to study interactions across epithelium, mesenchyme, and nerves. Here, we show that APMs and sympathetic nerves form a dual-component niche to modulate hair follicle stem cell (HFSC) activity. Sympathetic nerves form synapse-like structures with HFSCs and regulate HFSCs through norepinephrine, whereas APMs maintain sympathetic innervation to HFSCs. Without norepinephrine signaling, HFSCs enter deep quiescence by down-regulating the cell cycle and metabolism while up-regulating quiescence regulators Foxp1 and Fgf18. During development, HFSC progeny secretes Sonic Hedgehog (SHH) to direct the formation of this APM-sympathetic nerve niche, which in turn controls hair follicle regeneration in adults. Our results reveal a reciprocal interdependence between a regenerative tissue and its niche at different stages and demonstrate sympathetic nerves can modulate stem cells through synapse-like connections and neurotransmitters to couple tissue production with demands.

Nardilysin controls cardiac sympathetic innervation patterning through regulation of p75 neurotrophin receptor.

Cardiac sympathetic innervation is critically involved in the regulation of circulatory dynamics. However, the molecular mechanism for the innervation patterning has remained elusive. Here, we demonstrate that nardilysin (NRDC, Nrdc), an enhancer of ectodomain shedding, regulates cardiac sympathetic innervation. Nardilysin-deficient (Nrdc-/- ) mice show hypoplastic hearts, hypotension, bradycardia, and abnormal sympathetic innervation patterning. While the innervation of left ventricle (LV) of wild-type mice is denser in the subepicardium than in the subendocardium, Nrdc-/- LV lacks such a polarity and is uniformly and more abundantly innervated. At the molecular level, the full-length form of p75 neurotrophin receptor (p75NTR , Ngfr) is increased in Nrdc-/- LV due to the reduced ectodomain shedding of p75NTR . Importantly, the reduction of p75NTR rescued the abnormal innervation phenotype of Nrdc-/- mice. Moreover, sympathetic neuron-specific, but not cardiomyocyte-specific deletion of Nrdc recapitulated the abnormal innervation patterning of Nrdc-/- mice. In conclusion, neuronal nardilysin critically regulates cardiac sympathetic innervation and circulatory dynamics via modulation of p75NTR .

Microbiota modulate sympathetic neurons via a gut-brain circuit.

Connections between the gut and brain monitor the intestinal tissue and its microbial and dietary content1, regulating both physiological intestinal functions such as nutrient absorption and motility2,3, and brain-wired feeding behaviour2. It is therefore plausible that circuits exist to detect gut microorganisms and relay this information to areas of the central nervous system that, in turn, regulate gut physiology4. Here we characterize the influence of the microbiota on enteric-associated neurons by combining gnotobiotic mouse models with transcriptomics, circuit-tracing methods and functional manipulations. We find that the gut microbiome modulates gut-extrinsic sympathetic neurons: microbiota depletion leads to increased expression of the neuronal transcription factor cFos, and colonization of germ-free mice with bacteria that produce short-chain fatty acids suppresses cFos expression in the gut sympathetic ganglia. Chemogenetic manipulations, translational profiling and anterograde tracing identify a subset of distal intestine-projecting vagal neurons that are positioned to have an afferent role in microbiota-mediated modulation of gut sympathetic neurons. Retrograde polysynaptic neuronal tracing from the intestinal wall identifies brainstem sensory nuclei that are activated during microbial depletion, as well as efferent sympathetic premotor glutamatergic neurons that regulate gastrointestinal transit. These results reveal microbiota-dependent control of gut-extrinsic sympathetic activation through a gut-brain circuit.

A novel postsynaptic signal pathway of sympathetic neural regulation of murine colonic motility.

Transcriptome data revealed α1 adrenoceptors (ARs) expression in platelet-derived growth factor receptor α+ cells (PDGFRα+ cells) in murine colonic musculature. The role of PDGFRα+ cells in sympathetic neural regulation of murine colonic motility was investigated. Norepinephrine (NE), via α1A ARs, activated a small conductance Ca2+ -activated K+ (SK) conductance, evoked outward currents and hyperpolarized PDGFRα+ cells (the α1A AR-SK channel signal pathway). α1 AR agonists increased intracellular Ca2+ transients in PDGFRα+ cells and inhibited spontaneous phasic contractions (SPCs) of colonic muscle through activation of a SK conductance. Sympathetic nerve stimulation inhibited both contractions of distal colon and propulsive contractions represented by the colonic migrating motor complexes (CMMCs) via the α1A AR-SK channel signal pathway. Postsynaptic signaling through α1A ARs in PDGFRα+ cells is a novel mechanism that conveys part of stress responses in the colon. PDGFRα+ cells appear to be a primary effector of sympathetic neural regulation of murine colonic motility.

Maintenance of CaV2.2 channel-current by PIP2 unveiled by neomycin in sympathetic neurons of the rat.

Membrane lipids are key determinants in the regulation of voltage-gated ion channels. Phosphatidylinositol 4,5-bisphosphate (PIP2), a native membrane phospholipid, has been involved in the maintenance of the current amplitude and in the voltage-independent regulation of voltage-gated calcium channels (VGCC). However, the nature of the PIP2 regulation on VGCC has not been fully elucidated. This work aimed to investigate whether the interacting PIP2 electrostatic charges may account for maintaining the current amplitude of CaV2.2 channels. Furthermore, we tested whether charge shielding of PIP2 mimics the voltage-independent inhibition induced by M1 muscarinic acetylcholine receptor (M1R) activation. Therefore, neomycin, a polycation that has been shown to block electrostatic interactions of PIP2, was intracellularly dialyzed in superior cervical ganglion (SCG) neurons of the rat. Consistently, neomycin time-dependently diminished the calcium current amplitude letting the channel exhibit the hallmarks of the voltage-independent regulation. These results support that interacting PIP2 charges not only underly the maintenance of the channel-current but also that charge screening of PIP2 by itself unveils the voltage-independent features of CaV2.2 channels in SCG neurons.

Towards a clearer view of sympathetic innervation of cardiac and skeletal muscles.

It is well appreciated that autonomic neurons have a central role in the homeostatic regulation of organs and systems and participate to the pathogenesis of several disease conditions. As such, the function and signalling pathways activated by sympathetic neurons (SNs) in different cell types and organs have become a matter of intense investigation throughout the years of modern biomedical research. This review is focused on the methods used to address sympathetic innervation of cardiac and skeletal muscles which, quite surprisingly, has remained incompletely understood, mainly due to the technical limitations of the traditional methodologies. The current review provides a summary of the existing literature and, putting together the results obtained with different methodological approaches, provides a comprehensive view of the complexity of the SN network in striated muscles.

Age-Related Dopaminergic Innervation Augments T Helper 2-Type Allergic Inflammation in the Postnatal Lung.

Young children are more susceptible to developing allergic asthma than adults. As neural innervation of the peripheral tissue continues to develop after birth, neurons may modulate tissue inflammation in an age-related manner. Here we showed that sympathetic nerves underwent a dopaminergic-to-adrenergic transition during post-natal development of the lung in mice and humans. Dopamine signaled through a specific dopamine receptor (DRD4) to promote T helper 2 (Th2) cell differentiation. The dopamine-DRD4 pathway acted synergistically with the cytokine IL-4 by upregulating IL-2-STAT5 signaling and reducing inhibitory histone trimethylation at Th2 gene loci. In murine models of allergen exposure, the dopamine-DRD4 pathway augmented Th2 inflammation in the lungs of young mice. However, this pathway operated marginally after sympathetic nerves became adrenergic in the adult lung. Taken together, the communication between dopaminergic nerves and CD4+ T cells provides an age-related mechanism underlying the susceptibility to allergic inflammation in the early lung.

Chromaffin Cells of the Adrenal Medulla: Physiology, Pharmacology, and Disease.

Chromaffin cells (CCs) of the adrenal gland and the sympathetic nervous system produce the catecholamines (epinephrine and norepinephrine; EPI and NE) needed to coordinate the bodily "fight-or-flight" response to fear, stress, exercise, or conflict. EPI and NE release from CCs is regulated both neurogenically by splanchnic nerve fibers and nonneurogenically by hormones (histamine, corticosteroids, angiotensin, and others) and paracrine messengers [EPI, NE, adenosine triphosphate, opioids, γ-aminobutyric acid (GABA), etc.]. The "stimulus-secretion" coupling of CCs is a Ca2+ -dependent process regulated by Ca2+ entry through voltage-gated Ca2+ channels, Ca2+ pumps, and exchangers and intracellular organelles (RE and mitochondria) and diffusible buffers that provide both Ca2+ -homeostasis and Ca2+ -signaling that ultimately trigger exocytosis. CCs also express Na+ and K+ channels and ionotropic (nAChR and GABAA ) and metabotropic receptors (mACh, PACAP, ß-AR, 5-HT, histamine, angiotensin, and others) that make CCs excitable and responsive to autocrine and paracrine stimuli. To maintain high rates of E/NE secretion during stressful conditions, CCs possess a large number of secretory chromaffin granules (CGs) and members of the soluble NSF-attachment receptor complex protein family that allow docking, fusion, and exocytosis of CGs at the cell membrane, and their recycling. This article attempts to provide an updated account of well-established features of the molecular processes regulating CC function, and a survey of the as-yet-unsolved but important questions relating to CC function and dysfunction that have been the subject of intense research over the past 15 years. Examples of CCs as a model system to understand the molecular mechanisms associated with neurodegenerative diseases are also provided. Published 2019. Compr Physiol 9:1443-1502, 2019.

Changes of the Expression of Neuronal NO-Synthase in Rat Sympathetic Ganglia during Ontogeny.

Expression of neuronal NO synthase in the sympathetic cranial cervical ganglion and stellate ganglion in rats during postnatal ontogeny was studied by immunohistochemistry and Western blotting. In the sympathetic ganglia, neuronal NO synthase-immunoreactive neurons were absent in all rats. In the stellate and cranial cervical ganglia, the expression of neuronal NO synthase and the density of immunoreactive fibers increased in early postnatal ontogeny from the moment of birth to the age of 30 days and then decreased. Thus, we observed heterochroneous expression of neuronal NOS in the preganglionic somata in the spinal cord and in the preganglionic fibers in the sympathetic ganglia during ontogeny.

Dual Sympathetic Input into Developing Salivary Glands.

Neuronal signaling is known to be required for salivary gland development, with parasympathetic nerves interacting with the surrounding tissues from early stages to maintain a progenitor cell population and control morphogenesis. In contrast, postganglionic sympathetic nerves arrive late in salivary gland development to perform a secretory function; however, no previous report has shown their role during development. Here, we show that a subset of neuronal cells within the parasympathetic submandibular ganglion (PSG) express the catecholaminergic marker tyrosine hydroxylase (TH) in developing murine and human submandibular glands. This sympathetic phenotype coincided with the expression of transcription factor Hand2 within the PSG from the bud stage (E12.5) of mouse embryonic salivary gland development. Hand2 was previously associated with the decision of neural crest cells to become sympathetic in other systems, suggesting a role in controlling neuronal fate in the salivary gland. The PSG therefore provides a population of TH-expressing neurons prior to the arrival of the postganglionic sympathetic axons from the superior cervical ganglion at E15.5. In culture, in the absence of nerves from the superior cervical ganglion, these PSG-derived TH neurons were clearly evident forming a network around the gland. Chemical ablation of dopamine receptors in explant culture with the neurotoxin 6-hydroxydopamine at early stages of gland development resulted in specific loss of the TH-positive neurons from the PSG, and subsequent branching was inhibited. Taken altogether, these results highlight for the first time the detailed developmental time course of TH-expressing neurons during murine salivary gland development and suggest a role for these neurons in branching morphogenesis.

MicroRNAs are Necessary for BMP-7-induced Dendritic Growth in Cultured Rat Sympathetic Neurons.

Neuronal connectivity is dependent on size and shape of the dendritic arbor. However, mechanisms controlling dendritic arborization, especially in the peripheral nervous system, are not completely understood. Previous studies have shown that bone morphogenetic proteins (BMPs) are important initiators of dendritic growth in peripheral neurons. In this study, we examined the hypothesis that post-transcriptional regulation mediated by microRNAs (miRNAs) is necessary for BMP-7-induced dendritic growth in these neurons. To examine the role of miRNAs in BMP-7-induced dendritic growth, microarray analyses was used to profile miRNA expression in cultured sympathetic neurons from the superior cervical ganglia of embryonic day 21 rat pups at 6 and 24 h after treatment with BMP-7 (50 ng/mL). Our data showed that BMP-7 significantly regulated the expression of 43 of the 762 miRNAs. Of the 43 miRNAs, 22 showed robust gene expression; 14 were upregulated by BMP-7 and 8 were downregulated by BMP-7. The expression profile for miR-335, miR-664-1*, miR-21, and miR-23b was confirmed using qPCR analyses. Functional studies using morphometric analyses of dendritic growth in cultured sympathetic neurons transfected with miRNA mimics and inhibitors indicated that miR-664-1*, miR-23b, and miR-21 regulated early stages of BMP-7-induced dendritic growth. In summary, our data provide evidence for miRNA-mediated post-transcriptional regulation as important downstream component of BMP-7 signaling during early stages of dendritic growth in sympathetic neurons.

Innervation of thermogenic adipose tissue via a calsyntenin 3ß-S100b axis.

The sympathetic nervous system drives brown and beige adipocyte thermogenesis through the release of noradrenaline from local axons. However, the molecular basis of higher levels of sympathetic innervation of thermogenic fat, compared to white fat, has remained unknown. Here we show that thermogenic adipocytes express a previously unknown, mammal-specific protein of the endoplasmic reticulum that we term calsyntenin 3ß. Genetic loss or gain of expression of calsyntenin 3ß in adipocytes reduces or enhances functional sympathetic innervation, respectively, in adipose tissue. Ablation of calsyntenin 3ß predisposes mice on a high-fat diet to obesity. Mechanistically, calsyntenin 3ß promotes endoplasmic-reticulum localization and secretion of S100b-a protein that lacks a signal peptide-from brown adipocytes. S100b stimulates neurite outgrowth from sympathetic neurons in vitro. A deficiency of S100b phenocopies deficiency of calsyntenin 3ß, and forced expression of S100b in brown adipocytes rescues the defective sympathetic innervation that is caused by ablation of calsyntenin 3ß. Our data reveal a mammal-specific mechanism of communication between thermogenic adipocytes and sympathetic neurons.